JP2010246398A - New antibacterial peptide - Google Patents
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本発明は、発酵食品等の保蔵時に発生するラクトバチルス属細菌などに起因する酸敗防止などに有用な新規抗菌性ペプチドに関する。 The present invention relates to a novel antibacterial peptide useful for prevention of rancidity caused by Lactobacillus genus bacteria generated during storage of fermented foods and the like.
食品として常に摂取しているもののなかには天然の抗菌物質を含むものがあり、このような天然の抗菌物質を含むものを用いて食品を保蔵することは食品の安心、安全を担保する上で重要視されている。天然の抗菌物質のなかで食品の保存・保蔵に利用されているものの例としては、わさび、唐辛子をはじめとする香辛料抽出物、ホップなどが挙げられるが、これらのものは添加量を増やすと食品本来の味を損ねるため、十分な抗菌力を発揮させることができない。一方、乳酸菌は、古来より醤油、味噌、漬け物、日本酒等の様々な発酵食品や発酵飲料の生産に利用されている有用な微生物の一つである。乳酸菌を発酵食品等の製造過程で用いることにより、乳酸発酵が行われて産生された乳酸によって系のｐＨが低下したり、これらの乳酸菌が抗菌性物質（抗菌性ペプチド）を産生したりすることで、その製造過程及び得られる製品中での雑菌等の生育を阻害することが可能となり、製品の腐敗や品質の低下を防ぐことができることが知られている。 Some foods that are constantly ingested contain natural antibacterial substances, and preserving foods using such natural antibacterial substances is important for ensuring food safety and security. Has been. Examples of natural antibacterial substances that are used for the preservation and storage of foods include wasabi, chili and other spice extracts, and hops. Since the original taste is impaired, sufficient antibacterial power cannot be exhibited. On the other hand, lactic acid bacteria are one of useful microorganisms that have been used for the production of various fermented foods and fermented beverages such as soy sauce, miso, pickles, and sake since ancient times. By using lactic acid bacteria in the manufacturing process of fermented foods, the pH of the system is lowered by lactic acid produced by lactic acid fermentation, or these lactic acid bacteria produce antibacterial substances (antibacterial peptides) Thus, it is known that it is possible to inhibit the production process and the growth of germs and the like in the obtained product, and to prevent the product from being spoiled or from being deteriorated in quality.
このような乳酸菌の性質を利用して、かつ食品に使用できる安全な抗菌性素材として、種々の乳酸菌とそれらの生産する抗菌性物質の利用が検討されている。例えば、ラクトコッカス ラクティスに属する乳酸菌やエンテロコッカス属に属する乳酸菌の生産物である抗菌性ペプチドがこれらの目的に提案されているが、これらはいずれも抗菌スペクトルが狭いこと、温度安定性やｐＨ安定性が十分でないことなどのために未だ実用されるには至っていない。（例えば、特許文献１〜４参照） Utilization of various lactic acid bacteria and antibacterial substances produced by them as a safe antibacterial material that can be used for foods by utilizing such properties of lactic acid bacteria has been studied. For example, lactic acid bacteria belonging to Lactococcus lactis and lactic acid bacteria belonging to the genus Enterococcus have been proposed for these purposes, but they all have a narrow antibacterial spectrum, temperature stability and pH stability. Has not yet been put into practical use due to the lack of (For example, see Patent Documents 1 to 4)
現在のところ唯一実用されている乳酸菌に由来する抗菌性ぺプチドとしてナイシン（Ｎｉｓｉｎ）がある。これはある程度の抗菌活性を示す抗菌性ぺプチドとして食品用の防腐剤などの用途に広く利用されている。しかし、このナイシンもその抗菌スペクトルからある範囲の細菌類に対しては効果を有するが、食品中に多く見られる細菌類であっても満足な抗菌性を示さないものがあり、特にラクトバチルス属の細菌に対しては十分満足な抗菌活性を示すことができないという問題があった。（例えば、非特許文献１参照） Nisin is the only antibacterial peptide derived from lactic acid bacteria that is currently in practical use. This is widely used as a preservative for foods as an antibacterial peptide exhibiting a certain degree of antibacterial activity. However, this nisin is also effective against a range of bacteria from its antibacterial spectrum, but some of the bacteria found in foods do not show satisfactory antibacterial properties, especially Lactobacillus spp. There was a problem that it was not possible to exhibit a sufficiently satisfactory antibacterial activity against these bacteria. (For example, see Non-Patent Document 1)
このように乳酸菌が産生する抗菌性ペプチドは、一般的に消化酵素で分解されることから安全な抗菌物質として期待されているが、現在実用的に利用されているものは、ナイシンがその唯一の例であり、しかも、ナイシンはその抗菌スペクトル、中性域での熱安定性などにおいて必ずしも満足すべきものではない。
本発明は、古くから食品及び食品加工に利用されている乳酸菌を用いて、優れた抗菌活性と高い安全性を有するとともに、食品に添加した場合に添加した食品の本来の味、風味、色調などを損なうことがなく、かつ優れた抗菌活性を有する天然の抗菌物質を提供することを目的とする。
Antibacterial peptides produced by lactic acid bacteria are expected to be safe antibacterial substances because they are generally decomposed by digestive enzymes. However, nisin is the only one currently in practical use. In addition, nisin is not always satisfactory in its antibacterial spectrum, thermal stability in the neutral range, and the like.
The present invention uses lactic acid bacteria that have been used for food and food processing for a long time, has excellent antibacterial activity and high safety, and the original taste, flavor, color tone, etc. of food added when added to food It is an object of the present invention to provide a natural antibacterial substance that does not impair the antibacterial activity and has an excellent antimicrobial activity.
本発明者らは、上記のような課題を解決すべく、種々の発酵食品から乳酸菌のスクリーニングを行い、これらの菌が生産する抗菌性ペプチドについて検討を行った。その結果、従来報告されているものとは異なったアミノ酸配列を持ち、食品の酸敗で常に問題となるラクトバチルス属およびエンテロコッカス属の乳酸菌の生育を特異的に抑える抗菌性ペプチドを見出し、本発明を完成した。 In order to solve the above-mentioned problems, the present inventors screened lactic acid bacteria from various fermented foods and examined antibacterial peptides produced by these bacteria. As a result, an antibacterial peptide that has an amino acid sequence different from that conventionally reported and that specifically suppresses the growth of Lactobacillus and Enterococcus lactic acid bacteria, which is always a problem in the rancidity of foods, has been found. completed.
即ち、本発明は、以下の内容をその要旨とするものである。
（１）配列表の配列番号１で表されるアミノ酸配列を有する新規抗菌性ペプチドＡ−１。
（２）配列表の配列番号２にて表わされる塩基配列を有する、前記（１）記載の新規抗菌性ペプチドＡ−１のアミノ酸配列をコードする領域を含むＤＮＡ。
That is, the gist of the present invention is as follows.
(1) A novel antibacterial peptide A-1 having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing.
( 2 ) A DNA comprising a region encoding the amino acid sequence of the novel antibacterial peptide A-1 according to (1) , which has the base sequence represented by SEQ ID NO: 2 in the sequence listing.
本発明の抗菌性ペプチドＡ−１は、例えばラクトバチルス・プランタラムＡ−１株（Lactobacillus plantarum A-1）から産生される新規な抗菌性ペプチドである。この本発明の抗菌性ペプチドＡ−１は熱安定性が高いと同時に、従来食品保存剤として一般的に使用されている有機酸やナイシンに対して耐性を示すことから食品の保存の際に問題となっていたラクトバチルス属乳酸菌などに対しても、その増殖を抑制することができるという特徴を有する。従って、食品に本発明の抗菌性ペプチドＡ−１を加えることによって、食品の日持ちをより向上させることができる。特に、漬け物や加熱加工食品でしばしば発生するラクトバチルス属乳酸菌に起因する酸敗を効果的に抑制することができる。 The antibacterial peptide A-1 of the present invention is a novel antibacterial peptide produced from, for example, Lactobacillus plantarum A-1 ( Lactobacillus plantarum A-1). This antibacterial peptide A-1 of the present invention has high heat stability and at the same time exhibits resistance to organic acids and nisin that have been generally used as food preservatives, and therefore has a problem when storing food. It also has the feature that its growth can be suppressed against Lactobacillus lactic acid bacteria. Therefore, the shelf life of the food can be further improved by adding the antimicrobial peptide A-1 of the present invention to the food. In particular, it is possible to effectively suppress the rancidity caused by Lactobacillus lactic acid bacteria frequently occurring in pickles and heat-processed foods.
本発明者らは、種々の発酵食品を分離源として多数の乳酸菌を分離し、これから抗菌性ペプチドを産生する菌株のスクリーニングを行った。即ち、分離したこれらの乳酸菌から、漬物の酸敗の主因となる細菌であるラクトバチルス・プランタラム（ＪＣＭ１０５７）に対する抗菌活性を指標として、抗菌性物質の生産能を有する乳酸菌を選択した。その結果、このラクトバチルス・プランタラム（ＪＣＭ１０５７）に対して優れた抗菌活性を有する物質を生産する乳酸菌として、ラクトバチルス・プランタラムＡ−１株（Lactobacillus plantarum A-1）を見出し、更にこのラクトバチルス・プランタラムＡ−１株の培養液から本発明の新規抗菌性ペプチドＡ−１を得たものである。 The present inventors have isolated a large number of lactic acid bacteria using various fermented foods as a separation source, and screened for strains that produce antimicrobial peptides therefrom. That is, from these isolated lactic acid bacteria, lactic acid bacteria having the ability to produce antibacterial substances were selected using as an index the antibacterial activity against Lactobacillus plantarum (JCM1057), which is the main cause of pickled sourness. As a result, Lactobacillus plantarum A-1 was found as a lactic acid bacterium producing a substance having excellent antibacterial activity against this Lactobacillus plantarum (JCM1057). The novel antibacterial peptide A-1 of the present invention is obtained from the culture solution of Bacillus plantarum A-1.
まず、スクリーニングのための乳酸菌は、以下のようにして分離し、取得した。即ち、各種の発酵食品から採取した試料を、０．５％の炭酸カルシウムを含むＭＲＳ培地（メルク社製）を用いて培養し、必要に応じて数回の継代培養を行った。次いで、この培養液をＭＲＳ寒天培地に塗抹培養し、ここに生じたコロニーから乳酸菌を採取した。尚、ＭＲＳ培地の組成は表１に示すとおりである。 First, lactic acid bacteria for screening were isolated and obtained as follows. That is, samples collected from various fermented foods were cultured using an MRS medium (manufactured by Merck) containing 0.5% calcium carbonate, and subcultured several times as necessary. Subsequently, this culture broth was smeared on an MRS agar medium, and lactic acid bacteria were collected from the colonies formed there. The composition of the MRS medium is as shown in Table 1.
このようにして分離した乳酸菌から、ラクトバチルス・プランタラム（ＪＣＭ１０５７）に対す抗菌活性を指標として抗菌性物質の生産能が特に強い乳酸菌１株を選択した。この選択した乳酸菌の１６ＳリボソームＤＮＡの塩基配列と、現在報告されている乳酸菌のＤＮＡライブラリーについて、相同性解析を行った。その結果、ラクトバチルス・プランタラム ＷＣＦＳ1と９９％の相同性を示した。また、この選択した乳酸菌の糖質の発酵性がラクトバチルス・プランタラムの発酵性とほぼ一致したことから、本菌株をラクトバチルス・プランタラムＡ−１株（Lactobacillus plantarum A-1）と命名した。本菌株は、２００４年１１月９日に、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（〒305-8566 日本国茨城県つくば市東１丁目１番地１中央第６）へ寄託し、その寄託番号はＦＥＲＭ Ｐ−２０２９４である。 From the lactic acid bacteria thus isolated, one lactic acid bacterium strain having a particularly strong ability to produce an antibacterial substance was selected using the antibacterial activity against Lactobacillus plantarum (JCM1057) as an index. Homology analysis was performed on the nucleotide sequence of the selected 16S ribosomal DNA of lactic acid bacteria and the currently reported DNA library of lactic acid bacteria. As a result, it showed 99% homology with Lactobacillus plantarum WCFS1. Moreover, since the fermentability of the saccharides of the selected lactic acid bacteria was almost the same as that of Lactobacillus plantarum , this strain was named Lactobacillus plantarum A-1 ( Lactobacillus plantarum A-1). . This strain was deposited on November 9, 2004 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st, 1st East, 1-chome, Tsukuba, Ibaraki 305-8566, Japan). The number is FERM P-20294.
次に、本発明の新規な抗菌性ペプチドである抗菌性ペプチドＡ−１について説明する。
本発明の抗菌性ペプチドＡ−１は、例えば、ラクトバチルス・プランタラムＡ−１株をＭＲＳ培地を用いて所定の条件で培養し、その培養液から分離・精製することによって得ることができる。即ち、ラクトバチルス・プランタラムＡ−１株をＭＲＳ培地で３０〜４０℃の培養温度で培養することにより、２４時間程度の培養で十分な生育が認められる。抗菌性ペプチドの取得を目的として本菌を大量に培養する場合は、培地としてコーン培地（組成：１リットルあたりコーンスティープリカー（庄野澱粉社製）１０ｇ、酵母エキス（オリエンタル酵母社製）２ｇ、グルコース（和光純薬杜製）１０ｇ）を用いることが好ましく、この培地を用いて３０〜４０℃の培養温度で嫌気的に２４〜４８時間培養すればよい。
本発明の抗菌性ペプチドＡ−１のラクトバチルス・プランタラム（ＪＣＭ１０５７）に対する抗菌活性は、３０℃付近で２４〜４８時間培養するときに最大となる。
Next, antibacterial peptide A-1 which is a novel antibacterial peptide of the present invention will be described.
The antibacterial peptide A-1 of the present invention can be obtained, for example, by culturing Lactobacillus plantarum A-1 strain using MRS medium under predetermined conditions, and separating and purifying it from the culture solution. That is, when the Lactobacillus plantarum A-1 strain is cultured in an MRS medium at a culture temperature of 30 to 40 ° C., sufficient growth is observed for about 24 hours. When cultivating this bacterium in large quantities for the purpose of obtaining antibacterial peptides, corn medium (composition: 10 g corn steep liquor (manufactured by Shono Starch)), 2 g yeast extract (manufactured by Oriental Yeast), glucose, as a medium (Wako Pure Chemical Industries, Ltd.) 10 g) is preferably used, and this medium can be anaerobically cultured at a culture temperature of 30 to 40 ° C. for 24 to 48 hours.
The antibacterial activity of the antibacterial peptide A-1 of the present invention against Lactobacillus plantarum (JCM1057) is maximized when cultured at around 30 ° C. for 24-48 hours.
次に、培養液から目的とする抗菌性ペプチドＡ−１を採取し、精製する方法について説明する。ラクトバチルス・プランタラムＡ−１株の生産する抗菌性ペプチドの分離、精製は常法によって行うことができる。一例を挙げると、培養液を遠心分離した上清を逆相カラムクロマトグラフィーにかけて抗菌活性を示す画分を分画し、分画した画分を濃縮した後、再度逆相カラムクロマトグラフィーにかけて抗菌活性画分を分画する（この操作を２回繰り返す）。次いでＳＤＳ−ポリアクリルアミドゲル電気泳動法で上記画分を泳動させた後、抗菌活性を示すバンドを切り出し、切り出したゲルから抗菌性ペプチドを回収する。こうして得られた精製ペプチドは、ＳＤＳ−ポリアクリルアミドゲル電気泳動において単一バンドを示す。 Next, a method for collecting and purifying the target antibacterial peptide A-1 from the culture solution will be described. Separation and purification of the antibacterial peptide produced by the Lactobacillus plantarum A-1 strain can be performed by conventional methods. For example, the supernatant obtained by centrifuging the culture solution is subjected to reverse phase column chromatography to fractionate a fraction showing antibacterial activity, and the fraction is concentrated and then subjected to reverse phase column chromatography again to obtain antibacterial activity. Fraction the fraction (repeat this operation twice). Subsequently, after the fraction is migrated by SDS-polyacrylamide gel electrophoresis, a band showing antibacterial activity is cut out, and the antibacterial peptide is recovered from the cut out gel. The purified peptide thus obtained shows a single band in SDS-polyacrylamide gel electrophoresis.
このようにして得られる抗菌性ペプチドＡ−１のアミノ酸配列は以下のようにして決定する。まず、エドマン分解法によりこの抗菌性ペプチドのＮ−末端側から全アミノ酸配列の約７割を決定する。次に、上記アミノ酸配列を基に設計したＤＮＡプローブを用いて、ラクトバチルス・プランタラムＡ−１株のＤＮＡのHindIII消化物から抗菌性ペプチドをコードする約２ｋｂのＤＮＡ断片をサザンブロット法により選び出し、このＤＮＡ断片を大腸菌ベクター系で増幅した後、その塩基配列を常法により決定する。このようにして得られるＤＮＡ塩基配列のなかから前述のエドマン分解法によって決定した抗菌性ペプチドＡ−１のＮ末端側から３２残基のアミノ酸をコードする配列表の配列番号３に示すＤＮＡ配列を見出し、これより下流の（３´側の）ＤＮＡ配列より未決定だったＣ末端側残り部分のアミノ酸配列を決定する。かくして得られる抗菌性ペプチドＡ−１の全アミノ酸配列を配列表の配列番号１に、それをコードするＤＮＡ塩基配列を配列表の配列番号２に示す。
即ち、本発明の抗菌性ペプチドＡ−１は、この配列表の配列番号１に示す４３個のアミノ酸配列を有するペプチドである。
The amino acid sequence of the antibacterial peptide A-1 thus obtained is determined as follows. First, about 70% of the total amino acid sequence is determined from the N-terminal side of this antibacterial peptide by Edman degradation. Next, using a DNA probe designed on the basis of the amino acid sequence, a DNA fragment of about 2 kb encoding an antibacterial peptide was selected by Southern blotting from HindIII digest of Lactobacillus plantarum A-1 DNA. After amplifying this DNA fragment with an E. coli vector system, its base sequence is determined by a conventional method. The DNA sequence shown in SEQ ID NO: 3 in the sequence listing encoding the amino acid of 32 residues from the N-terminal side of the antibacterial peptide A-1 determined by the aforementioned Edman degradation method from the DNA base sequence thus obtained is The amino acid sequence of the remaining portion of the C-terminal side that has not been determined from the DNA sequence downstream (3 ′ side) of the heading is determined. The entire amino acid sequence of the antibacterial peptide A-1 thus obtained is shown in SEQ ID NO: 1 in the sequence listing, and the DNA base sequence encoding it is shown in SEQ ID NO: 2 in the sequence listing.
That is, the antibacterial peptide A-1 of the present invention is a peptide having 43 amino acid sequences shown in SEQ ID NO: 1 in this sequence listing.
このようにして得られる本発明の抗菌性ペプチドＡ−１は、既に実用化されている抗菌性ペプチドであるナイシンにはみられない次のような特徴を有する。即ち、本発明の抗菌性ペプチドＡ−１は、ラクトバチルス属乳酸菌に対し、ナイシンより広い抗菌スペクトルを示し、エンテロコッカス属乳酸菌の一部に対しても抗菌活性を示す。また、本発明の抗菌性ペプチドＡ−１は、ナイシンよりも中性域における熱安定性が高く、ｐＨ７．０において、ナイシンが８０℃の加熱で活性が５０％以下に低下するのに対し、本発明の抗菌性ペプチドＡ−１は、ｐＨ７．０において８０℃の加熱後でも８０％以上の活性を維持する。 The antibacterial peptide A-1 of the present invention thus obtained has the following characteristics not found in nisin, an antibacterial peptide that has already been put into practical use. That is, the antibacterial peptide A-1 of the present invention exhibits a broader antibacterial spectrum than nisin against Lactobacillus lactic acid bacteria, and also exhibits antibacterial activity against some of the Enterococcus lactic acid bacteria. The antibacterial peptide A-1 of the present invention has higher thermal stability in the neutral range than nisin, whereas at pH 7.0, the activity of nisin is reduced to 50% or less by heating at 80 ° C., whereas The antibacterial peptide A-1 of the present invention maintains an activity of 80% or more even after heating at 80 ° C. at pH 7.0.
さらに、本発明の新規抗菌性ペプチドＡ−１は、消化酵素であるトリプシンまたはプロテナーゼを添加すると容易に分解され、その活性を失う。従って、本発明の新規抗菌性ペプチドＡ−１は、消化管内で容易に分解されアミノ酸となると考えられ、極めて安全性に優れた抗菌素材であるということができる。 Furthermore, the novel antibacterial peptide A-1 of the present invention is easily decomposed and loses its activity when trypsin or proteinase, which is a digestive enzyme, is added. Therefore, it can be said that the novel antibacterial peptide A-1 of the present invention is easily decomposed in the gastrointestinal tract to become an amino acid, and is an antibacterial material excellent in safety.
本発明の抗菌性ペプチドＡ−１は、乳酸菌に由来することから安全性が高く、畜肉製品中にしばしばみられ、ｐＨを低下させる主因となるラクトバチルス属やエンテロコッカス属の乳酸菌に対して抗菌作用があり、熱安定性に優れることから、加熱加工食品でしばしば起こるラクトバチルス属乳酸菌による酸敗を効果的に抑制することができる抗菌性の素材として食品産業への広い応用が期待できる。 The antibacterial peptide A-1 of the present invention is highly safe because it is derived from lactic acid bacteria , and is antibacterial against lactic acid bacteria belonging to the genus Lactobacillus or Enterococcus, which is frequently found in livestock meat products and is a major cause of lowering the pH. Because of its excellent thermal stability, it can be expected to be widely applied to the food industry as an antibacterial material that can effectively suppress the rancidity caused by Lactobacillus lactic acid bacteria often occurring in heat-processed foods.
次に、以下に実施例を挙げて本発明をさらに詳細に説明するが、本発明はこれらによって何ら限定されるものではない。尚、各実施例中「％」は特に注記しない限り質量基準である。 Next, although an Example is given to below and this invention is demonstrated still in detail, this invention is not limited at all by these. In each example, “%” is based on mass unless otherwise noted.
(1) ラクトバチルス・プランタラムＡ−１株の取得
乳酸菌の分離源として、種々の発酵食品 1０ｇを採取し、０．５％の炭酸カルシウムを含む前記表１に示す組成からなるＭＲＳ培地（メルク社製）を用いて、３７℃で４日間培養した。得られた培養液を更に新しいＭＲＳ培地に添加して同様の条件での培養を行い、この操作を２回繰り返した。それぞれの試料について、このようにして得られた培養液をＭＲＳ寒天培地（組成：表１に示す培地組成に寒天１．５％を添加したもの）に塗抹培養し、ここに生じたコロニーから乳酸菌を採取した。このようにして分離した種々の標準菌体又は発酵食品から得た乳酸菌について、ラクトバチルス・プランタラム（ＪＣＭ１０５７）に対す抗菌活性を指標として抗菌活性を調べ、抗菌性物質の生産能が最も強いラクトバチルス・プランタラムＡ−１を選択した。
(1) Acquisition of Lactobacillus plantarum A-1 strain As an isolation source of lactic acid bacteria, 10 g of various fermented foods were collected and MRS medium (Merck) comprising the composition shown in Table 1 above containing 0.5% calcium carbonate was obtained. For 4 days at 37 ° C. The obtained culture broth was further added to a new MRS medium and cultured under the same conditions, and this operation was repeated twice. For each sample, the culture solution thus obtained was smear-cultured on an MRS agar medium (composition: 1.5% agar added to the medium composition shown in Table 1). Were collected. Lactic acid bacteria obtained from various standard bacterial cells or fermented foods isolated in this way were examined for antibacterial activity using the antibacterial activity against Lactobacillus plantarum (JCM1057) as an index, and the lactose with the strongest ability to produce antibacterial substances. Bacillus plantarum A-1 was selected.
(2) 選択した乳酸菌の相同性解析
(1)で選択したラクトバチルス・プランタラムＡ−１について、１６ＳリボソームＤＮＡ（ｒＤＮＡ）の塩基配列の相同性解析を行った。現在ＤＮＡデータベースに登録されている１００株以上の乳酸菌の塩基配列と比較した結果、相同性の高かった上位２０株のうち１６株がラクトバチルス・プランタラムであった。最も相同性の高かったラクトバチルス・プランタラム ＷＣＦＳ１とは９９％の相同性を示した。
(2) Homology analysis of selected lactic acid bacteria
The Lactobacillus plantarum A-1 selected in (1) was subjected to homology analysis of the base sequence of 16S ribosomal DNA (rDNA). As a result of comparison with the base sequences of more than 100 lactic acid bacteria currently registered in the DNA database, 16 strains out of the top 20 strains with high homology were Lactobacillus plantarum. It showed 99% homology with Lactobacillus plantarum WCFS1, which had the highest homology.
(3) ラクトバチルス・プランタラムＡ−１株の菌学的性質
このラクトバチルス・プランタラムＡ−１の菌学的性質は、以下に示すように、一般的なラクトバチルス・プランタラムの性質とほぼ一致した。
（ａ）形態的性質
細胞の形及び大きさ：桿菌 短径 約０．５μｍ
細胞の多形性の有無： 無、
運動性の有無： 無、
胞子の有無： 無
（ｂ）培養的性質
肉汁寒天平板培養：生育良、淡黄色の光沢のある小コロニーを形成
肉汁液体培養：生育良、表面発育なし
肉汁ゼラチン：生育良、ゼラチンの液化なし
リトマスミルク：産生可
（ｃ）生理学的性質
グラム染色： 陽性、
好気条件での生育： ＋、
嫌気条件での生育： ＋、
オキシダーゼ： −、
カタラーゼ： −、
ＯＦテスト： Ｆ
(3) Bacteriological properties of Lactobacillus plantarum A-1 strain The bacteriological properties of Lactobacillus plantarum A-1 are as follows: Almost matched.
(A) Morphological properties Shape and size of cells: Neisseria gonorrhoeae, short diameter: about 0.5 μm
Presence or absence of cell polymorphism: None,
Existence of mobility: None,
Presence or absence of spore: None (b) Culture properties Meat broth agar plate culture: good growth, forming a light yellow shiny small colony Meat broth liquid culture: good growth, no surface growth Meat juice gelatin: good growth, no gelatin liquefaction Litmus Milk: production possible (c) physiological properties Gram staining: positive,
Growth under aerobic conditions: +,
Growth under anaerobic conditions: +,
Oxidase:-,
Catalase:-,
OF test: F
(1) 新規抗菌性ペプチドＡ−１粗粉末の調製
実施例１で得たラクトバチルス・プランタラムＡ−１株のスラントからＴＹＧ培地｛トリプトン １％、酵母エキス ０．５％、ブドウ糖 １％、食塩 ０．５％｝１００ｍＬに菌を接種し、３０℃で１日間培養して前培養液を得た。次いで、コーン培地｛コーンスティープリカー（庄野澱粉社製）１％、酵母エキス（オリエンタル酵母社製）０．２％、ブドウ糖（和光純薬杜製）１％、食塩 ０．２％、硫酸マンガン ０．０２％（ｐＨ６．５に調整）｝１０リットルにこの前培養液を加え、３０℃で下限ｐＨ４．５にて２日間培養し、抗菌性ペプチドＡ−１を含む培養液を得た。この培養液を８０℃で３０分間加熱殺菌した後冷却し、濾過助剤を用いて不溶物を濾別した後、濾液を噴霧乾燥して本発明の抗菌性ペプチドＡ−１の粗粉末約５００ｇを得た。
(1) Preparation of New Antibacterial Peptide A-1 Crude Powder From the slant of Lactobacillus plantarum A-1 strain obtained in Example 1, TYG medium {tryptone 1%, yeast extract 0.5%, glucose 1%, Salt 0.5%} 100 mL was inoculated with bacteria and cultured at 30 ° C. for 1 day to obtain a precultured solution. Next, corn medium {corn steep liquor (manufactured by Shono Starch Co., Ltd.) 1%, yeast extract (manufactured by Oriental Yeast Co., Ltd.) 0.2%, glucose (manufactured by Wako Pure Chemical Industries, Ltd.) 1%, salt 0.2%, manganese sulfate 0 0.02% (adjusted to pH 6.5)} This preculture was added to 10 liters, and cultured at 30 ° C. at a lower limit of pH 4.5 for 2 days to obtain a culture containing antibacterial peptide A-1. The culture broth was sterilized by heating at 80 ° C. for 30 minutes, cooled, filtered to remove insolubles using a filter aid, and then the filtrate was spray-dried to obtain about 500 g of the antibacterial peptide A-1 crude powder of the present invention. Got.
(2) 新規抗菌性ペプチドＡ−１の精製
(1)で得た抗菌性ペプチドＡ−１粗粉末の２％水溶液を調製し、不溶物を濾過した後、次の条件で逆相カラムクロマトグラフィーによって抗菌活性を示す画分を分画した。
カラム： 資生堂 Capcell Pak C18MG 4.6mm×150mm
溶離液Ａ：０．１％リン酸
溶離液Ｂ：９０％アセトニトリル
溶離液Ｂは２０％→４０％の４０分リニアグラディエント
検 出： ２２０ｎｍ
１回の注入量：０．０８ｍＬ
得られた抗菌活性をもつ画分を集めて濃縮した後、再度逆相カラムクロマトグラフィーで抗菌活性画分を分画した（この操作を２回繰り返した）。次に、ＳＤＳ−ポリアクリルアミドゲル電気泳動法で泳動し、抗菌活性を示すバンド（分子量約７０００Ｄａ）のバンドを切り出し、ゲルから抗菌性ペプチドＡ−１を回収した。こうして精製した抗菌性ペプチドＡ−１は前記したＳＤＳ−ポリアクリルアミドゲル電気泳動において単一バンドを示した。
(2) Purification of novel antibacterial peptide A-1
After preparing a 2% aqueous solution of the antibacterial peptide A-1 crude powder obtained in (1) and filtering insoluble matter, a fraction showing antibacterial activity was fractionated by reverse phase column chromatography under the following conditions.
Column: Shiseido Capcell Pak C18MG 4.6mm × 150mm
Eluent A: 0.1% phosphoric acid Eluent B: 90% acetonitrile
Eluent B is 20% → 40% 40 minute linear gradient detection: 220nm
Single injection volume: 0.08 mL
The obtained fractions having antibacterial activity were collected and concentrated, and then the antibacterial activity fractions were fractionated again by reversed-phase column chromatography (this operation was repeated twice). Next, electrophoresis was performed by SDS-polyacrylamide gel electrophoresis, a band having antibacterial activity (molecular weight of about 7000 Da) was cut out, and antibacterial peptide A-1 was recovered from the gel. The purified antibacterial peptide A-1 showed a single band in the above-described SDS-polyacrylamide gel electrophoresis.
(3) 新規抗菌性ペプチドＡ−１の抗菌スペクトル
表１に示す組成のＭＲＳ寒天培地に次の表３に示す各種の被検菌を混釈し、ここに上記(1)で得られた抗菌性ペプチドＡ−１粉末の０．５％水溶液、及び、ナイシン（シグマアルドリッチ製試薬）の１００ｐｐｍ水溶液を０．０２ｍＬスポットし、３０℃で２日間培養し、生育阻止円の有無を確認した。なお、ナイシン１００ｐｐｍ水溶液というは、一般的に食品類の保存に使用する濃度であり、抗菌性ペプチドＡ−１粉末の０．５％水溶液はほぼこれに対応する濃度である。
種々の乳酸菌の場合の結果を表３に、乳酸菌以外の食品腐敗菌として一般的に見られるいくつかの細菌での結果を表４に示す。「＋」が生育阻止円ありを、「−」が生育阻止円なしを意味する。
(3) Antibacterial spectrum of novel antibacterial peptide A-1 The MRS agar medium having the composition shown in Table 1 was mixed with various test bacteria shown in Table 3 below, and the antibacterial obtained in (1) above was mixed therewith. 0.02 mL of a 0.5% aqueous solution of the soluble peptide A-1 powder and a 100 ppm aqueous solution of nisin (Sigma Aldrich reagent) were spotted and cultured at 30 ° C. for 2 days to confirm the presence or absence of a growth inhibition circle. Nisin 100 ppm aqueous solution is a concentration generally used for storage of foods, and a 0.5% aqueous solution of antibacterial peptide A-1 powder has a concentration corresponding to this.
The results for various lactic acid bacteria are shown in Table 3, and the results for some bacteria commonly found as food spoilage bacteria other than lactic acid bacteria are shown in Table 4. “+” Means that there is a growth inhibition circle, and “−” means that there is no growth inhibition circle.
上記の表３において、菌株の番号が１〜１６のものについては、正確に菌株の特定を行ったものではないため未同定と記載しているが、菌の形状、生育が抑制されるｐＨ条件などからほぼ間違いなくラクトバチルス属の乳酸菌であると考えられる。 In Table 3 above, those with strain numbers 1 to 16 are described as unidentified because they were not accurately identified, but the shape of the fungus, pH conditions under which growth is suppressed From these, it is almost certain that it is a lactic acid bacterium belonging to the genus Lactobacillus.
表３の結果からわかるように、本発明の新規抗菌性ペプチドＡ−１は、ラクトバチルス属乳酸菌に対し広い抗菌スペクトルを有し、例えば、ＩＡＭ１０４１やＴＩＳＴＲ３９０などのようなナイシンには耐性を示すいくつかのラクトバチルス属菌株に対しても抗菌活性を示す。エンテロコッカス属、ロイコノストック属の一部の菌株に対しても抗菌作用を示す。
また、表４に示すように、本発明の新規抗菌性ペプチドＡ−１は、乳酸菌以外の細菌に対しては抗菌作用を示さない。
As can be seen from the results in Table 3, the novel antibacterial peptide A-1 of the present invention has a broad antibacterial spectrum against lactic acid bacteria of the genus Lactobacillus, and for example, some resistance to nisin such as IAM1041 and TISTR390. It also exhibits antibacterial activity against these Lactobacillus strains. It also exhibits antibacterial activity against some strains of the genus Enterococcus and Leuconostoc.
Moreover, as shown in Table 4, the novel antibacterial peptide A-1 of the present invention does not exhibit antibacterial action against bacteria other than lactic acid bacteria.
(4) 新規抗菌性ペプチドＡ−１の熱安定性
上記の(1)で得た新規抗菌性ペプチドＡ−１粉末の１％水溶液、及びナイシン（シグマアルドリッチ製試薬）３００ｐｐｍの水溶液を調製し、それぞれの水溶液を、ｐＨ５．０、７．０、８．５に調整した後、７０℃、８０℃、９０℃、１００℃で１５分間加熱した。これらの被検液をあらかじめラクトバチルス・プランタラム（ＪＣＭ１０５７）を混釈したＭＲＳ寒天培地上に０．０２ｍＬスポットし、３０℃で２日間培養し、生育阻止円の面積を測定した。加熱前の被検液のｐＨ５．０における抗菌活性を１００として、各被検液についてその相対的な抗菌活性を生育阻止円の面積の値から求めた。その結果を表５及び表６に示す。
(4) Thermal stability of novel antibacterial peptide A-1 A 1% aqueous solution of the novel antibacterial peptide A-1 powder obtained in (1) above and a 300 ppm aqueous solution of nisin (Sigma Aldrich reagent) were prepared. Each aqueous solution was adjusted to pH 5.0, 7.0, 8.5 and then heated at 70 ° C., 80 ° C., 90 ° C., and 100 ° C. for 15 minutes. 0.02 mL of these test solutions was spotted on an MRS agar medium previously mixed with Lactobacillus plantarum (JCM1057), cultured at 30 ° C. for 2 days, and the area of the growth inhibition circle was measured. Assuming that the antibacterial activity at pH 5.0 of the test solution before heating was 100, the relative antibacterial activity of each test solution was determined from the value of the area of the growth inhibition circle. The results are shown in Tables 5 and 6.
表５、表６の結果から分かるように、本発明の新規抗菌性ペプチドＡ−１は、ナイシンに比較して高い耐熱性を有しており、ｐＨ７．０やｐＨ８．５においてナイシンに比較して非常に優れた耐熱性を示している。また、ｐＨ５．０では８０℃でも９０％以上という優れた耐熱性を示す。一方、ナイシンは表６の結果に示すように、特にｐＨ７．０、ｐＨ８．５のような中性からアルカリ領域においては非常に熱安定性が悪く、大きく活性が低下している。また、未加熱のものであっても、表６の未加熱の欄に示すように、試験用の試料を調製してそのまま室温に放置したものでも活性の低下が見られた。 As can be seen from the results in Tables 5 and 6, the novel antibacterial peptide A-1 of the present invention has higher heat resistance compared to nisin, and compared to nisin at pH 7.0 and pH 8.5. It has excellent heat resistance. Further, at pH 5.0, excellent heat resistance of 90% or more is exhibited even at 80 ° C. On the other hand, as shown in the results of Table 6, nisin is very poor in heat stability especially in neutral to alkaline regions such as pH 7.0 and pH 8.5, and its activity is greatly reduced. Moreover, even if it was an unheated thing, as shown in the unheated column of Table 6, the activity fall was seen even if it prepared the sample for a test and left it as it was at room temperature.
(5) 新規抗菌性ペプチドＡ−１の消化酵素による分解
上記の(1)で得た新規抗菌性ペプチドＡ−１粉末の１％水溶液を調製し、トリプシンまたはプロナーゼ０．０１％を加え、３７℃で１時間インキュベートした。これらのサンプルについて、上記(3)と同様の方法によって抗菌活性を測定した。
その結果、トリプシンまたはプロナーゼのいずれを添加した場合にも生育阻止円はまったく観察されず、抗菌性ペプチドＡ−１はトリプシンまたはプロナーゼ処理により抗菌活性を失ったことがわかった。
(5) Degradation of novel antibacterial peptide A-1 by digestive enzyme A 1% aqueous solution of the novel antibacterial peptide A-1 powder obtained in (1) above is prepared, and trypsin or pronase 0.01% is added. Incubated for 1 hour at ° C. For these samples, antibacterial activity was measured by the same method as in (3) above.
As a result, no growth inhibition circle was observed when either trypsin or pronase was added, and it was found that antibacterial peptide A-1 lost antibacterial activity by trypsin or pronase treatment.
本発明の新規抗菌性ペプチドＡ−１により、ラクトバチルス属やエンテロコッカス属の細菌の増殖を制御することが可能となり、特に、加熱加工食品でしばしば起こるラクトバチルス属細菌による酸敗などを防止する素材を食品産業へ提供することが可能となり、食品または食品素材を取り扱う食品産業、流通産業などにおいて特に有用である。 With the novel antibacterial peptide A-1 of the present invention, it becomes possible to control the growth of bacteria of the genus Lactobacillus or Enterococcus, and in particular, a material that prevents acid spoilage caused by the Lactobacillus bacterium often occurring in heat-processed foods It can be provided to the food industry, and is particularly useful in the food industry, the distribution industry, etc. that handle food or food materials.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2017217350A1 (en) * | 2016-06-13 | 2017-12-21 | 株式会社村田製作所 | Antimicrobial and antiviral drug, antimicrobial and antiviral member, and method for producing antimicrobial and antiviral drug |
| CN111411057A (en) * | 2020-04-22 | 2020-07-14 | 福州大学 | Lactobacillus corynebacterium for producing broad-spectrum antibacterial peptide and application of antibacterial peptide thereof |
| CN114276951A (en) * | 2021-12-01 | 2022-04-05 | 江南大学 | Lactobacillus plantarum capable of producing antibacterial peptide with broad-spectrum antibacterial activity |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017217350A1 (en) * | 2016-06-13 | 2017-12-21 | 株式会社村田製作所 | Antimicrobial and antiviral drug, antimicrobial and antiviral member, and method for producing antimicrobial and antiviral drug |
| JPWO2017217350A1 (en) * | 2016-06-13 | 2019-02-21 | 株式会社村田製作所 | Antibacterial antiviral agent, antibacterial antiviral member, and method for producing antibacterial antiviral agent |
| JP2019038832A (en) * | 2016-06-13 | 2019-03-14 | 株式会社村田製作所 | Antibacterial antiviral agent, antibacterial antiviral member, and method for producing antibacterial antiviral agent |
| US11944651B2 (en) | 2016-06-13 | 2024-04-02 | Murata Manufacturing Co., Ltd. | Antimicrobial and antiviral agent, antimicrobial and antiviral member, and method for producing antimicrobial and antiviral agent |
| CN111411057A (en) * | 2020-04-22 | 2020-07-14 | 福州大学 | Lactobacillus corynebacterium for producing broad-spectrum antibacterial peptide and application of antibacterial peptide thereof |
| CN111411057B (en) * | 2020-04-22 | 2021-09-28 | 福州大学 | Lactobacillus corynebacterium for producing broad-spectrum antibacterial peptide and application of antibacterial peptide thereof |
| CN114276951A (en) * | 2021-12-01 | 2022-04-05 | 江南大学 | Lactobacillus plantarum capable of producing antibacterial peptide with broad-spectrum antibacterial activity |
| CN114276951B (en) * | 2021-12-01 | 2023-10-03 | 江南大学 | Lactobacillus plantarum capable of producing antibacterial peptide with broad-spectrum antibacterial activity |
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