JP6052485B2 - Immune activated lactic acid bacteria composition and immune activated lactic acid fermented food - Google Patents
Immune activated lactic acid bacteria composition and immune activated lactic acid fermented food Download PDFInfo
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Description
本発明は、免疫賦活作用を有する新規な免疫活性化乳酸菌組成物及び免疫活性化乳酸発酵食品に関し、特に、各種なれずし食品から単離される特定の乳酸菌の菌体等を含有する免疫活性化乳酸菌組成物及び免疫活性化乳酸発酵食品に関する。 The present invention relates to a novel immunostimulatory lactic acid bacterium composition and immunostimulatory lactic acid fermented food having an immunostimulatory action, and in particular, immunostimulation containing cells of specific lactic acid bacteria isolated from various kinds of foods The present invention relates to a lactic acid bacteria composition and an immunostimulated lactic acid fermented food.
消化管や呼吸器粘膜に分布する免疫システムは、免疫系全体の６０％以上を占め、その特徴的な免疫活動により、外来異物の認識・排除を行い、生命活動を維持するのに極めて重要な役割を担っている。 The immune system distributed in the gastrointestinal tract and respiratory mucosa occupies more than 60% of the immune system, and it is extremely important for recognizing and eliminating foreign substances and maintaining vital activities through its characteristic immune activity. Have a role.
粘膜免疫システムの中でも消化管免疫は，食物からの栄養源の取り込みを監視する他、微生物や食物由来の成分、薬物などの影響を受け、腸内環境の保全や食物アレルギー応答の制御、免疫寛容、造血などの生体応答と密接に関連している。この特徴的な消化管免疫反応は、パイエル板という腸の特殊なリンパ組織によって担われ、このリンパの機能を制御するアジュバント活性様のメカニズムが経口可能なワクチンや抗アレルギー薬開発のための新規な薬理学的作用機序として注目されている（例えば、非特許文献１を参照）。 Among the mucosal immune systems, gastrointestinal immunity monitors the intake of nutrients from food, is affected by microorganisms, food-derived components, drugs, etc., protects the intestinal environment, controls food allergic responses, and immune tolerance It is closely related to biological responses such as hematopoiesis. This characteristic gastrointestinal immune response is borne by a special lymphoid tissue of the intestine called Peyer's patch, and an adjuvant-like mechanism that controls the function of this lymph is a novel novel for the development of orally available vaccines and antiallergic drugs. It attracts attention as a pharmacological mechanism of action (see, for example, Non-Patent Document 1).
一方で、石川県下で造られる発酵食品には、あじなれずし、かぶらずし、だいこんずし、いかこうじ漬け等、独自性のある発酵食品が多く、この発酵プロセスには、多くの乳酸菌が関与している。 On the other hand, fermented foods made in Ishikawa Prefecture have many unique fermented foods such as Ajinarezushi, Kaburazushi, Daikonzushi, and pickled squid, and this fermentation process involves many lactic acid bacteria. Is involved.
前記なれずしの遊離アミノ酸には、γ−アミノ酪酸を多く含むものがあり、このγ−アミノ酪酸の人等の哺乳動物に対する代表的な生理作用として、脳機能改善、精神安定化、血圧上昇抑制等が知られている。また、なれずしの有機酸量の主成分である乳酸は、乳酸菌によって生成されるが、乳酸菌は、生菌、死菌であっても、その菌体成分が生体に吸収されるか、あるいは腸内細菌を介して、免疫機能の増強や肝機能の促進等に有利に働くとされている。 Some of the free amino acids in the above-described natural amino acids contain a large amount of γ-aminobutyric acid. As typical physiological actions of γ-aminobutyric acid on mammals such as humans, brain function improvement, mental stabilization, blood pressure increase Suppression and the like are known. In addition, lactic acid, which is the main component of the organic acid amount of Nazushishi, is produced by lactic acid bacteria, but even if lactic acid bacteria are live or dead bacteria, their cell components are absorbed by the living body, or Via intestinal bacteria, it is said to be advantageous for enhancing immune function and promoting liver function.
前述のなれずし等の乳酸発酵食品は、腸内環境を改善し、生体の健康維持や増進に役立つものと考えられるが、免疫に対する機能性等については、未だ十分に解明されていない。 The aforementioned lactic acid fermented foods such as Narezushi are considered to be useful for improving the intestinal environment and maintaining and promoting the health of the living body, but the functionality for immunity has not been fully elucidated.
本発明は、このような実情に鑑みて提案されたものであり、石川県下で造られる発酵食品の粘膜応答に及ぼす影響や作用機序を遺伝子レベルで解明し、腸管機能に優れ、ＩＦＮ−γ産生増強作用やＩＬ−２産生増強作用を持った乳酸菌を見出すことにより、健康維持に役立つ乳酸菌組成物や乳酸発酵食品を提供することを目的とする。 The present invention has been proposed in view of such circumstances, elucidating the effect and mechanism of action on the mucosal response of fermented foods produced in Ishikawa Prefecture at the gene level, having excellent intestinal function, and IFN-γ. An object of the present invention is to provide a lactic acid bacteria composition and a lactic acid fermented food useful for maintaining health by finding lactic acid bacteria having a production enhancing action and an IL-2 production enhancing action .
本発明者らは、石川県の特産の発酵食品について、発酵菌の菌叢解析を行うとともに、ＤＮＡマイクロアレイ法によって腸管の機能を評価することで、免疫活性化に優れた菌を見出すに至った。本発明は、ＩＦＮ−γ産生増強作用やＩＬ−２産生増強作用が見出された乳酸菌を用いて、免疫活性化に優れた乳酸菌組成物及び乳酸発酵食品を提供するものである。 The inventors of the present invention have conducted a bacterial flora analysis of fermented bacteria for specially fermented foods in Ishikawa Prefecture, and have also found bacteria with excellent immune activation by evaluating the function of the intestinal tract by the DNA microarray method. . The present invention provides a lactic acid bacterium composition and a lactic acid fermented food excellent in immune activation using a lactic acid bacterium that has been found to have an IFN-γ production enhancing action or an IL-2 production enhancing action .
すなわち、本発明のＩＦＮ−γ産生増強剤は、あじなれずし食品由来のラクトバチルス・ブレビス（Lactobacillus brevis）、ぶりなれずし食品由来のラクトバチルス・パラブチネリ（Lactobacillus parabuchneri）から選択される少なくとも１種の菌体の乾燥粉末体を含有するＩＦＮ−γ産生増強剤であって、
前記あじなれずし食品由来の前記ラクトバチルス・ブレビス（Lactobacillus brevis）が、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 受託番号ＮＩＴＥ Ｐ−１２６０で寄託されたラクトバチルス・ブレビス（Lactobacillus brevis）ＳＢ１０９であり、前記ぶりなれずし食品由来のラクトバチルス・パラブチネリ（Lactobacillus parabuchneri）が、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 受託番号ＮＩＴＥ Ｐ−１２５８で寄託されたラクトバチルス・パラブチネリ（Lactobacillus parabuchneri）ＢＮ１−２であって、
前記ラクトバチルス・ブレビス（Lactobacillus brevis）ＳＢ１０９、前記ラクトバチルス・パラブチネリ（Lactobacillus parabuchneri）ＢＮ１−２は、サイトカイン選択的にＩＦＮ−γ産生を制御する用途に使用されることを特徴とする。
That is, the IFN-γ production enhancer of the present invention is at least one selected from Lactobacillus brevis derived from Aji naizushi food and Lactobacillus parabuchneri derived from Lactobacillus parabuchneri. An IFN-γ production enhancer containing a dry powder of bacterial cells of a species,
Lactobacillus brevis (Lactobacillus brevis) SB109, which was deposited under the accession number NITE P-1260 of the National Institute of Technology and Evaluation for the Lactobacillus brevis derived from the hydrangea food The Lactobacillus parabuchneri derived from the above-mentioned food product is the Lactobacillus parabuchneri deposited at the National Institute of Technology and Evaluation Patent Microorganism Depositary Accession Number NITE P-1258. ) BN1-2,
The Lactobacillus brevis SB109 and the Lactobacillus parabuchneri BN1-2 are used for applications that selectively control IFN-γ production.
また、本発明のＩＬ−２産生増強剤は、あじなれずし食品由来のラクトバチルス・ブチネリ（Lactobacillus buchneri）から選択される少なくとも１種の菌体の乾燥粉末体を含有するＩＬ−２産生増強剤であって、
前記あじなれずし食品由来のラクトバチルス・ブチネリ（Lactobacillus buchneri）が、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 受託番号ＮＩＴＥ Ｐ−１１２３で寄託されたラクトバチルス・ブチネリ（Lactobacillus buchneri）ＡＮ１−１であり、
前記ラクトバチルス・ブチネリ（Lactobacillus buchneri）ＡＮ１−１は、サイトカイン選択的にＩＬ−２産生を制御する用途に使用されることを特徴とする。
また、本発明の乳酸発酵食品は、これらのＩＦＮ−γ産生増強剤、ＩＬ−２産生増強剤を用い、食品原料を乳酸発酵させたことを特徴とする。
In addition, the IL-2 production enhancer of the present invention enhances IL-2 production containing a dry powder of at least one microbial cell selected from Lactobacillus buchneri derived from Aji naizushi food An agent,
Lactobacillus buchneri derived from the hydrangea food is Lactobacillus buchneri AN1-deposited under the National Institute for Product Evaluation Technology Patent Microorganism Depositary Accession Number NITE P-1123 1 and
The Lactobacillus buchneri AN1-1 is used for the purpose of controlling IL-2 production selectively with cytokines.
Further, the lactic acid fermented food of the present invention is characterized in that the food material is lactic acid fermented using these IFN-γ production enhancer and IL-2 production enhancer.
あじなれずし食品由来のラクトバチルス・ブチネリ（Lactobacillus buchneri）やラクトバチルス・ブレビス（Lactobacillus brevis）、ラクトバチルス・プランタラム（Lactobacillus plantarum）、さらには、ぶりなれずし食品由来のラクトバチルス・パラブチネリ（Lactobacillus parabuchneri）は、腸内での抗体産生や、ＩＦＮ−γ産生増強作用やＩＬ−２産生増強作用等の免疫活性化作用を有する。したがって、これらを含む乳酸菌組成物や、これら乳酸菌を用いて乳酸発酵された乳酸発酵食品は、免疫賦活化作用に優れ、身体の健康維持に役立つ。 Lactobacillus buchneri (Lactobacillus buchneri) and Lactobacillus brevis (Lactobacillus plantarum) derived from Aji nalesushi food, and Lactobacillus plantarum (Lactobacillus plantarum) Lactobacillus parabuchneri) has and antibody production in the gut, immune activating effect, such as IFN-gamma production enhancing action and IL-2 production-enhancing effects. Therefore, lactic acid bacteria compositions containing these and lactic acid fermented foods that have been lactic acid fermented using these lactic acid bacteria are excellent in immunostimulatory action and are useful for maintaining the health of the body.
本発明によれば、ＩＦＮ−γ産生増強作用やＩＬ−２産生増強作用等の免疫活性化作用に優れ、健康維持に有用な乳酸菌組成物を提供することができ、また、これを用いて乳酸発酵させることで、同じく免疫活性作用に優れ、健康維持に有用な乳酸発酵食品を提供することが可能である。
According to the present invention, it is possible to provide a lactic acid bacterium composition that is excellent in immune activation effects such as IFN-γ production enhancing action and IL-2 production enhancing action, and is useful for maintaining health. By fermenting, it is possible to provide a lactic acid fermented food that is also excellent in immune activity and useful for maintaining health.
以下、本発明を適用した乳酸菌組成物及び乳酸発酵食品の実施形態について詳細に説明する。 Hereinafter, embodiments of a lactic acid bacteria composition and a lactic acid fermented food to which the present invention is applied will be described in detail.
本発明の免疫活性化乳酸菌組成物は、「あじなれずし食品」由来のラクトバチルス・ブチネリ（Lactobacillus buchneri）、ラクトバチルス・ブレビス（Lactobacillus
brevis）、ラクトバチルス・プランタラム（Lactobacillus plantarum）、「ぶりなれずし食品」由来のラクトバチルス・パラブチネリ（Lactobacillus parabuchneri）から選択される少なくとも１種を含むことを特徴事項とするものであり、これにより免疫活性を向上させるという機能を有する。
The immunostimulatory lactic acid bacteria composition of the present invention comprises Lactobacillus buchneri and Lactobacillus brevis (Lactobacillus) derived from “Aji nalesushi food”.
brevis), Lactobacillus plantarum, and Lactobacillus parabuchneri derived from “Baburi Nazushi Foods”. It has a function of improving immune activity.
「なれずし」は、塩蔵した魚を米飯と共に漬け込み熟成させた発酵食品であり、いわゆる「ほんなれ」と「なまなれ」とがある。「ほんなれ」は、熟成期間が長く、米飯が粥状になるまでじっくりと熟成させているため、魚だけが食される。「なまなれ」は、熟成期間が短く、発酵の初期段階を利用するため、米飯は食用可能な形状となっており、魚とともに食する。 “Narezushi” is a fermented food that is made by immersing salted fish with rice and aging, and there are so-called “honare” and “namare”. “Honnari” has a long ripening period and is carefully ripened until the rice is in a bowl shape, so only fish is eaten. Namamare has a short ripening period and uses the initial stage of fermentation, so the cooked rice is edible and eaten with fish.
能登地方の「なれずし」は、市販品や一般家庭で作られたもの等があるが、原魚に各種魚が利用されており、熟成期間は約２ヶ月間で、「なまなれ」に類する。この能登地方のアジ（鯵）を用いた「なれずし」が、本発明で言うところの「あじなれずし食品」である。同じく、ぶり（鰤）を用いた「なれずし」が「ぶりなれずし食品」である。 “Naruzushi” in the Noto region includes commercial products and products made in ordinary households, but various fish are used as raw fish, and the aging period is about two months. Similar to This “Naruzushi” using horse mackerel from the Noto region is the “Aji nalesushi food” referred to in the present invention. Similarly, “Narezushi” using a bowl is a “buri nazushi food”.
前述の「なれずし」の発酵プロセスには、多くの乳酸菌が関与しているが、本発明者らは、乳酸菌の菌叢解析を行うとともに、ＤＮＡマイクロアレイ法によりその機能を評価した。その結果、「あじなれずし食品」の乳酸発酵に関与するラクトバチルス・ブチネリ（Lactobacillus buchneri）やラクトバチルス・ブレビス（Lactobacillus
brevis）、ラクトバチルス・プランタラム（Lactobacillus plantarum）、「ぶりなれずし食品」の乳酸発酵に関与するラクトバチルス・パラブチネリ（Lactobacillus parabuchneri）が、腸での抗体産生等、免疫活性化に関して、顕著な活性を有することを見出した。
Although many lactic acid bacteria are involved in the above-mentioned “Naruzushi” fermentation process, the present inventors have analyzed the bacterial flora of lactic acid bacteria and evaluated its function by the DNA microarray method. As a result, Lactobacillus buchneri and Lactobacillus brevis (Lactobacillus) are involved in lactic acid fermentation of “Aji nalesushi food”
brevis), Lactobacillus plantarum, and Lactobacillus parabuchneri, which is involved in lactic acid fermentation of “buri nareshi food”, are prominent in terms of immune activation such as antibody production in the intestine. It was found to have activity.
本発明の乳酸菌組成物において使用する前記ラクトバチルス・ブチネリ（Lactobacillus buchneri）やラクトバチルス・ブレビス（Lactobacillus
brevis）、ラクトバチルス・プランタラム（Lactobacillus plantarum）、ラクトバチルス・パラブチネリ（Lactobacillus parabuchneri）は、菌体そのものであってもよいし、菌体成分や菌体由来成分、菌体処理物等であってもよい。例えば、前記菌体成分は、水やアルコールで抽出することにより得ることができる。
The Lactobacillus buchneri or Lactobacillus brevis (Lactobacillus) used in the lactic acid bacteria composition of the present invention
brevis), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus parabuchneri (Lactobacillus parabuchneri) may be a cell itself, or a cell component, a cell-derived component, a cell-treated product, etc. Also good. For example, the cell component can be obtained by extraction with water or alcohol.
また、前記ラクトバチルス・ブチネリ（Lactobacillus buchneri）やラクトバチルス・ブレビス（Lactobacillus brevis）、ラクトバチルス・プランタラム（Lactobacillus
plantarum）、ラクトバチルス・パラブチネリ（Lactobacillus parabuchneri）は、それぞれ単独で用いてもよいし、これらのうちの２種以上を混合して用いることも可能であり、他の乳酸菌等を添加することも可能である。
In addition, the Lactobacillus buchneri, Lactobacillus brevis, Lactobacillus plantarum (Lactobacillus
plantarum), Lactobacillus parabuchneri (Lactobacillus parabuchneri) may be used alone, or two or more of these may be used in combination, or other lactic acid bacteria may be added. It is.
さらに、本発明の乳酸菌組成物を薬剤等の形態で供する場合には、薬理上許容し得る担体、賦活剤、各種添加物等を加えることも可能である。 Furthermore, when the lactic acid bacteria composition of the present invention is provided in the form of a drug or the like, it is possible to add a pharmacologically acceptable carrier, an activator, various additives and the like.
一方、本発明の免疫活性化乳酸発酵食品は、前記免疫活性化機能を有するラクトバチルス・ブチネリ（Lactobacillus buchneri）やラクトバチルス・ブレビス（Lactobacillus
brevis）、ラクトバチルス・プランタラム（Lactobacillus plantarum）、ラクトバチルス・パラブチネリ（Lactobacillus parabuchneri）を用い、各種食品原料を発酵させたものである。これら免疫活性化機能を有する乳酸菌を予め仕込んでおくことで、特定の乳酸菌（前記ラクトバチルス・ブチネリやラクトバチルス・ブレビス、ラクトバチルス・プランタラム、ラクトバチルス・パラブチネリ）が増殖し、免疫活性化機能に優れた乳酸発酵食品を提供することが可能となる。
On the other hand, the immunostimulated lactic acid fermented food of the present invention includes Lactobacillus buchneri and Lactobacillus brevis (Lactobacillus) having the above-mentioned immunostimulatory function.
brevis), Lactobacillus plantarum, and Lactobacillus parabuchneri, and fermented various food materials. By preparing these lactic acid bacteria having an immune activation function in advance, specific lactic acid bacteria (the aforementioned Lactobacillus butinelli, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus parabutinelli) proliferate, and the immune activation function It is possible to provide a lactic acid fermented food excellent in the quality.
乳酸発酵させる食品原料は任意であり、あらゆる種類の発酵食品に適用することが可能である。例えば、あじなれずしにも適用することができるし、かぶらずし、だいこんずし、いかこうじ漬け等にも適用することができる。勿論、これらに限らず、世の中に知られている乳酸発酵食品の全てに適用することが可能である。発酵に際しては、例えば前記ラクトバチルス・ブチネリやラクトバチルス・ブレビス、ラクトバチルス・プランタラム、ラクトバチルス・パラブチネリを豊富に含む菌叢を利用して発酵食品を造り、製品とすればよい。 The raw material for food to be subjected to lactic acid fermentation is arbitrary, and can be applied to all types of fermented foods. For example, the present invention can be applied to hydrangea sushi, and can also be applied to kabazuzushi, daikon sushi and pickled squid. Of course, the present invention is not limited to these and can be applied to all lactic acid fermented foods known in the world. For fermentation, for example, fermented foods may be made using the above-mentioned bacterial flora rich in Lactobacillus butinelli, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus parabutinelli, and used as a product.
以上のように、本発明の免疫活性化乳酸菌組成物や免疫活性化乳酸発酵食品は、免疫活性化機能を有するラクトバチルス・ブチネリやラクトバチルス・ブレビス、ラクトバチルス・プランタラム、ラクトバチルス・パラブチネリを含有しているので、健康を維持する上で極めて有用なものである。 As described above, the immunostimulatory lactic acid bacteria composition and the immunostimulatory lactic acid fermented food of the present invention include Lactobacillus butinelli, Lactobacillus brevis, Lactobacillus plantarum, and Lactobacillus parabutinelli having an immune activation function. Since it contains, it is very useful in maintaining health.
以下、本発明を適用した具体的な実施例について、実験結果を基に説明する。 Hereinafter, specific examples to which the present invention is applied will be described based on experimental results.
実験方法
数種の「なれずし」から発酵に関係する菌を採取して菌叢解析を実施し、単離された菌をマウスに経口投与し、小腸のリンパ節（パイエル板）細胞が産生するサイトカインのＩＬ−２とＩＦＮ−γの産生を指標に免疫応答に及ぼす影響を調べると共に，小腸のリンパ節を含む組織を採取してｔｏｔａｌ ＲＮＡを採取し、これを２万個の発現遺伝子が解析可能なＤＮＡマイクロアレイに付し、有益な遺伝子群の変動を指標に健康に有用な菌を探索した。
Experimental method Bacteria related to fermentation were collected from several kinds of "Narezushi" and analyzed for flora, and the isolated bacteria were orally administered to mice, and lymph nodes (Peier's plate) of the small intestine were administered. ) Examining the effect of IL-2 and IFN-γ, cytokines produced by cells, on immune responses, collecting tissues including small intestinal lymph nodes and collecting total RNA, 20,000 DNA microarrays that can analyze the expressed genes were used to search for useful bacteria for health using the variation of beneficial genes as an index.
（１）サンプルの作製
菌体を濾過後に凍結乾燥に付し、乾燥粉末体を得た。これをサンプルとして以下の実験に用いた。
（２）実験動物
Ｃ５７ＢＬ／６Ｎ（雄，５ｗ，日本チャールズリバー）を実験時に適宜購入し、金沢大学自然研動物飼育施設で１週間以上飼育、馴化させてから実験に使用した。
(1) Preparation of sample The bacterial cells were subjected to freeze-drying after filtration to obtain a dry powder. This was used as a sample in the following experiment.
(2) Experimental animal C57BL / 6N (male, 5w, Charles River, Japan) was appropriately purchased at the time of the experiment, and was used for the experiment after being bred and acclimatized for more than a week at the Kanazawa University Nature Research Animal Breeding Facility.
（３）サンプルの調製と投与
（１）の方法で得られたサンプルについて、最終投与量が３０ｍｇ／ｋｇ／ｄａｙになるよう、用時、精製水に懸濁して投与サンプルを調製した。この投与サンプルを（２）の条件で飼育したマウスに０．１ｍＬ／１０ｇで１日１回（午前１０時）、連続的に５日間、経口投与した。
(3) Preparation and administration of sample A sample obtained by the method of (1) was prepared by suspending in purified water at the time of use so that the final dose was 30 mg / kg / day. This administration sample was orally administered to mice bred under the condition (2) at 0.1 mL / 10 g once a day (10 am) for 5 consecutive days.
（４）パイエル板構成細胞の採取
初回のサンプル投与開始から７日後に、マウスを過剰のエーテルにより致死せしめ、無菌条件下に小腸を摘出して、散布するパイエル板を実体顕微鏡下に摘出した。得られたパイエル板を冷えたリン酸緩衝液に入れて洗浄後、７５０Ｕ／ｍＬのコラゲナーゼ（ｔｙｐｅＩ：シグマ・アルドリッチ社製）完全培地（ＲＰＭＩ−１６４０）に投入し、１時間インキュベートした。インキュベート終了後、分離したパイエル板構成リンパ球を２００μｍのナイロンメッシュを通してシングルセル懸濁液として細胞数を計測した。
(4) Collection of Peyer's patch constituent cells Seven days after the start of the first sample administration, the mice were killed with excess ether, the small intestine was removed under aseptic conditions, and the Peyer's patch to be sprayed was removed under a stereomicroscope. The obtained Peyer's board was washed in a cold phosphate buffer, and then placed in 750 U / mL collagenase (type I: Sigma-Aldrich) complete medium (RPMI-1640) and incubated for 1 hour. After completion of the incubation, the number of cells was counted as a single cell suspension of the separated Peyer's plate lymphocytes through a 200 μm nylon mesh.
パイエル板構成細胞を３×１０６ｃｅｌｌｓ／ｍＬの濃度となるように５％ＦＣＳ化ＲＰＭＩ−１６４０培地に再浮遊して調製し、２４ウエル培養プレートに１ｍＬずつ播種した。５％ＣＯ２気流下、３７℃で１時間の安定培養を行った後に、最終濃度が５μｇ／ｍＬのConcanavalin
A（ＣｏｎＡ），あるいは１μｇ／ｍＬのlipopolysaccharide（ＬＰＳ）を各ウエルに添加し、４８〜１４４時間、静置培養した。
Peyer's plate constituting cells were prepared by resuspending in 5% FCS-modified RPMI-1640 medium to a concentration of 3 × 10 6 cells / mL, and seeding 1 mL each in a 24-well culture plate. After stable culture at 37 ° C. for 1 hour in a 5% CO 2 stream, Concanavalin with a final concentration of 5 μg / mL
A (ConA) or 1 μg / mL lipopolysaccharide (LPS) was added to each well, followed by static culture for 48 to 144 hours.
（５）培養液上清中のＴｈ１およびＴｈ２関連サイトカインの測定
ＣｏｎＡによる刺激培養終了後に、培養液上清を回収し、ここに含まれるＴヘルパー細胞による免疫調節因子であるＩＬ−２とＩＦＮ−γ（Ｔｈ１）、ＩＬ−４とＩＬ−５（Ｔｈ２）を市販のＥＬＩＳＡ ｋｉｔ（Ｂｉｏｓｃｉｅｎｃｅ社製）を用いて測定した。測定方法は、ｋｉｔの添付文書に従い、形成する発色色素の吸光度を測定し、標準物質による検量線からそれぞれのサイトカイン産生量を算定した。
(5) Measurement of Th1 and Th2-related cytokines in culture supernatant After completion of stimulation culture with ConA, the culture supernatant is recovered, and IL-2 and IFN- which are immunoregulatory factors by T helper cells contained therein γ (Th1), IL-4, and IL-5 (Th2) were measured using a commercially available ELISA kit (manufactured by Bioscience). The measuring method was to measure the absorbance of the chromogenic dye to be formed according to the package insert of the kit, and to calculate the amount of each cytokine produced from the calibration curve of the standard substance.
発酵食品から単離できた乳酸菌
（数種のあじなれずし食品由来）
AN1-1: Lactobacillus buchneri
AN1-3: L. alimentarius
AN1-5: L. brevis
AN1-7: L. casei
AN2-2: L. brevis = AN1-5
AN2-3: L. plantarum
AN2-5: L. alimentarius = AN1-3
AN3-2: L. plantarum = AN2-3
AN3-5: L. brevis = AN1-5, AN2-2
AN4-1: L. plantarum = AN2-3, AN3-2
AN5-1: L. paraplantarum
AN5-3: L. plantarum =
AN2-3, AN3-2,
AN4-1
ANP1-4: L. brevis
ANP7-1: L. plantarum
ANP7-4: L. casei
ANP7-6: L. brevis
SB108: Pediococcus ethanolidurans
SB109: L. brevis
Lactic acid bacteria isolated from fermented food (derived from several types of Aji nalesushi food)
AN1-1: Lactobacillus buchneri
AN1-3: L. alimentarius
AN1-5: L. brevis
AN1-7: L. casei
AN2-2: L. brevis = AN1-5
AN2-3: L. plantarum
AN2-5: L. alimentarius = AN1-3
AN3-2: L. plantarum = AN2-3
AN3-5: L. brevis = AN1-5, AN2-2
AN4-1: L. plantarum = AN2-3, AN3-2
AN5-1: L. paraplantarum
AN5-3: L. plantarum =
AN2-3, AN3-2,
AN4-1
ANP1-4: L. brevis
ANP7-1: L. plantarum
ANP7-4: L. casei
ANP7-6: L. brevis
SB108: Pediococcus ethanolidurans
SB109: L. brevis
（ぶりなれずし食品由来）
BN1-2: L. parabuchneri
(Derived from burari nazushi food)
BN1-2: L. parabuchneri
（さばなれずし食品由来）
SN1-7: L. alimentarius
SN1-8: L. parabuchneri
（いかこうじ漬け食品由来）
SB103: Enterococcus faecalis
（いか黒造り食品由来）
SB105: Weissella hellenica
(Saba nalesushi food origin)
SN1-7: L. alimentarius
SN1-8: L. parabuchneri
(From squid koji pickled food)
SB103: Enterococcus faecalis
(From squid black food)
SB105: Weissella hellenica
上記に示される乳酸菌のうち、ラクトバチラス・ブチネリ（Lactobacillus
buchneri） AN1-1、ラクトバチルス・ブレビス（Lactobacillus brevis） AN1-5、AN3-5およびSB109、ラクトバチルス・プランタラム (Lactobacillus
plantarum) AN3-2およびANP7-1、ラクトバチルス・パラブチネリ（Lactobacillus parabuchneri） BN1-2、エンテロコッカス・フェカリス（Enterococcus faecalis） SB103、ワイセラ・ヘレニカ（Weissella hellenica）SB105及びぺディオコッカス・エタノリデュランス(Pediococcus ethanolidurans) SB108は、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に寄託申請し、以下の受託番号で受託された。
Among the lactic acid bacteria shown above, Lactobacillus butinelli (Lactobacillus
buchneri) AN1-1, Lactobacillus brevis AN1-5, AN3-5 and SB109, Lactobacillus plantarum (Lactobacillus
plantarum) AN3-2 and ANP7-1, Lactobacillus parabuchneri BN1-2, Enterococcus faecalis SB103, Weissella hellenica SB105 and Pediococcus etanocodudus ccus SB108 applied for deposit at the Patent Microorganism Depositary Center for Product Evaluation Technology, Japan, and was deposited with the following deposit number.
Lactobacillus buchneri AN1-1（受託番号：NITE P-1123）
Lactobacillus brevis AN1-5（受託番号：NITE P-1124）
Lactobacillus plantarum AN3-2（受託番号：NITE P-1255）
Lactobacillus brevis AN3-5（受託番号：NITE P-1256）
Lactobacillus parabuchneri BN1-2（受託番号：NITE P-1258）
Lactobacillus plantarum ANP7-1（受託番号：NITE P-1224）
Enterococcus faecalis SB103（受託番号：NITE P-1127）
Weissella hellenica SB105（受託番号：NITE P-1128）
Pediococcus ethanolidurans SB108（受託番号：NITE P-1228）
Lactobacillus brevis SB109（受託番号：NITE P-1260）
Lactobacillus buchneri AN1-1 (Accession number: NITE P-1123)
Lactobacillus brevis AN1-5 (Accession number: NITE P-1124)
Lactobacillus plantarum AN3-2 (Accession number: NITE P-1255)
Lactobacillus brevis AN3-5 (Accession number: NITE P-1256)
Lactobacillus parabuchneri BN1-2 (Accession number: NITE P-1258)
Lactobacillus plantarum ANP7-1 (Accession number: NITE P-1224)
Enterococcus faecalis SB103 (Accession number: NITE P-1127)
Weissella hellenica SB105 (Accession number: NITE P-1128)
Pediococcus ethanolidurans SB108 (Accession number: NITE P-1228)
Lactobacillus brevis SB109 (Accession number: NITE P-1260)
単離乳酸菌の生物活性の要約
あじなれずしから単離された乳酸菌のうちＡＮ１−１（Lactobacillus
buchneri）、ＳＢ１０９（Lactobacillus
brevis）およびＡＮＰ７−１（L. plantarum）、ぶりなれずしから単離されたＢＮ１−２（Lactobacillus parabuchneri）については、抗体産生など免疫活性化の活性があった。さらに、「あじなれずし由来」のＡＮ１−１およびＡＮＰ７−１については、腸管の免疫賦活化活性の他に、ＤＮＡマイクロアレイの結果において、interferon-induced protein with tetratricopeptide repeats 1、interferon activated gene set、guanylate nucleotide binding proteins、2'-5' oligoadenylate synthetases、およびchemokine (C-C motif) ligand等のウイルス感染の防御に役立つ遺伝子が強く上昇していた。
Summary of biological activity of isolated lactic acid bacteria AN1-1 (Lactobacillus) among lactic acid bacteria isolated from Aji naizushi
buchneri), SB109 (Lactobacillus
brevis), ANP7-1 (L. plantarum), and BN1-2 (Lactobacillus parabuchneri) isolated from Buri nazushi, had immune activation activities such as antibody production. Further, for AN1-1 and ANP7-1 derived from “Ajinarezushi”, in addition to intestinal immunostimulatory activity, in the results of DNA microarray, interferon-induced protein with tetratricopeptide repeats 1, interferon activated gene set, Genes useful for protection against viral infection such as guanylate nucleotide binding proteins, 2'-5 'oligoadenylate synthetases, and chemokine (CC motif) ligand were strongly elevated.
また、ぶり，あるいはあじのなれずしから単離された乳酸菌のうち、ＢＮ１−２（Lactobacillus
parabuchneri）とＳＢ１０９（Lactobacillus brevis）の２種は、消化管免疫関連サイトカインのうち、ＩＬ−２産生には影響せず、ＩＦＮ−γの産生を選択的に増加させた。このことから、これらの乳酸菌は、腸における単球系の免疫を活性化する作用があり、これらの菌および菌叢を豊富に含む食品や、これらの菌を用いて発酵させた食材は、食中毒の防御や消化管における感染症を緩和するのに役立つ機能性食品となるものと考えられる。
In addition, among lactic acid bacteria isolated from yellowtail or hydrangea, BN1-2 (Lactobacillus
Parabuchneri) and SB109 (Lactobacillus brevis), among gastrointestinal immunity related cytokines, did not affect IL-2 production and selectively increased IFN-γ production. Therefore, these lactic acid bacteria have the effect of activating monocyte-based immunity in the intestines, and foods rich in these bacteria and bacterial flora and foods fermented using these bacteria are food poisoning. It is considered to be a functional food that is useful for protecting the body and relieving infections in the digestive tract.
小腸におけるサイトカイン産生の結果（免疫賦活作用）
（あじなれずし食品由来の乳酸菌について）
「あじなれずし製品」から単離された乳酸菌でＡＮおよびＡＮＰシリーズの１６種について、培養菌体をマウスに経口投与してパイエル板構成細胞によるＴｈサイトカイン産生に及ぼす影響を調べた。その結果、Ｔｈ１サイトカイン産生のうちＩＬ−２あるいはＩＦＮ−γのいずれかの産生を変化させたのは、ＡＮ１−１およびＡＮＰ７−１の２種の菌であった。
Results of cytokine production in the small intestine (immunostimulatory effect)
(About lactic acid bacteria derived from Aji Nalesushi Food)
With regard to 16 types of AN and ANP series of lactic acid bacteria isolated from “Aji nalesushi product”, cultured cells were orally administered to mice to examine the effects on the production of Th cytokines by Peyer's patch constituent cells. As a result, two types of bacteria, AN1-1 and ANP7-1, changed the production of either IL-2 or IFN-γ among the Th1 cytokine production.
先ず、ＡＮ１−１（L. buchneri）であるが、図２に示す通り、３０ｍｇ／ｋｇ／ｄａｙの用量のＡＮ１−１をマウスに経口投与することにより、ＩＬ−２で１．５倍の有意な産生上昇が見られた。なお、ＡＮ１−１の経口投与はパイエル板のＩＦＮ−γ産生に影響しない（図１）。また、データには示さないが、Ｔｈ２のサイトカイン、ＩＬ−４とＩＬ−５産生には、ＡＮ１−１は影響しないことがわかった。他のＡＮ１−３（L. alimentarius）、ＡＮ１−５（L. brevis）の乳酸菌は、Ｔｈ１のサイトカイン産生には影響を及ぼさなかった。 First, AN1-1 (L. buchneri), and as shown in FIG. 2, by administering AN1-1 at a dose of 30 mg / kg / day to mice orally, it was 1.5 times significant in IL-2. A significant increase in production was observed. In addition, oral administration of AN1-1 does not affect IFN-γ production of Peyer's patches (FIG. 1). Although not shown in the data, it was found that AN1-1 does not affect the production of Th2 cytokines, IL-4 and IL-5. Other AN1-3 (L. alimentarius) and AN1-5 (L. brevis) lactic acid bacteria did not affect Th1 cytokine production.
次に、ＡＮＰ７−１（L. plantarum ）であるが、３０ｍｇ／ｋｇ／ｄａｙの用量のＡＮＰ７−１をマウスに５日間経口投与すると、ＩＦＮ−γで１．８倍の有意な産生上昇が見られ（図３参照）、ＩＬ−２においても有意差はないが、１．３倍の産生増強作用が認められた（図４参照）。 Next, regarding ANP7-1 (L. plantarum), when ANP7-1 at a dose of 30 mg / kg / day was orally administered to mice for 5 days, a significant increase in production by 1.8 times was observed with IFN-γ. Although there was no significant difference in IL-2, a 1.3-fold production enhancing action was observed (see FIG. 4).
以上のことから、あじなれずし由来の２種の乳酸菌［ＡＮ１−１（L. buchneri）とＡＮＰ７−１（L. plantarum ）］について、消化管免疫の中でも腸内環境の改善や感染予防、及びアレルギーの緩和等の作用を示すものと考えられる。 Based on the above, two types of lactic acid bacteria [AN1-1 (L. buchneri) and ANP7-1 (L. plantarum)] derived from Aji naizushi have improved intestinal environment and prevent infection, In addition, it is considered to exhibit actions such as allergy relief.
（ぶりなれずし食品由来及びさばなれずし食品由来の乳酸菌について）
「ぶりなれずし食品」及び「さばなれずし食品」の製品から単離された乳酸菌のＢＮ，ＳＮの２シリーズ３種について、マウスパイエル板構成細胞によるサイトカイン産生によるスクリーニングを行った。
(About lactic acid bacteria derived from burabarezushi food and sabarashizushi food)
Two types of BN and SN of the lactic acid bacteria isolated from the products of “Baburi Nazushi Food” and “Sabana Nazushi Food” were screened by cytokine production by mouse Peyer's patch constituent cells.
ＢＮ１−２（L.parabuchneri）は、先のＡＮＰ７−１等とは分離された食品が異なり、「ぶりなれずし」由来である。ＢＮ１−２菌は、図５に示す通り、ＡＮＰ７−１と同様にＩＦＮ−γ産生を増強させるが、その活性はＡＮＰ７−１よりもはるかに強く、１５倍のＩＦＮ−γ産生増強活性を認めた。しかしながら、図６に示す通り、ＩＬ−２産生には影響を与えなかった。 BN1-2 (L. parabuchneri) is different from the above-mentioned ANP7-1 and the like, and is derived from “Kabari Nazushi”. As shown in FIG. 5, BN1-2 enhances IFN-γ production in the same manner as ANP7-1, but its activity is much stronger than ANP7-1, and 15-fold increase in IFN-γ production was observed. It was. However, as shown in FIG. 6, it did not affect IL-2 production.
ＳＮ１−７（L.alimentarius）については、ＳＮ１−７の３０ｍｇ／ｋｇ／ｄａｙをマウスに経口投与することにより、ＩＦＮ−γ産生は２倍に増加した。なお、ＩＬ−２産生に対してＳＮ１−７は影響しなかった。 For SN1-7 (L. alimentarius), IFN-γ production was increased 2-fold by orally administering 30 mg / kg / day of SN1-7 to mice. In addition, SN1-7 did not affect IL-2 production.
（あじなれずし食品由来の他の乳酸菌について）
ＳＢ１０８（Pediococcus ethanolidurans）とＳＢ１０９（L.brevis）の２種は、いずれもあじなれずしより分離された乳酸菌で、ＳＢ１０９はＡＮシリーズのＡＮＰ７−６と同種であり、先の検討ではＴｈサイトカインに影響を与えなかった。しかしながら、同じあじなれずし由来のＳＢ１０９は、図５に示したように、何も処理しないマウスに比較して１６倍以上の強いＩＦＮ−γ産生を示した。一方、ＳＢ１０８は、転じてＩＦＮγ産生を約４０％有意に抑制する活性が認められた。なお、図６に示す通り、ＳＢ１０８も１０９もパイエル板細胞によるＩＬ−２産生には影響を及ぼさなかった。
(About other lactic acid bacteria derived from Aji Nalesushi Food)
SB108 (Pediococcus ethanolidurans) and SB109 (L. brevis) are both lactic acid bacteria isolated from hydrangea, and SB109 is the same as AN series ANP7-6. There was no effect. However, as shown in FIG. 5, SB109 derived from the same hydrangea showed strong IFN-γ production of 16 times or more compared to mice not treated with anything. On the other hand, SB108, in turn, was found to have an activity of significantly suppressing IFNγ production by about 40%. As shown in FIG. 6, neither SB108 nor 109 affected IL-2 production by Peyer's patch cells.
「ぶり」あるいは「あじ」をベースとする「なれずし」から単離した乳酸菌（ＢＮ１−２及びＳＢ１０９）は、強弱の差こそあれ、強いＩＦＮ−γ産生の誘導活性が認められた。あじなれずし由来のＡＮシリーズに認められたのと同様にＩＬ−２の産生には影響しないことから、サイトカイン選択的な制御活性があることが判明した。 Lactic acid bacteria (BN1-2 and SB109) isolated from “Narezushi” based on “Buri” or “Aji” showed strong IFN-γ production-inducing activity, regardless of strength. Since it did not affect the production of IL-2 in the same manner as that observed in the AN series derived from Ajinarizushi, it was found that there is a cytokine-selective regulatory activity.
Claims (3)
前記あじなれずし食品由来の前記ラクトバチルス・ブレビス（Lactobacillus brevis）が、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 受託番号ＮＩＴＥ Ｐ−１２６０で寄託されたラクトバチルス・ブレビス（Lactobacillus brevis）ＳＢ１０９であり、前記ぶりなれずし食品由来のラクトバチルス・パラブチネリ（Lactobacillus parabuchneri）が、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 受託番号ＮＩＴＥ Ｐ−１２５８で寄託されたラクトバチルス・パラブチネリ（Lactobacillus parabuchneri）ＢＮ１−２であり、
前記ラクトバチルス・ブレビス（Lactobacillus brevis）ＳＢ１０９および前記ラクトバチルス・パラブチネリ（Lactobacillus parabuchneri）ＢＮ１−２は、サイトカイン選択的にＩＦＮ−γ産生を制御する用途に使用されることを特徴とするＩＦＮ−γ産生増強剤。 IFN- containing a dry powder of at least one bacterial cell selected from Lactobacillus brevis derived from Aji nalesushi food and Lactobacillus parabuchneri derived from food a γ production enhancer,
Lactobacillus brevis (Lactobacillus brevis) SB109, which was deposited under the accession number NITE P-1260 of the National Institute of Technology and Evaluation for the Lactobacillus brevis derived from the hydrangea food The Lactobacillus parabuchneri derived from the above-mentioned food product is the Lactobacillus parabuchneri deposited at the National Institute of Technology and Evaluation Patent Microorganism Depositary Accession Number NITE P-1258. ) BN1-2,
The Lactobacillus brevis SB109 and the Lactobacillus parabuchneri BN1-2 are used for the purpose of selectively controlling IFN-γ production, wherein IFN-γ production is characterized. Enhancer.
前記あじなれずし食品由来のラクトバチルス・ブチネリ（Lactobacillus buchneri）が、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 受託番号ＮＩＴＥ Ｐ−１１２３で寄託されたラクトバチルス・ブチネリ（Lactobacillus buchneri）ＡＮ１−１であり、Lactobacillus buchneri derived from the hydrangea food is Lactobacillus buchneri AN1-deposited under the National Institute for Product Evaluation Technology Patent Microorganism Depositary Accession Number NITE P-1123 1 and
前記ラクトバチルス・ブチネリ（Lactobacillus buchneri）ＡＮ１−１は、サイトカイン選択的にＩＬ−２産生を制御する用途に使用されることを特徴とするＩＬ−２産生増強剤。The Lactobacillus buchneri AN1-1 is used for the purpose of controlling IL-2 production selectively with cytokines, and is an IL-2 production enhancer.
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