JP2011148775A - Antibacterial substance, mold preventive, food preservative, yeast extract, bactericide, method for inhibiting proliferation of mold and/or yeast, and method for inhibiting growth of yeast and/or bacterium - Google Patents
Antibacterial substance, mold preventive, food preservative, yeast extract, bactericide, method for inhibiting proliferation of mold and/or yeast, and method for inhibiting growth of yeast and/or bacterium Download PDFInfo
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
Abstract
Description
本発明は、新規な抗菌性物質、該抗菌性物質を含有する防カビ剤、食品保存料、酵母エキス、殺菌剤、該抗菌性物質を用いる、カビ及び／又は酵母の増殖を阻害する方法、及び酵母及び／又は細菌の生育を阻害する方法に関する。また、本発明は、キャンディダ（Ｃａｎｄｉｄａ）属菌、サッカロマイセス（Ｓａｃｃｈａｒｏｍｙｃｅｓ）属菌、ピキア（Ｐｉｃｈｉａ）属菌、ロドトルラ（Ｒｈｏｄｏｔｏｒｕｌａ）属菌、クラビスポラ（Ｃｌａｖｉｓｐｏｒａ）属菌、及びトルラスポラ（Ｔｏｒｕｌａｓｐｏｒａ）属菌からなる群より選ばれる１の酵母が産生する、揮発性の抗菌性物質、該抗菌性物質を含有する防カビ剤、食品保存料、酵母エキス、殺菌剤、該抗菌性物質又は該酵母を用いる、カビ及び／又は酵母の増殖を阻害する方法、及び酵母及び／又は細菌の生育を阻害する方法に関する。 The present invention relates to a novel antibacterial substance, a fungicide containing the antibacterial substance, a food preservative, a yeast extract, a fungicide, a method for inhibiting the growth of mold and / or yeast using the antibacterial substance, And a method for inhibiting the growth of yeast and / or bacteria. The present invention also relates to the genus Candida, the genus Saccharomyces, the genus Pichia, the genus Rhodotorula, the genus Clavispora, and the genus Torulaspora. A volatile antibacterial substance produced by one yeast selected from the group consisting of, a fungicide containing the antibacterial substance, a food preservative, a yeast extract, a fungicide, the antibacterial substance or the yeast, The present invention relates to a method for inhibiting the growth of mold and / or yeast, and a method for inhibiting the growth of yeast and / or bacteria.
微生物による腐敗や変敗を防止し、食品の保存性を高めるため、様々な抗菌性物質が、食品保存料として用いられている。食品の保存性を向上させることにより、流通過程における食品の腐敗等が効率的に防止されるため、製造コストの改善が期待でき、また、食品の安全性上も好ましい。このため、優れた抗菌性物質の開発が望まれている。ここで、抗菌性物質は、主に、化学合成物質と、微生物等から得られた天然由来の物質とに分類できるが、食品保存料として用いる場合には、安全性の点から、天然由来の物質であることが好ましい。 Various antibacterial substances are used as food preservatives in order to prevent spoilage and deterioration due to microorganisms and to enhance the preservation of food. By improving the preservability of food, it is possible to efficiently prevent the food from being spoiled in the distribution process, and therefore it is possible to expect an improvement in manufacturing cost, and it is also preferable from the viewpoint of food safety. For this reason, the development of an excellent antibacterial substance is desired. Here, antibacterial substances can be classified mainly into chemically synthesized substances and naturally-derived substances obtained from microorganisms, etc., but when used as food preservatives, they are naturally derived from the viewpoint of safety. A substance is preferred.
近年、天然由来の抗菌性物質として、ナイシン等の、乳酸菌由来の様々な種類のバクテリオシンが知られている（例えば、非特許文献１参照。）。バクテリオシンは、通常、ペプチド若しくはタンパク質であり、消化酵素等により容易に分解されるため、非常に安全性が高い。また、主にグラム陽性菌に対し優れた抗菌活性を有することが知られている。 In recent years, various types of bacteriocin derived from lactic acid bacteria, such as nisin, have been known as natural antibacterial substances (for example, see Non-patent Document 1). Bacteriocin is usually a peptide or protein, and is easily decomposed by digestive enzymes or the like, so it is very safe. It is also known to have excellent antibacterial activity mainly against gram-positive bacteria.
その他、グラム陽性菌とグラム陰性菌の両方に対し優れた抗菌活性を有する抗菌性物質を含む食品保存料の製造方法として、例えば、（１）酵母の増殖に必要な栄養素、無機塩類と炭素源としてグルコースを含む培地で酵母を定常期に達するまで増殖させた後、酵母を除去した培地に必要ならば炭素源を加え、再び定常期に達するまで増殖させる繰り返し培養を２回以上行って得られた水溶液であることを特徴とする酵母が産生する抗菌性物質を含む食品保存料の製造方法が開示されている（例えば、特許文献１参照。）。
また、天然由来の抗菌性物質として、カビ由来の揮発性抗菌物質も開示されている（例えば、非特許文献２参照）。
Other methods for producing food preservatives containing antibacterial substances having excellent antibacterial activity against both gram-positive and gram-negative bacteria include, for example, (1) nutrients, inorganic salts and carbon sources necessary for yeast growth As described above, the yeast is grown in a medium containing glucose until reaching the stationary phase, and then, if necessary, a carbon source is added to the medium from which the yeast has been removed, and repeated cultivation is repeated twice or more until the stationary phase is reached. A method for producing a food preservative containing an antibacterial substance produced by yeast, characterized by being an aqueous solution, is disclosed (for example, see Patent Document 1).
In addition, mold-derived volatile antibacterial substances are also disclosed as natural antibacterial substances (see, for example, Non-Patent Document 2).
グラム陽性菌等のバクテリア（細菌）のみならず、カビや酵母等の真菌も、食品の腐敗等を引き起こす。特にカビは、多くの食品の腐敗の主要な要因であることから、カビの増殖を抑制することができる抗真菌活性を有する物質を、食品保存料や抗菌剤として使用できることが好ましい。
また、空気中には種々のカビ胞子が浮遊しているため、カビ胞子の付着を完全に防ぐことは困難である。そこで、特に食品では、製造段階又は食品開封後の、食品へのカビ胞子の付着を防ぐことと同時に、喫食時点まで、付着したカビ胞子の発芽を抑制できることが好ましい。
しかしながら、ナイシン等のバクテリオシンは、一般にグラム陽性菌にのみ抗菌活性を示し、カビや酵母等の真菌の増殖を抑制することができないという問題がある。また、上記（１）の方法で得られる食品保存料は、酵母由来の抗菌性物質であるため、食品に対し安全に使用することができ、また、耐熱性も有しているが、特許文献１には、カビに対する抗菌活性については一切記載がない。
一方、非特許文献２に開示された抗菌性物質は、カビ由来であるため、食品に対して安全に使用することができるかどうかは定かではない。また、上記のように胞子の発芽を抑制することは食品等の分野において非常に重要であるにも関わらず、非特許文献２には、菌糸の成長を阻害するとの記載はあるが、胞子の発芽を阻害するかどうかについては一切記載がない。
Not only bacteria such as gram-positive bacteria (bacteria) but also fungi such as mold and yeast cause food spoilage and the like. In particular, since mold is a major factor in the decay of many foods, it is preferable that a substance having antifungal activity capable of suppressing mold growth can be used as a food preservative or antibacterial agent.
Further, since various mold spores are floating in the air, it is difficult to completely prevent the mold spores from adhering. Therefore, particularly for foods, it is preferable to prevent the adhesion of mold spores to the food at the production stage or after opening the food, and to suppress germination of the attached mold spores until the time of eating.
However, bacteriocin such as nisin generally has antibacterial activity only against gram-positive bacteria, and has a problem that it cannot inhibit the growth of fungi such as mold and yeast. Moreover, since the food preservative obtained by the method (1) is an antibacterial substance derived from yeast, it can be used safely for food and has heat resistance. No. 1 describes nothing about antibacterial activity against mold.
On the other hand, since the antibacterial substance disclosed in Non-Patent Document 2 is derived from mold, it is not certain whether it can be safely used for food. In addition, although suppression of spore germination as described above is very important in the field of foods and the like, Non-Patent Document 2 describes that it inhibits mycelial growth. There is no mention of whether to inhibit germination.
本発明は、安全であり、且つ、中性においてカビや酵母等の真菌の胞子の発芽及び増殖や、酵母や細菌の生育を抑制することのできる、新規な抗菌性物質を提供することを目的とする。
また、本発明は、該抗菌性物質を含有する防カビ剤、食品保存料、酵母エキス及び殺菌剤、並びに、該抗菌性物質又は該抗菌性物質を産生する微生物を用いるカビ及び／又は酵母の増殖を阻害する方法及び酵母及び／又は細菌の生育を阻害する方法を提供することを目的とする。
An object of the present invention is to provide a novel antibacterial substance that is safe and can neutrally inhibit germination and growth of fungi spores such as mold and yeast, and growth of yeast and bacteria. And
The present invention also provides a fungicide, a food preservative, a yeast extract and a fungicide containing the antibacterial substance, and a fungus and / or yeast using the antibacterial substance or a microorganism that produces the antibacterial substance. It is an object of the present invention to provide a method for inhibiting growth and a method for inhibiting the growth of yeast and / or bacteria.
本発明者らは、上記課題を解決すべく鋭意研究した結果、食品由来の微生物が産生する抗真菌性物質であれば、食品保存料としても安全に用いることができると考え、様々な種類のチーズから分離した微生物の中から、ｐＨ７．０において、アスペルギルス・ニガー（Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ）又はリゾパス・ストロニファー（Ｒｈｉｚｏｐｕｓ ｓｔｏｌｏｎｉｆｅｒ）の増殖を抑制することのできる抗真菌活性を有する微生物を見出し、さらに、該微生物が産生する抗真菌性物質、及び、同様の抗真菌活性を有する該抗真菌性物質の類縁体を見出すことにより、本発明を完成させた。
さらに、本発明者らは、該抗真菌性物質が、抗真菌活性のみならず、酵母や細菌の生育を阻害する抗菌活性を有することを見出し、本発明を完成させた。
As a result of earnest research to solve the above problems, the present inventors believe that any antifungal substance produced by food-derived microorganisms can be safely used as a food preservative, and various types of Among microorganisms isolated from cheese, a microorganism having antifungal activity capable of suppressing the growth of Aspergillus niger or Rhizopus stroniffer at pH 7.0, and The present invention was completed by finding antifungal substances produced by microorganisms and analogs of the antifungal substances having similar antifungal activity.
Furthermore, the present inventors have found that the antifungal substance has not only antifungal activity but also antibacterial activity that inhibits the growth of yeast and bacteria, and completed the present invention.
すなわち、本発明の第一の態様は、下記（１）〜（４）の特徴を有する抗菌性物質である。
(１)下記一般式（１）で表される抗菌性物質。
That is, the first aspect of the present invention is an antibacterial substance having the following features (1) to (4).
(1) An antibacterial substance represented by the following general formula (1).
（２）前記一般式（１）において、Ｒ２が炭素数７〜９のアルキル基である前記（１）の抗菌性物質。
（３）前記抗菌性物質が、酢酸ｎ−オクチル、２−ノナノン、２−デカノン、又は２−ウンデカノンである前記（２）の抗菌性物質。
（４）前記抗菌性物質が、揮発により胞子の発芽を阻害する前記（１）〜（３）のいずれかの抗菌性物質。
（５）前記抗菌性物質が、揮発により酵母及び／又は細菌の生育を阻害する（１）〜（３）のいずれかの抗菌性物質。
本発明の第二〜第七の態様は、それぞれ、下記（６）〜（１１）の特徴を有する防カビ剤、食品保存料、酵母エキス、殺菌剤、カビ及び／又は酵母の増殖を阻害する方法、酵母及び／又は細菌の生育を阻害する方法である。
（６）前記（１）〜（５）のいずれかの抗菌性物質を含有する防カビ剤。
（７）前記（１）〜（５）のいずれかの抗菌性物質を含有する食品保存料。
（８）前記（１）〜（５）のいずれかの抗菌性物質を含有する酵母エキス。
（９）前記（１）〜（５）のいずれかの抗菌性物質を含有する殺菌剤。
（１０）前記（１）〜（５）のいずれかの抗菌性物質を用いることを特徴とする、カビ及び／又は酵母の増殖を阻害する方法。
（１１）前記（１）〜（５）のいずれかの抗菌性物質を用いることを特徴とする、酵母及び／又は細菌の生育を阻害する方法。
本発明の第八の態様は、下記（１２）〜（１９）の特徴を有する抗菌性物質である。
（１２）キャンディダ（Ｃａｎｄｉｄａ）属菌、サッカロマイセス（Ｓａｃｃｈａｒｏｍｙｃｅｓ）属菌、ピキア（Ｐｉｃｈｉａ）属菌、ロドトルラ（Ｒｈｏｄｏｔｏｒｕｌａ）属菌、クラビスポラ（Ｃｌａｖｉｓｐｏｒａ）属菌、及びトルラスポラ（Ｔｏｒｕｌａｓｐｏｒａ）属菌からなる群より選ばれる１の酵母が産生する、揮発性の抗菌性物質。
（１３）前記抗菌性物質が、酢酸イソアミルである前記（１２）の抗菌性物質。
（１４）前記キャンディダ属菌が、キャンディダ・マルトーサ（Ｃａｎｄｉｄａ ｍａｌｔｏｓａ）、キャンディダ・トロピカリス（Ｃａｎｄｉｄａ ｔｒｏｐｉｃａｌｉｓ）、又はキャンディダ・シュードインターメディア（Ｃａｎｄｉｄａ ｐｓｅｕｄｉｎｔｅｒｍｅｄｉａ）である前記（１２）又は（１３）の抗菌性物質。
（１５）前記キャンディダ属菌が、キャンディダ・マルトーサＯ９−ＮＰ９（受託番号 ＮＩＴＥ ＢＰ−２９７）、キャンディダ・マルトーサＩＡＭ１２２４７、キャンディダ・マルトーサＩＡＭ１２２４８、キャンディダ・トロピカリスＩＡＭ４９６５、又はキャンディダ・シュードインターメディアＩＡＭ１２５１０である前記（１２）又は（１３）の抗菌性物質。
（１６）前記サッカロマイセス属菌がサッカロマイセス・セレビシエ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）であり、前記ピキア属菌がピキア・フェルメンタス（Ｐｉｃｈｉａ ｆｅｒｍｅｎｔａｎｓ）であり、前記ロドトルラ属菌がロドトルラ・アクタ（Ｒｈｏｄｏｔｏｒｕｌａ ａｃｕｔａ）であり、前記クラビスポラ属菌がクラビスポラ・ルシタニエ（Ｃｌａｖｉｓｐｏｒａ ｌｕｓｉｔａｎｉａｅ）であり、前記トルラスポラ属菌がトルラスポラ・デルブレッキー（Ｔｏｒｕｌａｓｐｏｒａ ｄｅｌｂｒｕｅｃｋｉｉ）である前記（１２）又は（１３）の抗菌性物質。
（１７）前記ピキア属菌がピキア・フェルメンタスＪＣＭ１８２４であり、前記ロドトルラ属菌がロドトルラ・アクタＪＣＭ９４９４であり、前記サッカロマイセス属菌がサッカロマイセス・セレビシエＨ−１（受託番号 ＮＩＴＥ ＢＰ−４７３）であり、前記クラビスポラ属菌がクラビスポラ・スピーシーズＰ−５（Ｃｌａｖｉｓｐｏｒａ ｓｐ． Ｐ−５、受託番号 ＮＩＴＥ ＢＰ−４７４）又はクラビスポラ・ルシタニエＮＢＲＣ１００５９であり、前記トルラスポラ属菌がトルラスポラ・デルブルッキーＮＢＲＣ０９５５である前記（１２）又は（１３）の抗菌性物質。
（１８）前記抗菌性物質が、揮発により胞子の発芽を阻害する（１２）〜（１７）のいずれかの抗菌性物質。
（１９）前記抗菌性物質が、揮発により酵母及び／又は細菌の生育を阻害することを特徴とする（１２）〜（１７）のいずれかの抗菌性物質。
さらに、本発明の第九〜第十四の態様は、それぞれ、下記（２０）〜（２７）の特徴を有する防カビ剤、食品保存料、酵母エキス、殺菌剤、カビ及び／又は酵母の増殖を阻害する方法、及び酵母及び／又は細菌の生育を阻害する方法である。
（２０）前記（１２）〜（１９）のいずれかの抗菌性物質を含有する防カビ剤。
（２１）前記（１２）〜（１９）のいずれかの抗菌性物質を含有する食品保存料。
（２２）前記（１２）〜（１９）のいずれかの抗菌性物質を含有する酵母エキス。
（２３）前記（１２）〜（１９）のいずれかの抗菌性物質を含有する殺菌剤。
（２４）前記（１２）〜（１９）のいずれかの抗菌性物質を用いることを特徴とする、カビ及び／又は酵母の増殖を阻害する方法。
（２５）前記（１２）〜（１９）のいずれかの抗菌性物質を用いることを特徴とする、酵母及び／又は細菌の生育を阻害する方法。
（２６）前記（１２）〜（１９）のいずれかの抗菌性物質を産生する酵母を用いることを特徴とする、カビ及び／又は酵母の増殖を阻害する方法。
（２７）前記（１２）〜（１９）のいずれかの抗菌性物質を産生する酵母を用いることを特徴とする、酵母及び／又は細菌の生育を阻害する方法。
(2) The antibacterial substance according to (1), wherein, in the general formula (1), R 2 is an alkyl group having 7 to 9 carbon atoms.
(3) The antibacterial substance according to (2), wherein the antibacterial substance is n-octyl acetate, 2-nonanone, 2-decanone, or 2-undecanone.
(4) The antibacterial substance according to any one of (1) to (3), wherein the antibacterial substance inhibits germination of spores by volatilization.
(5) The antibacterial substance according to any one of (1) to (3), wherein the antibacterial substance inhibits the growth of yeast and / or bacteria by volatilization.
The second to seventh aspects of the present invention inhibit the growth of fungicides, food preservatives, yeast extracts, fungicides, fungi and / or yeasts having the following characteristics (6) to (11), respectively. A method of inhibiting the growth of yeast and / or bacteria.
(6) A fungicide containing the antibacterial substance according to any one of (1) to (5).
(7) A food preservative containing the antibacterial substance according to any one of (1) to (5).
(8) A yeast extract containing the antibacterial substance according to any one of (1) to (5).
(9) A bactericide containing the antibacterial substance according to any one of (1) to (5).
(10) A method for inhibiting the growth of mold and / or yeast, comprising using the antibacterial substance according to any one of (1) to (5).
(11) A method for inhibiting the growth of yeast and / or bacteria, comprising using the antibacterial substance according to any one of (1) to (5).
The eighth aspect of the present invention is an antibacterial substance having the following features (12) to (19).
(12) Candida spp., Saccharomyces spp., Pichia spp., Rhodotorula spp., Clavispora spp, and Torulaspora spp. Volatile antibacterial substance produced by one selected yeast.
(13) The antibacterial substance according to (12), wherein the antibacterial substance is isoamyl acetate.
(14) The above (12) or (13), wherein the genus Candida is Candida maltosa, Candida tropicalis, or Candida pseudointermedia. Antibacterial substances.
(15) The genus Candida is Candida maltosa O9-NP9 (Accession number NITE BP-297), Candida maltosa IAM12247, Candida maltosa IAM12248, Candida tropicalis IAM4965, or Candida pseudo The antibacterial substance according to (12) or (13), which is Intermedia IAM12510.
(16) The Saccharomyces spp. Is Saccharomyces cerevisiae, the Pichia spp. Is Pichia fermentans, and the Rhodotorula spp. Is Rhodotorula Actor. The antibacterial substance according to (12) or (13), wherein the genus Clavispora is Clavispora lusitanie, and the genus Torraspora is Toruspora delbrueckii.
(17) The Pichia spp. Is Pichia fermentas JCM1824, the Rhodotorula spp. Is Rhodotorula acta JCM9494, and the Saccharomyces spp. Is Saccharomyces cerevisiae H-1 (Accession Number NITE BP-473). The clavispora sp. Is Clavispora sp. P-5 (accession number NITE BP-474) or clavis pora lucitanie NBRC10059, and the torula spora delbrucky NBRC0955 is (12) Or the antibacterial substance of (13).
(18) The antibacterial substance according to any one of (12) to (17), wherein the antibacterial substance inhibits spore germination by volatilization.
(19) The antibacterial substance according to any one of (12) to (17), wherein the antibacterial substance inhibits the growth of yeast and / or bacteria by volatilization.
Furthermore, the ninth to fourteenth aspects of the present invention are, respectively, fungicides, food preservatives, yeast extracts, fungicides, fungi and / or yeast growth having the following characteristics (20) to (27): And a method of inhibiting the growth of yeast and / or bacteria.
(20) A fungicide containing the antibacterial substance according to any one of (12) to (19).
(21) A food preservative containing the antibacterial substance according to any one of (12) to (19).
(22) A yeast extract containing the antibacterial substance according to any one of (12) to (19).
(23) A bactericide containing the antibacterial substance according to any one of (12) to (19).
(24) A method for inhibiting the growth of mold and / or yeast, comprising using the antibacterial substance according to any one of (12) to (19).
(25) A method for inhibiting the growth of yeast and / or bacteria, comprising using the antibacterial substance according to any one of (12) to (19).
(26) A method for inhibiting the growth of mold and / or yeast, comprising using a yeast that produces the antibacterial substance according to any one of (12) to (19).
(27) A method for inhibiting the growth of yeast and / or bacteria, comprising using a yeast that produces the antibacterial substance according to any one of (12) to (19).
本発明の第一の態様及び第八の態様の抗菌性物質は、効果的にカビや酵母等の真菌の胞子の発芽及び増殖を抑制することができる。
本発明の第一の態様及び第八の態様の抗菌性物質により、胞子の発芽を阻害することで、食品等の保存期間を安全に延長することができるため、製造コストの改善も期待される。また、本発明の第一の態様及び第八の態様の抗菌性物質は、揮発によりカビや酵母等の真菌の活性を抑制するため、様々な用途に用いることができる。
また、本発明の第一の態様及び第八の態様の抗菌性物質により、酵母や細菌の生育を阻害することで、食品等の保存期間を安全に延長することができるため、製造コストの改善も期待される。また、本発明の第一の態様及び第八の態様の抗菌性物質は、揮発により酵母や真菌等の菌類の生育を抑制するため、様々な用途に用いることができる。
さらに、本発明の第六の態様及び第十二の態様のカビ及び／又は酵母の増殖を阻害する方法により、安全かつ簡便に、カビや酵母等の真菌の増殖を抑制することができる。
本発明の第七の態様及び第十三の態様の酵母及び／又は細菌の生育を阻害する方法により、安全且つ簡便に、酵母や細菌の生育を阻害することができる。
The antibacterial substance of the first aspect and the eighth aspect of the present invention can effectively suppress germination and growth of fungi spores such as mold and yeast.
By inhibiting the germination of spores by the antibacterial substance of the first aspect and the eighth aspect of the present invention, it is possible to safely extend the storage period of foods and the like, and thus an improvement in manufacturing cost is also expected. . Moreover, since the antimicrobial substance of the 1st aspect and 8th aspect of this invention suppresses the activity of fungi, such as mold | fungi and yeast, by volatilization, it can be used for various uses.
In addition, the antimicrobial substance of the first aspect and the eighth aspect of the present invention can safely extend the storage period of foods and the like by inhibiting the growth of yeast and bacteria, thus improving the production cost Is also expected. Moreover, since the antimicrobial substance of the 1st aspect and 8th aspect of this invention suppresses growth of fungi, such as yeast and a fungus, by volatilization, it can be used for various uses.
Furthermore, by the method for inhibiting the growth of mold and / or yeast according to the sixth aspect and the twelfth aspect of the present invention, the growth of fungi such as mold and yeast can be suppressed safely and easily.
By the method for inhibiting the growth of yeast and / or bacteria according to the seventh and thirteenth aspects of the present invention, the growth of yeast and bacteria can be inhibited safely and simply.
本発明における抗真菌活性とは、カビや酵母等の真菌の増殖を抑制する活性のうち、胞子の発芽を阻害する活性を意味する。胞子の発芽を阻害することにより、効果的にカビ等の発生及び増殖を防止することができる。
本発明における抗菌活性とは、上記抗真菌活性と、酵母等の真菌や、細菌の生育を阻害し、殺菌する活性との両方を意味する。
The antifungal activity in the present invention means an activity of inhibiting spore germination among the activities of suppressing the growth of fungi such as mold and yeast. By inhibiting spore germination, generation and growth of mold and the like can be effectively prevented.
The antibacterial activity in the present invention means both the above antifungal activity and the activity of inhibiting fungicides such as yeast and bacteria and killing bacteria.
本発明の第八の態様の抗菌性物質は、食品から得られた微生物が産生するものであって、ｐＨ７．０において、抗真菌活性及び抗菌活性を有するものである。このような抗菌性物質であれば、食品等に安全に使用することができ、かつ、中性の食品等に対しても効果を発揮することができるためである。 The antibacterial substance according to the eighth aspect of the present invention is produced by a microorganism obtained from food and has antifungal activity and antibacterial activity at pH 7.0. This is because such an antibacterial substance can be safely used for foods and the like, and can also be effective for neutral foods and the like.
本発明の抗菌性物質を産生する微生物は、例えば、各種の食品から分離した微生物の中から、ｐＨ７．０において、カビ等の増殖を抑制することができる抗菌活性を有する菌株を選択することにより得ることができる。ｐＨ７．０において抗真菌活性及び抗菌活性を有する菌株は、本発明の抗菌性物質を産生していると考えられるためである。以下、本発明の第一の態様及び第八の態様の抗菌性物質、及び該第八の態様の抗菌性物質を産生する微生物を得る方法を、さらに詳細に説明する。 The microorganism that produces the antibacterial substance of the present invention can be selected from, for example, microorganisms isolated from various foods by selecting a strain having antibacterial activity capable of suppressing the growth of mold and the like at pH 7.0. Obtainable. This is because a strain having antifungal activity and antibacterial activity at pH 7.0 is considered to produce the antibacterial substance of the present invention. Hereinafter, the antibacterial substance of the first aspect and the eighth aspect of the present invention, and the method for obtaining the microorganism that produces the antibacterial substance of the eighth aspect will be described in more detail.
１．抗真菌性物質を産生する微生物の取得
本発明者らは、抗真菌性物質を産生する微生物を食品中から取得するにあたり、良好な発酵食品の一つであるチーズに着目し、常法により、１４種類の市販されているチーズから微生物を分離したところ、カスピアンチーズ（イラン製）由来のＯ−９菌群中に、アスペルギルス・ニガー等の増殖を抑制する抗真菌性物質を産生する菌が存在していることを見出した。そして、アスペルギルス・ニガーを用いた抗真菌活性の測定の結果、Ｏ−９菌群には、ｐＨ７．０において抗真菌活性を有する菌、すなわち、本発明の抗真菌性物質を産生する菌が存在していることがわかった。
Ｏ−９菌群から単離された、本発明の抗真菌性物質を産生する菌として、キャンディダ・マルトーサＯ９−ＮＰ９株（受託番号 ＮＩＴＥ ＢＰ−２９７）、及びクラビスポラ・スピーシーズＰ−５株（受託番号 ＮＩＴＥ ＢＰ−４７４）が同定された。また、上記と同様にして、別のロットのカスピアンチーズから、本発明の抗真菌性物質を産生する菌として、サッカロマイセス・セレビシエＨ−１株（受託番号 ＮＩＴＥ ＢＰ−４７３）が同定された。上記菌株は、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された。上記菌株の遺伝学的性質及び菌学的性質は、特開２００８−２３９６０４号公報に開示された通りである。
1. Acquisition of microorganisms that produce antifungal substances The present inventors focused on cheese, which is one of the good fermented foods, to obtain microorganisms that produce antifungal substances from foods. When microorganisms were isolated from 14 kinds of commercially available cheeses, bacteria that produce antifungal substances that suppress the growth of Aspergillus niger etc. exist in the group of O-9 bacteria derived from Caspian cheese (made in Iran). I found out. As a result of measuring the antifungal activity using Aspergillus niger, the O-9 fungus group has a fungus having antifungal activity at pH 7.0, that is, a fungus producing the antifungal substance of the present invention. I found out.
Candida maltosa O9-NP9 strain (Accession No. NITE BP-297) and Clavispora species P-5 strain (isolated from the O-9 fungus group and producing the antifungal substance of the present invention) Accession Number NITE BP-474) has been identified. Moreover, Saccharomyces cerevisiae H-1 strain (accession number NITE BP-473) was identified as a microbe which produces the antifungal substance of this invention from the caspian cheese of another lot like the above. The above strains were deposited with the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation. The genetic and mycological properties of the strain are as disclosed in Japanese Patent Application Laid-Open No. 2008-239604.
２．抗真菌性物質の形体の検討
上記１で得られた菌の産生する抗真菌性物質の形体を、キャンディダ・マルトーサＯ９−ＮＰ９株を用いて、後記実施例１に記載の方法により検討した。
この結果、コントロールでは、アスペルギルス・ニガーの増殖が観察されたが、Ｏ９−ＮＰ９株の気相と接していたサンプルでは、アスペルギルス・ニガーの胞子の発芽及び増殖は観察されなかった。したがって、Ｏ９−ＮＰ９株の産生する抗真菌性物質は、該Ｏ９−ＮＰ９株と真菌とが気相を介して接触した際に抗真菌活性を有するものであり、揮発性の抗真菌性物質であることが明らかである。
2. Examination of Form of Antifungal Substance The form of the antifungal substance produced by the fungus obtained in 1 above was examined by the method described in Example 1 below using Candida maltosa O9-NP9 strain.
As a result, the growth of Aspergillus niger was observed in the control, but the spore germination and proliferation of Aspergillus niger was not observed in the sample in contact with the gas phase of the O9-NP9 strain. Therefore, the antifungal substance produced by the O9-NP9 strain has antifungal activity when the O9-NP9 strain and the fungus are contacted via the gas phase, and is a volatile antifungal substance. It is clear that there is.
３．抗真菌性物質候補の同定
上記２において揮発性の抗真菌性物質を産生することが判明したＯ９−ＮＰ９株を用いて、後記実施例２及び実施例３に記載の方法により、Ｏ９−ＮＰ９株の産生する揮発性物質を同定した。
この結果、Ｏ９−ＮＰ９株が産生する抗真菌性物質候補として、酢酸イソアミル、イソアミルアルコール、フェネチルアルコール、及びｎ−ペンタノールが同定された。
3. Identification of Antifungal Substance Candidates Using the O9-NP9 strain that was found to produce a volatile antifungal substance in 2 above, the method described in Examples 2 and 3 below describes the O9-NP9 strain. Volatile substances produced by this were identified.
As a result, isoamyl acetate, isoamyl alcohol, phenethyl alcohol, and n-pentanol were identified as candidate antifungal substances produced by the O9-NP9 strain.
４．抗真菌性物質候補の抗真菌活性の測定
上記３で得られた抗真菌性物質候補の抗真菌活性を、後記実施例４に記載の方法により検討した。
この結果から、少なくとも標準品の酢酸イソアミルは、マイクロカップに添加した２０μＬが全て揮発した際に、抗真菌活性を有することが分かった。
4). Measurement of Antifungal Activity of Antifungal Substance Candidate The antifungal activity of the antifungal substance candidate obtained in 3 above was examined by the method described in Example 4 below.
From this result, it was found that at least standard isoamyl acetate had antifungal activity when all 20 μL added to the microcup was volatilized.
５．酢酸イソアミル類縁体の抗真菌活性の測定
酢酸イソアミル類縁体が、酢酸イソアミルと同様の抗真菌活性を有するかどうかを、後記実施例５に記載の方法により検討した。
図６〜８は、酢酸イソアミル及び／又は酢酸イソアミル類縁体の、胞子発芽阻止最小添加量と、該最小添加量における揮発量と、ケト基又はエステルに結合したアルキル基の長さとを示した図である。図６は、酢酸イソアミルの酸側構造の類縁体、図７は、酢酸イソアミルのアルコール側構造の類縁体（酢酸エステル類）、図８は、酢酸イソアミル類縁体（２−ケトン類）の結果を示す。
図６〜８及び後記実施例５に示すように、酢酸イソアミルのみならず、酢酸イソアミルの類縁体である、蟻酸イソアミル、プロピオン酸イソアミル、ｎ−酪酸イソアミル、酢酸ｎ−プロピル、酢酸イソブチル、酢酸ｎ−ブチル、酢酸２−メチルブチル、酢酸ｎ−ペンチル、酢酸ｎ−ヘキシル、酢酸ｎ−ヘプチル、酢酸ｎ−オクチル、２−ペンタノン、２−ヘキサノン、５−メチル−２−ヘキサノン、２−ヘプタノン、２−オクタノン、２−ノナノン、２−デカノン、及び２−ウンデカノンが、本発明の抗真菌活性を有することが明らかである。また、酢酸ｎ−オクチル、２−ノナノン、２−デカノン、及び２−ウンデカノンは、添加量が少なく、揮発量が少ない場合にも真菌の胞子の発芽及び増殖を阻害するため、本発明の抗真菌活性が高いことが明らかである。
また、これら本発明の抗真菌活性を有する酢酸イソアミル及び酢酸イソアミル類縁体の構造から、下記一般式（１）で表される化合物が、本発明の抗真菌活性を有することが分かった。
5. Measurement of Antifungal Activity of Isoamyl Acetate Analogue Whether or not the isoamyl acetate analog has the same antifungal activity as isoamyl acetate was examined by the method described in Example 5 below.
FIGS. 6 to 8 are views showing the minimum addition amount of spore germination inhibition, the volatilization amount at the minimum addition amount, and the length of the alkyl group bonded to the keto group or ester of isoamyl acetate and / or isoamyl acetate analog. It is. Fig. 6 shows the result of isoamyl acetate acid side structure, Fig. 7 shows the result of isoamyl acetate alcohol side structure (acetate ester), and Fig. 8 shows the result of isoamyl acetate analog (2-ketones). Show.
As shown in FIGS. 6 to 8 and Example 5 described later, not only isoamyl acetate, but also isoamyl acetate, isoamyl formate, isoamyl propionate, isoamyl n-butyrate, n-propyl acetate, isobutyl acetate, n-acetate -Butyl, 2-methylbutyl acetate, n-pentyl acetate, n-hexyl acetate, n-heptyl acetate, n-octyl acetate, 2-pentanone, 2-hexanone, 5-methyl-2-hexanone, 2-heptanone, 2- It is clear that octanone, 2-nonanone, 2-decanone, and 2-undecanone have the antifungal activity of the present invention. In addition, n-octyl acetate, 2-nonanone, 2-decanone, and 2-undecanone inhibit germination and growth of fungal spores even when the addition amount is small and the volatilization amount is small. It is clear that the activity is high.
Moreover, it turned out that the compound represented by following General formula (1) has the antifungal activity of this invention from the structure of the isoamyl acetate which has the antifungal activity of this invention, and the isoamyl acetate analog.
６．種々の真菌及び細菌に対する抗菌性の検討
酢酸イソアミルが、アスペルギルス・ニガーと同様に、他の真菌や細菌に対して抗菌活性を有するかどうかを、後記実施例６〜８に記載の方法により検討した。
その結果、酢酸イソアミルは、検討に用いたアスペルギルス・ニガーを含む糸状菌（カビ）１５種、酵母５種、グラム陽性細菌７種、及びグラム陰性細菌２種の全てに対して、抗菌活性を有することが分かった。
6). Examination of antibacterial activity against various fungi and bacteria Whether or not isoamyl acetate has antibacterial activity against other fungi and bacteria as in the case of Aspergillus niger was examined by the methods described in Examples 6 to 8 below. .
As a result, isoamyl acetate has antibacterial activity against all 15 types of fungi (mold) containing Aspergillus niger used in the study, 5 types of yeast, 7 types of gram-positive bacteria, and 2 types of gram-negative bacteria. I understood that.
また、酢酸イソアミルが各種菌類に与える影響を検討すべく、酢酸イソアミルに曝露したアスペルギルス・ニガー、エスケリキア・コリ（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）及びサッカロマイセス・セレビシエの形態を観察した。
その結果、アスペルギルス・ニガーでは、酢酸イソアミルに曝露下において胞子の発芽が阻害されるものの、酢酸イソアミルを除去すると胞子が発芽することから、酢酸イソアミルは胞子自体を死滅させる殺菌効果ではなく、胞子の発芽のみを阻害する静菌効果を有することが分かった。一方、エスケリキア・コリ及びサッカロマイセス・セレビシエでは、酢酸イソアミルにより細胞内構造の変化や崩壊が認められたことから、酢酸イソアミルはこれらの菌に対して細胞を死滅させる殺菌効果を有することが分かった。
さらに、蛍光ディファレンスゲル二次元電気泳動（２Ｄ−ＤＩＧＥ）を用いて、通常培養のエスケリキア・コリと、酢酸イソアミルに曝露したエスケリキア・コリとのタンパク質発現量解析を行ったところ、分子量２０ｋＤａ以上、且つ、酸性の領域に多くのタンパク質スポットが検出され、それらタンパク質スポットの中には、通常培養と酢酸イソアミル曝露とで発現量の異なるタンパク質スポットが複数観察された。
In order to examine the effect of isoamyl acetate on various fungi, the morphology of Aspergillus niger, Escherichia coli and Saccharomyces cerevisiae exposed to isoamyl acetate was observed.
As a result, in Aspergillus niger, spore germination is inhibited under exposure to isoamyl acetate, but when isoamyl acetate is removed, spores germinate, so isoamyl acetate is not a bactericidal effect that kills spores themselves. It was found to have a bacteriostatic effect that inhibits germination alone. On the other hand, in Escherichia coli and Saccharomyces cerevisiae, changes and disruption of the intracellular structure were observed by isoamyl acetate, and thus it was found that isoamyl acetate has a bactericidal effect to kill cells against these bacteria.
Furthermore, when the protein expression level analysis of Escherichia coli cultured in normal culture and Escherichia coli exposed to isoamyl acetate was performed using fluorescence difference gel two-dimensional electrophoresis (2D-DIGE), the molecular weight was 20 kDa or more. In addition, many protein spots were detected in the acidic region, and among these protein spots, a plurality of protein spots with different expression levels were observed between normal culture and exposure to isoamyl acetate.
本発明の第一の態様の抗菌性物質は、上記一般式（１）で表される抗菌性物質であって、胞子の発芽を阻害し、且つ、酵母や細菌の生育を阻害し、殺菌する抗菌性物質である。
上記式（１）中、Ｒ１は炭素数０〜３のアルキル基であり、炭素数１〜３であることが好ましい。該アルキル基は直鎖状であっても、分岐鎖状であってもよい。
上記式（１）中、Ｒ２は炭素数３〜９のアルキル基であり、炭素数４〜９であることが好ましく、炭素数５〜９であることがより好ましく、炭素数７〜９であることがさらに好ましい。該アルキル基は直鎖状であっても、分岐鎖状であってもよい。
また、本発明者らの検討により、上記式（１）中のケト基（Ｃ（＝Ｏ））は抗真菌活性及び抗菌活性に必須であり、ケト基を有しない同様の鎖長の炭化水素基（例えば、Ｃ８〜Ｃ１２の直鎖状又は分岐鎖状の炭化水素基）は、抗菌活性を有しないことが分かった。
The antibacterial substance of the first aspect of the present invention is an antibacterial substance represented by the above general formula (1), which inhibits germination of spores, inhibits the growth of yeasts and bacteria, and sterilizes. It is an antibacterial substance.
In the above formula (1), R 1 is an alkyl group having 0 to 3 carbon atoms, and preferably 1 to 3 carbon atoms. The alkyl group may be linear or branched.
In the above formula (1), R 2 is an alkyl group having 3 to 9 carbon atoms, preferably 4 to 9 carbon atoms, more preferably 5 to 9 carbon atoms, and 7 to 9 carbon atoms. More preferably it is. The alkyl group may be linear or branched.
Further, as a result of the study by the present inventors, the keto group (C (═O)) in the above formula (1) is essential for antifungal activity and antibacterial activity, and has a similar chain length hydrocarbon not having a keto group. It has been found that groups (eg, C8-C12 linear or branched hydrocarbon groups) do not have antimicrobial activity.
本発明の第一の態様の抗菌性物質は、酢酸イソアミル、蟻酸イソアミル、酢酸イソアミル、プロピオン酸イソアミル、ｎ−酪酸イソアミル、酢酸ｎ−プロピル、酢酸イソブチル、酢酸ｎ−ブチル、酢酸２−メチルブチル、酢酸ｎ−ペンチル、酢酸ｎ−ヘキシル、酢酸ｎ−ヘプチル、酢酸ｎ−オクチル、２−ペンタノン、２−ヘキサノン、５−メチル−２−ヘキサノン、２−ヘプタノン、２−オクタノン、２−ノナノン、２−デカノン、及び２−ウンデカノンからなる群から選ばれる１種以上であることが好ましく、酢酸ｎ−オクチル、２−ノナノン、２−デカノン、又は２−ウンデカノンであることがより好ましい。
本発明の第一の態様の抗菌性物質は、揮発により胞子の発芽を阻害する。抗菌性物質を揮発させる方法は特に限定されるものではなく、該抗菌性物質を含む溶液を常温下で静置する方法や、適度な熱をかけて揮発を促進する方法等の公知の方法により行うことができる。
本発明の第一の態様の抗菌性物質は、防カビ剤や食品保存料として用いることもできる。また、本発明の第一の態様の抗菌性物質は、殺菌剤として用いることもできる。
The antibacterial substance of the first aspect of the present invention is isoamyl acetate, isoamyl formate, isoamyl acetate, isoamyl propionate, isoamyl n-butyrate, n-propyl acetate, isobutyl acetate, n-butyl acetate, 2-methylbutyl acetate, acetic acid n-pentyl, n-hexyl acetate, n-heptyl acetate, n-octyl acetate, 2-pentanone, 2-hexanone, 5-methyl-2-hexanone, 2-heptanone, 2-octanone, 2-nonanone, 2-decanone And at least one selected from the group consisting of 2-undecanone, more preferably n-octyl acetate, 2-nonanone, 2-decanone, or 2-undecanone.
The antibacterial substance of the first aspect of the present invention inhibits spore germination by volatilization. The method for volatilizing the antibacterial substance is not particularly limited, and a known method such as a method of allowing the solution containing the antibacterial substance to stand at room temperature or a method of promoting volatilization by applying appropriate heat. It can be carried out.
The antibacterial substance of the first aspect of the present invention can also be used as a fungicide or a food preservative. The antibacterial substance of the first aspect of the present invention can also be used as a bactericidal agent.
本発明の第六の態様のカビ及び／又は酵母の増殖を阻害する方法は、本発明の第一の態様の抗菌性物質を用いる方法であって、好適にカビ及び／又は酵母の増殖を阻害できる方法であれば特に限定されるものではない。例えば、該抗菌性物質を揮発させ、食品等に原料として付着させる方法や、製造された食品に噴霧し揮発させる方法等がある。また、該抗菌性物質を産生する生物（菌体、植物等）を用いて、該生物の産生した揮発性物質を、食品等に原料として付着させる方法や、製造された食品に噴霧し揮発させる方法も用いることができる。また、抗菌性物質を産生する生物が食品添加物となり得るものの場合は、該生物自体や該生物の抽出エキス（例えば酵母エキス）を食品等に原料として添加し、産生された揮発性の抗菌性物質を利用してもよい。 The method for inhibiting the growth of mold and / or yeast according to the sixth aspect of the present invention is a method using the antibacterial substance according to the first aspect of the present invention, preferably inhibiting the growth of mold and / or yeast. It is not particularly limited as long as it can be performed. For example, there are a method of volatilizing the antibacterial substance and adhering it as a raw material to foods, a method of spraying and volatilizing manufactured foods, and the like. In addition, by using organisms (such as fungus bodies and plants) that produce the antibacterial substances, the volatile substances produced by the organisms can be attached as a raw material to foods, etc., or sprayed and volatilized on manufactured foods. Methods can also be used. In the case where an organism producing an antibacterial substance can be a food additive, the organism itself or an extract of the organism (for example, yeast extract) is added to food as a raw material, and the produced volatile antibacterial property Substances may be used.
本発明の第七の態様の酵母及び／又は細菌の生育を阻害する方法は、本発明の第一の態様の抗菌性物質を用いる方法であって、好適に酵母及び／又は細菌の生育を阻害し、殺菌できる方法であれば特に限定されるものではなく、上記第六の態様と同様に行うことができる。 The method for inhibiting the growth of yeast and / or bacteria according to the seventh aspect of the present invention is a method using the antibacterial substance according to the first aspect of the present invention, and preferably inhibits the growth of yeast and / or bacteria. And it will not specifically limit if it is a method which can be sterilized, It can carry out similarly to the said 6th aspect.
本発明の第八の態様の抗菌性物質は、キャンディダ属菌、サッカロマイセス属菌、ピキア属菌、ロドトルラ属菌、クラビスポラ属菌、及びトルラスポラ属菌からなる群より選ばれる１の酵母が産生する、胞子の発芽を阻害する揮発性の抗菌性物質である。 The antibacterial substance of the eighth aspect of the present invention is produced by one yeast selected from the group consisting of Candida spp., Saccharomyces spp., Pichia spp., Rhodotorula spp., Clavispora spp. It is a volatile antibacterial substance that inhibits spore germination.
本発明の第八の態様の抗菌性物質を産生するキャンディダ属菌は、キャンディダ・マルトーサ、キャンディダ・トロピカリス、又はキャンディダ・シュードインターメディアであることが好ましく、キャンディダ・マルトーサであることが特に好ましい。該キャンディダ・マルトーサには、例えば、キャンディダ・マルトーサＯ９−ＮＰ９、キャンディダ・マルトーサＩＡＭ１２２４７、及びキャンディダ・マルトーサＩＡＭ１２２４８等がある。該キャンディダ・トロピカリスには、例えば、キャンディダ・トロピカリスＩＡＭ４９６５等がある。該キャンディダ・シュードインターメディアには、例えば、キャンディダ・シュードインターメディアＩＡＭ１２５１０等がある。 The Candida spp. Producing the antibacterial substance of the eighth aspect of the present invention is preferably Candida maltosa, Candida tropicalis, or Candida pseudointermedia, and is Candida maltosa. It is particularly preferred. Examples of the Candida maltosa include Candida maltosa O9-NP9, Candida maltosa IAM12247, and Candida maltosa IAM12248. Examples of the Candida tropicalis include Candida tropicalis IAM4965. Examples of the Candida pseudo intermedia include Candida pseudo intermedia IAM12510.
本発明の第八の態様の抗菌性物質を産生するピキア属菌は、ピキア・フェルメンタスであることが好ましく、ピキア・フェルメンタスＪＣＭ１８２４であることが特に好ましい。また、本発明の抗菌性物質を産生するロドトルラ属菌は、ロドトルラ・アクタであることが好ましく、ロドトルラ・アクタＪＣＭ９４９４であることが特に好ましい。また、本発明の抗菌性物質を産生するサッカロマイセス属菌は、サッカロマイセス・セレビシエであることが好ましく、サッカロマイセス・セレビシエＨ−１であることが特に好ましい。また、本発明の抗菌性物質を産生するクラビスポラ属菌は、クラビスポラ・ルシタニエであることが好ましく、クラビスポラ・スピーシーズＰ−５又はクラビスポラ・ルシタニエＮＢＲＣ１００５９であることが特に好ましい。また、本発明の抗菌性物質を産生するトルラスポラ属菌は、トルラスポラ・デルブレッキーであることが好ましく、トルラスポラ・デルブルッキーＮＢＲＣ０９５５であることが特に好ましい。 The Pichia spp. Producing the antibacterial substance of the eighth aspect of the present invention is preferably Pichia fermentas, particularly preferably Pichia fermentas JCM1824. Further, Rhodotorula genus that produces the antibacterial substance of the present invention is preferably Rhodotorula acta, particularly preferably Rhodotorula acta JCM9494. The Saccharomyces spp. Producing the antibacterial substance of the present invention is preferably Saccharomyces cerevisiae, and particularly preferably Saccharomyces cerevisiae H-1. The clavispora spp. Producing the antibacterial substance of the present invention is preferably clavispora lucitanie, particularly preferably clavispora species p-5 or clavispora lucitanie NBRC10059. Moreover, it is preferable that the Torraspora genus microbe which produces the antibacterial substance of this invention is a Torulas pora delbrecky, and it is especially preferable that it is a Torraspora delbrucky NBRC0955.
本発明の酵母、すなわち、本発明の第八の態様の抗菌性物質を産生するキャンディダ属菌、サッカロマイセス属菌、ピキア属菌、ロドトルラ属菌、クラビスポラ属菌、及びトルラスポラ属菌は、常法により培養することができ、培地や温度等の培養条件は、通常、キャンディダ属菌等の酵母を培養する条件であれば、特に限定されるものではない。該培地として、例えば、ＹＰＤ培地、ＰＤ培地、ＹＭ培地、１０％（ｗ／ｗ）スキムミルク培地等がある。本発明の抗菌性物質の活性が阻害されるおそれが少ないため、ＹＰＤ培地、ＰＤ培地、又は１０％（ｗ／ｗ）スキムミルク培地であることが好ましい。また、培養温度は、２７〜３０℃が好ましい。 The yeast of the present invention, that is, the Candida spp., Saccharomyces spp., Pichia spp., Rhodotorula spp., Clavispora spp. And Torlaspora spp. The culture conditions such as medium and temperature are not particularly limited as long as they are conditions for culturing yeasts such as Candida. Examples of the medium include YPD medium, PD medium, YM medium, 10% (w / w) skim milk medium, and the like. Since there is little possibility that the activity of the antibacterial substance of the present invention is inhibited, a YPD medium, PD medium, or 10% (w / w) skim milk medium is preferable. The culture temperature is preferably 27 to 30 ° C.
本発明の第八の態様の抗菌性物質は、本発明の酵母を培養することにより得ることができる。例えば、本発明の酵母を培養したシャーレ等の培養容器中の気相を回収することにより、得ることができる。気相を回収する方法は、該気相中の抗菌性物質を回収できる条件であれば、特に限定されるものではないが、他の物質の混入を避けるため、密閉したシャーレ等の培養容器中からシリンジ等を用いて回収する方法や、密閉容器に多数のシャーレを入れ、真空ポンプで気相を吸引し途中に設置した吸着剤に吸着させる方法が好ましい。また、公知の方法により、回収された気相から抗菌性物質のみを濃縮することもできる。このようにして回収された気相中の抗菌性物質は、防カビ剤、食品保存料、該抗菌性物質を含有する殺菌剤等として用いることもできる。 The antibacterial substance of the eighth aspect of the present invention can be obtained by culturing the yeast of the present invention. For example, it can be obtained by recovering the gas phase in a culture vessel such as a petri dish in which the yeast of the present invention is cultured. The method for recovering the gas phase is not particularly limited as long as the antibacterial substance in the gas phase can be recovered. However, in order to avoid contamination with other substances, it can be contained in a closed culture vessel such as a petri dish. A method of collecting from a syringe using a syringe or a method of putting a large number of petri dishes in an airtight container, sucking the gas phase with a vacuum pump and adsorbing it on an adsorbent installed in the middle is preferable. Moreover, only an antibacterial substance can also be concentrated from the collect | recovered gaseous phase by a well-known method. The antibacterial substance in the gas phase thus recovered can be used as a fungicide, a food preservative, a bactericide containing the antibacterial substance, and the like.
また、本発明の第八の態様の抗菌性物質は、該抗菌性物質を含む酵母エキスの形態で、上記防カビ剤、食品保存料、殺菌剤等として用いることもできる。
本発明において酵母エキスとは、酵母が有する様々な成分を抽出したものであり、アミノ酸やペプチド、核酸、ミネラル等が含まれている。また、酵母の種類や培養条件、抽出条件によって、各種成分の含有比を調整することができる。
酵母エキスの抽出方法は、特に限定されるものではなく、酵母等の生物原料からエキスを抽出する際に通常用いられる方法のうち、いずれの方法を用いてもよい。該抽出方法として、例えば、自己消化法、酵素分解法等がある。ここで、自己消化法とは、酵母が本来有している酵素の働きにより、酵母を可溶化し、抽出する方法であり、遊離アミノ酸含有量の多い酵母エキスを得ることができる。一方、酵素分解法とは、熱処理等により、酵母が有する酵素等を不活性化した後、分解酵素を添加して酵母を可溶化し、抽出する方法である。外部から適当な酵素を添加することにより、酵素反応を簡便に制御し得るため、遊離アミノ酸や核酸の含有量を調整することができる。酵素分解法において用いられる酵素は、通常生体成分を分解する際に用いられる酵素であれば、特に限定されるものではなく、任意の酵素を用いることができる。該酵素として、例えば、酵母の細胞壁を分解し得る酵素、タンパク質分解酵素、核酸分解酵素等があり、これらを適宜併用することにより、酵母から各種成分を効率よく抽出することができる。
In addition, the antibacterial substance of the eighth aspect of the present invention can be used as the above-mentioned antifungal agent, food preservative, bactericidal agent and the like in the form of yeast extract containing the antibacterial substance.
In the present invention, the yeast extract is an extract of various components of yeast, and includes amino acids, peptides, nucleic acids, minerals, and the like. Moreover, the content ratio of various components can be adjusted according to the kind of yeast, culture conditions, and extraction conditions.
The method for extracting the yeast extract is not particularly limited, and any of the methods usually used for extracting the extract from biological materials such as yeast may be used. Examples of the extraction method include an autolysis method and an enzymatic decomposition method. Here, the self-digestion method is a method of solubilizing and extracting yeast by the action of an enzyme inherent in yeast, and a yeast extract having a high free amino acid content can be obtained. On the other hand, the enzymatic decomposition method is a method of inactivating an enzyme or the like possessed by yeast by heat treatment or the like and then adding a degrading enzyme to solubilize and extract the yeast. Since an enzyme reaction can be easily controlled by adding an appropriate enzyme from the outside, the content of free amino acids and nucleic acids can be adjusted. The enzyme used in the enzymatic decomposition method is not particularly limited as long as it is an enzyme that is usually used for decomposing biological components, and any enzyme can be used. Examples of the enzyme include an enzyme capable of degrading the cell wall of yeast, a proteolytic enzyme, a nucleolytic enzyme, and the like, and various components can be efficiently extracted from the yeast by appropriately using them together.
本発明の第八の態様の抗菌性物質が、喫食可能な食品（例えば、チーズ等）由来の酵母が産生する抗菌性物質である場合、該抗菌性物質を食品に対して安全に用いることができるのみならず、化粧品や医薬品等にも用いることができる。
また、第八の態様の抗菌性物質が、中性の食品由来の酵母、又は、中性で生存可能な酵母が産生する抗菌性物質である場合、中性の食品等においても効果的に抗菌活性を示すことができる。
When the antibacterial substance according to the eighth aspect of the present invention is an antibacterial substance produced by yeast derived from food that can be eaten (for example, cheese), the antibacterial substance can be used safely for food. It can be used not only for cosmetics and pharmaceuticals.
In addition, when the antibacterial substance of the eighth aspect is an antibacterial substance produced by a neutral food-derived yeast or a neutral and viable yeast, the antibacterial substance can be effectively antibacterial even in a neutral food. Activity can be demonstrated.
本発明の第十三の態様のカビ及び／又は酵母の増殖を阻害する方法は、本発明の抗菌性物質又は本発明の抗菌性物質を産生する酵母を用いる方法であって、本発明の抗菌性物質の抗真菌活性及び抗菌活性が得られる方法であれば、特に限定されるものではない。本発明の抗菌性物質を用いる方法として、例えば、該抗菌性物質を産生する酵母の培養液の気相を、食品等に原料として付着させる方法や、製造された食品に噴霧して揮発させる方法等がある。また、本発明の酵母を用いる方法として、例えば、発酵性食品の製造において、原料に本発明の酵母を添加した後、発酵させる方法等がある。該発酵性食品は、加熱処理を含まない発酵性食品であることが好ましい。 The method for inhibiting the growth of mold and / or yeast according to the thirteenth aspect of the present invention is a method using the antibacterial substance of the present invention or the yeast that produces the antibacterial substance of the present invention, and comprises the antibacterial agent of the present invention. The method is not particularly limited as long as the antifungal activity and antibacterial activity of the active substance can be obtained. As a method using the antibacterial substance of the present invention, for example, a method of adhering a gas phase of a culture solution of a yeast producing the antibacterial substance as a raw material, or a method of volatilizing sprayed manufactured food Etc. Moreover, as a method using the yeast of this invention, there exists the method of fermenting, for example, after adding the yeast of this invention to a raw material in manufacture of fermentable foodstuff. The fermentable food is preferably a fermentable food that does not include heat treatment.
本発明の第十四の態様の酵母及び／又は細菌の生育を阻害する方法は、本発明の抗菌性物質又は本発明の抗菌性物質を産生する酵母を用いる方法であって、本発明の抗菌物質の抗真菌活性や抗菌活性が得られる方法であれば、特に限定されるものではなく、上記第十三の態様と同様に行うことができる。 The method for inhibiting the growth of yeast and / or bacteria according to the fourteenth aspect of the present invention is a method using the antibacterial substance of the present invention or the yeast producing the antibacterial substance of the present invention. The method is not particularly limited as long as the antifungal activity and antibacterial activity of the substance can be obtained, and can be performed in the same manner as in the thirteenth aspect.
次に実施例を示して本発明をさらに詳細に説明するが、本発明は以下の実施例に限定されるものではない。 EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.
［参考例１］
アスペルギルス・ニガーの胞子懸濁液を調整した。
アスペルギルス・ニガーＮＢＲＣ９４５５株は、ＰＤＡ（ポテトデキストロース寒天）平板培地に塗布した後、２８℃で３日間培養して得られた胞子を、０．０５％ｔｗｅｅｎ８０添加生理食塩水に懸濁し、懸濁液を綿濾過後、−８０℃で凍結保存したものを用いた。該凍結保存後のアスペルギルス・ニガーＮＢＲＣ９４５５株を、生理食塩水を用いて希釈した後、サブロー培地を用いて、１．０×１０４ｓｐｏｒｅｓ／ｍＬのアスペルギルス・ニガー胞子懸濁液を調製した。
[Reference Example 1]
A spore suspension of Aspergillus niger was prepared.
Aspergillus niger NBRC 9455 strain was applied to a PDA (potato dextrose agar) plate medium and then cultured at 28 ° C. for 3 days. Suspension obtained by suspending the spore in 0.05% tween 80-added physiological saline Was filtered through cotton and stored frozen at -80 ° C. The Aspergillus niger NBRC 9455 strain after the cryopreservation was diluted with physiological saline, and then a 1.0 × 10 4 spores / mL Aspergillus niger spore suspension was prepared using Sabouraud medium.
［実施例１］
キャンディダ・マルトーサＯ９−ＮＰ９株の産生する抗真菌性物質の形体を検討した。
まず、キャンディダ・マルトーサＯ９−ＮＰ９株を、ＹＰＤアガー平板培地に塗布して２８℃で４８時間培養して、キャンディダ・マルトーサＯ９−ＮＰ９株が生育しているＹＰＤアガー平板培地（以下、「酵母混釈寒天培地」という。）含有シャーレを作製した。
一方、参考例１に記載の方法で調整した１．０×１０４ｓｐｏｒｅｓ／ｍＬのアスペルギルス・ニガーＮＢＲＣ９４５５株胞子懸濁液を混釈したＰＤＡ（ポテトデキストロースアガー）平板培地（以下、「胞子混釈寒天培地」という。）含有シャーレを作製した。
次に、酵母混釈寒天培地、及び胞子混釈寒天培地の気相同士が接触するように、それぞれのシャーレのふたを開け、胞子混釈寒天培地含有シャーレを上下逆にして、酵母混釈寒天培地含有シャーレの上に重ね合わせ、パラフィルムを用いて固定及び密閉し、２８℃で２日間培養した。コントロールとして、酵母混釈寒天培地の代わりにＰＤＡ培地のみを添加したシャーレを用いた。
この結果、コントロールでは、アスペルギルス・ニガーの増殖が観察された。一方、Ｏ９−ＮＰ９株を用いたサンプルでは、アスペルギルス・ニガーの増殖は観察されなかった。
該観察の結果から、Ｏ９−ＮＰ９株の産生する抗真菌性物質は、該Ｏ９−ＮＰ９株と真菌とが気相を介して接触した際に、真菌に対して抗真菌活性を有するものであるため、該抗真菌性物質は、揮発性であることが分かった。
[Example 1]
The form of the antifungal substance produced by Candida maltosa O9-NP9 strain was examined.
First, the Candida maltosa O9-NP9 strain was applied to a YPD agar plate medium and cultured at 28 ° C. for 48 hours to obtain a YPD agar plate medium (hereinafter referred to as “Candida maltosa O9-NP9 strain”). A yeast petri dish agar medium ”) was prepared.
On the other hand, a spore suspension of PDA (potato dextrose agar) prepared by pouring a 1.0 × 10 4 spores / mL Aspergillus niger NBRC 9455 spore suspension prepared by the method described in Reference Example 1 (hereinafter referred to as “spore blending”). A petri dish containing “agar medium” was prepared.
Next, open the lid of each petri dish so that the gas phase of the yeast pour agar medium and the spore pour agar medium are in contact with each other, and turn the petri dish containing the spore pour agar medium upside down. It was placed on a medium-containing petri dish, fixed and sealed with parafilm, and cultured at 28 ° C. for 2 days. As a control, a petri dish to which only a PDA medium was added instead of the yeast pour agar medium was used.
As a result, growth of Aspergillus niger was observed in the control. On the other hand, no growth of Aspergillus niger was observed in the sample using the O9-NP9 strain.
From the results of the observation, the antifungal substance produced by the O9-NP9 strain has an antifungal activity against the fungus when the O9-NP9 strain and the fungus are contacted via the gas phase. Thus, the antifungal substance was found to be volatile.
［実施例２］
Ｏ９−ＮＰ９株由来の揮発性の抗真菌性物質を同定するために、Ｏ９−ＮＰ９株の産生する揮発性物質を同定した。
まず、塗布後に２８℃で２４時間培養した以外は、上記実施例１と同様に調整した酵母混釈寒天培地含有シャーレを作製した。また、ＹＰＤ平板培地のみを含有するシャーレを作製した。その後、上記実施例１と同様に、これらのシャーレを重ね合わせて固定し、２８℃で４８時間培養した。このとき、ＹＰＤ平板培地のみ含有するシャーレを上下逆にして固定した。
次に、重ね合わせたシャーレの間から、ＳＰＭＥファイバーアセンブリー（ＰＤＭＳ膜厚１００μｍ）を挿入し、２枚のシャーレの間の気相内の揮発性物質を該ファイバーアセンブリーに１５分間吸着させた後、該ファイバーアセンブリーを用いて、ガスクロマトグラフィ−質量分析法（ＧＣ−ＭＳ法）による分析を行った。
ガスクロマトグラフィ、質量分析の条件はそれぞれ以下の通りである。
[ガスクロマトグラフィ]
・カラム：ＤＢ−ＷＡＸ（商品名、アジレント・テクノロジー社製、長さ３０ｍ、内径０．３２ｍｍ、膜厚０．２５μｍ）、
・注入口温度：２２０℃
・検出器温度：２２０℃
・オーブン温度：５０℃（５分間）〜４℃／分〜２２０℃（１２．５分間）
・キャリアガス：Ｈｅ
[質量分析]
・イオン化：ＥＩ
・イオン源温度：２３０℃
・イオン化エネルギー：７０ｅＶ
ガスクロマトグラフィによる分析の結果、図１に示すように、リテンションタイム４．４８分、７．４６分、２９．３２分において主要なピークが検出され、図３〜５に示す質量分析の結果、これらのピークは、それぞれ、酢酸イソアミル、イソアミルアルコール、フェネチルアルコールであった。
この結果から、Ｏ９−ＮＰ９株が、揮発性物質として、酢酸イソアミル、イソアミルアルコール、及びフェネチルアルコールを産生していることが分かった。
[Example 2]
In order to identify a volatile antifungal substance derived from the O9-NP9 strain, a volatile substance produced by the O9-NP9 strain was identified.
First, a petri dish containing a yeast pour-agar medium prepared in the same manner as in Example 1 was prepared except that the cells were cultured at 28 ° C. for 24 hours after application. Moreover, the petri dish containing only a YPD flat plate culture medium was produced. Thereafter, these petri dishes were stacked and fixed in the same manner as in Example 1, and cultured at 28 ° C. for 48 hours. At this time, the petri dish containing only the YPD flat plate medium was fixed upside down.
Next, an SPME fiber assembly (PDMS film thickness 100 μm) was inserted between the overlapping petri dishes, and the volatile substances in the gas phase between the two petri dishes were adsorbed to the fiber assembly for 15 minutes. Then, the analysis by gas chromatography-mass spectrometry (GC-MS method) was performed using this fiber assembly.
The conditions for gas chromatography and mass spectrometry are as follows.
[Gas chromatography]
Column: DB-WAX (trade name, manufactured by Agilent Technologies, length 30 m, inner diameter 0.32 mm, film thickness 0.25 μm),
・ Inlet temperature: 220 ° C
-Detector temperature: 220 ° C
Oven temperature: 50 ° C. (5 minutes) to 4 ° C./minute to 220 ° C. (12.5 minutes)
・ Carrier gas: He
[Mass spectrometry]
・ Ionization: EI
-Ion source temperature: 230 ° C
・ Ionization energy: 70 eV
As a result of analysis by gas chromatography, as shown in FIG. 1, major peaks were detected at retention times of 4.48 minutes, 7.46 minutes, and 29.32 minutes. As a result of mass spectrometry shown in FIGS. These peaks were isoamyl acetate, isoamyl alcohol, and phenethyl alcohol, respectively.
From this result, it was found that the O9-NP9 strain produced isoamyl acetate, isoamyl alcohol, and phenethyl alcohol as volatile substances.
［実施例３］
実施例２の揮発物質の回収方法を変更し、Ｏ９−ＮＰ９株由来の揮発性の抗真菌性物質の同定を再度試みた。
まず、上記実施例２と同様に調整した酵母混釈寒天培地含有シャーレ１１枚を、球形ガラス容器内に格納し、２８℃で４８時間培養した。
次に、球形ガラス容器内の気相をシリンジで５ｍＬ吸引し、ＧＳ−ＭＳ法により分析した。ガスクロマトグラフィ、質量分析の条件は上記実施例２と同様である。
ガスクロマトグラフィによる分析の結果、図２に示すように、リテンションタイム３．０８分においてピークが検出され、質量分析の結果、このピークはｎ−ペンタノールであった。
この結果から、Ｏ９−ＮＰ９株はさらに、揮発性物質として、ｎ−ペンタノールを産生していることが分かった。
[Example 3]
The recovery method of the volatile substance of Example 2 was changed, and identification of the volatile antifungal substance derived from the O9-NP9 strain was attempted again.
First, 11 petri dish containing yeast pour-agar medium prepared in the same manner as in Example 2 above was stored in a spherical glass container and cultured at 28 ° C. for 48 hours.
Next, 5 mL of the gas phase in the spherical glass container was sucked with a syringe and analyzed by the GS-MS method. The conditions for gas chromatography and mass spectrometry are the same as in Example 2 above.
As a result of analysis by gas chromatography, as shown in FIG. 2, a peak was detected at a retention time of 3.08 minutes. As a result of mass spectrometry, this peak was n-pentanol.
From this result, it was found that the O9-NP9 strain was further producing n-pentanol as a volatile substance.
[実施例４]
上記実施例２及び実施例３で得られた抗真菌性物質候補の、抗真菌活性を検討した。
上記実施例１と同様に、胞子混釈寒天培地を調整し、該培地を滅菌済みの型を用いてくりぬき、胞子が混釈されたアガープラグを作製した。該アガープラグは、縦、横、高さ共に３ｍｍの立方体状とした。
次に、直径９０ｍｍのガラスシャーレ内にＰＤＡ平板培地を作製し、該ＰＤＡ培地の中心点上に、滅菌済みのマイクロカップを設置して、その中に表１に示す抗真菌性物質候補の標準品を２０〜８０μＬ添加した。また、マイクロカップの周囲に、上記アガープラグを３つ設置し、ガラスシャーレを密閉して２８℃で４８時間培養した後、アガープラグ及びアガープラグ周囲のアスペルギルス・ニガーの胞子発芽及び増殖を観察した。
[Example 4]
The antifungal activity of the antifungal substance candidates obtained in Example 2 and Example 3 was examined.
In the same manner as in Example 1, a spore-mixed agar medium was prepared, and the medium was cut out using a sterilized mold to prepare an agar plug mixed with spores. The agar plug was in the form of a cube having a length, width and height of 3 mm.
Next, a PDA plate medium is prepared in a glass petri dish having a diameter of 90 mm, a sterilized microcup is placed on the center point of the PDA medium, and antifungal substance candidate standards shown in Table 1 therein. 20-80 μL of the product was added. Three agar plugs were placed around the microcup, the glass petri dish was sealed and cultured at 28 ° C. for 48 hours, and then spore germination and growth of Aspergillus niger around the agar plug and agar plug were observed. .
表１は、観察の結果に基づき、各抗真菌性物質候補において真菌（アスペルギルス・ニガー）の増殖抑制が観察されたサンプルのうち、最も少ない溶液添加量、すなわち、胞子発芽阻害最小添加量を示したものである。表中、「２０μＬ」の記載は、抗真菌性物質候補溶液を２０μＬ以上添加したサンプルにおいて、真菌の増殖抑制が観察されたことを示している。また、表中の「−」の記載は、８０μＬの抗真菌性物質候補溶液を添加した場合においても、抗真菌活性が観察されなかったことを示している。
また、上記検討において、マイクロカップ中の酢酸イソアミル２０μＬは全て揮発していることを確認した。
この結果から、標準品の酢酸イソアミルは、少なくとも、マイクロカップに添加した２０μＬが全て揮発した際に、抗真菌活性を有することが分かった。
Table 1 shows the smallest solution addition amount, that is, the minimum addition amount of spore germination inhibition among the samples in which the growth inhibition of fungi (Aspergillus niger) was observed in each antifungal substance candidate based on the observation results. It is a thing. In the table, the description “20 μL” indicates that fungal growth suppression was observed in the sample to which 20 μL or more of the antifungal substance candidate solution was added. Moreover, the description of “-” in the table indicates that no antifungal activity was observed even when 80 μL of the antifungal substance candidate solution was added.
Moreover, in the said examination, it confirmed that all 20 microliters of isoamyl acetate in a microcup was volatilizing.
From this result, it was found that the standard isoamyl acetate had antifungal activity when at least 20 μL added to the microcup was volatilized.
[実施例５]
酢酸イソアミル類縁体が、酢酸イソアミルと同様の抗真菌活性を有するかどうかを検討した。
上記実施例４と同様に、ガラスシャーレ内のマイクロカップの中に、表２に示す酢酸イソアミル類縁体の標準品を５〜８０μＬ添加し、２８℃で４８時間培養した後、アガープラグ及びアガープラグ周囲のアスペルギルス・ニガーの胞子発芽及び増殖を観察した。表２中、「＜５μＬ」の記載は、マイクロカップに５μＬの試料を添加した場合に、十分に胞子の発芽が阻害されたことを意味する。
また、揮発量は、上記実施例４と同様のガラスシャーレ内のマイクロカップに試料を一定量添加し、２８℃で４８時間静置することにより試験を行った。試験終了後マイクロカップの重量を秤量し、試験前後の重量差から容積差を算出し、揮発量とした。表２中、「＞８０μＬ」の記載は、マイクロカップ中の８０μＬが全て揮発したことを意味する。
[Example 5]
It was investigated whether isoamyl acetate analogs have antifungal activity similar to isoamyl acetate.
As in Example 4 above, 5 to 80 μL of a standard product of isoamyl acetate analog shown in Table 2 was added to a microcup in a glass petri dish and cultured at 28 ° C. for 48 hours, and then an agar plug and an agar plug were used. Spore germination and growth of the surrounding Aspergillus niger was observed. In Table 2, “<5 μL” means that spore germination was sufficiently inhibited when 5 μL of the sample was added to the microcup.
Further, the volatilization amount was tested by adding a certain amount of a sample to a microcup in the same glass petri dish as in Example 4 and allowing to stand at 28 ° C. for 48 hours. After completion of the test, the weight of the microcup was weighed, and the volume difference was calculated from the weight difference before and after the test to obtain the volatilization amount. In Table 2, “> 80 μL” means that all 80 μL in the microcup was volatilized.
表２の結果から、酢酸イソアミルのみならず、酢酸イソアミルの類縁体である、蟻酸イソアミル、プロピオン酸イソアミル、ｎ−酪酸イソアミル、酢酸ｎ−プロピル、酢酸イソブチル、酢酸ｎ−ブチル、酢酸２−メチルブチル、酢酸ｎ−ペンチル、酢酸ｎ−ヘキシル、酢酸ｎ−ヘプチル、酢酸ｎ−オクチル、２−ペンタノン、２−ヘキサノン、５−メチル−２−ヘキサノン、２−ヘプタノン、２−オクタノン、２−ノナノン、２−デカノン、及び２−ウンデカノンが、本発明の抗真菌活性を有することが明らかである。また、酢酸ｎ−オクチル、２−ノナノン、２−デカノン、及び２−ウンデカノンは、添加量が少なく、揮発量が少ない場合にも真菌の胞子の発芽及び増殖を阻害するため、本発明の抗真菌活性が高いことが分かった。 From the results in Table 2, not only isoamyl acetate, but also isoamyl acetate, isoamyl formate, isoamyl propionate, isoamyl n-butyrate, n-propyl acetate, isobutyl acetate, n-butyl acetate, 2-methylbutyl acetate, N-pentyl acetate, n-hexyl acetate, n-heptyl acetate, n-octyl acetate, 2-pentanone, 2-hexanone, 5-methyl-2-hexanone, 2-heptanone, 2-octanone, 2-nonanone, 2- It is clear that decanone and 2-undecanone have the antifungal activity of the present invention. In addition, n-octyl acetate, 2-nonanone, 2-decanone, and 2-undecanone inhibit germination and growth of fungal spores even when the addition amount is small and the volatilization amount is small. It was found that the activity was high.
[実施例６]
酢酸イソアミルの様々な真菌や細菌に対する抗菌活性について検討した。
具体的には、上記実施例４におけるアスペルギルス・ニガーに代えて表３記載の糸状菌（真菌）の胞子懸濁液、又は、酵母又は細菌の懸濁液を用い、且つ、酢酸イソアミルの標準品を表３に示す量添加した以外は実施例４と同様にして、アガープラグ及びアガープラグ周囲の糸状菌、酵母又は細菌の増殖を観察し、以下の評価条件にて評価を行った。
−:糸状菌の胞子の発芽、又は、酵母若しくは細菌の生育が全く阻害されなかった。
−／＋：糸状菌の胞子の発芽、又は、酵母若しくは細菌の生育がほぼ阻害されなかった。
＋／−：糸状菌の胞子の発芽、又は、酵母若しくは細菌の生育が僅かに阻害された。
＋：糸状菌の胞子の発芽、又は、酵母若しくは細菌の生育の一部が阻害された。
＋＋：糸状菌の胞子の発芽、又は、酵母若しくは細菌の生育の大部分が阻害された。
＋＋＋：糸状菌の胞子の発芽、又は、酵母若しくは細菌の生育が完全に阻害された。
[Example 6]
The antibacterial activity of isoamyl acetate against various fungi and bacteria was investigated.
Specifically, instead of Aspergillus niger in Example 4 above, a spore suspension of fungus (fungus) described in Table 3, or a yeast or bacterial suspension, and a standard product of isoamyl acetate In the same manner as in Example 4 except that the amount shown in Table 3 was added, the growth of filamentous fungi, yeast or bacteria around the agar plug and the agar plug was observed and evaluated under the following evaluation conditions.
-: Germination of spores of filamentous fungi or growth of yeast or bacteria was not inhibited at all.
− / +: Germination of filamentous fungal spores or growth of yeast or bacteria was hardly inhibited.
+/-: Sprouting of filamentous fungi spores or growth of yeast or bacteria was slightly inhibited.
+: Spore germination of filamentous fungi, or part of the growth of yeast or bacteria was inhibited.
++: Sprouting of filamentous fungi spores, or most of the growth of yeast or bacteria was inhibited.
+++: Germination of spores of filamentous fungi or growth of yeast or bacteria was completely inhibited.
表３の結果から、酢酸イソアミルは表３記載の糸状菌１５菌株の全てにおいて胞子の発芽を阻害し、且つ、表３記載の酵母５菌株、グラム陽性菌７菌株、及びグラム陰性菌２菌株の全てにおいてその生育を阻害した。よって、本発明の抗菌物質は、カビ、酵母、グラム陽性細菌、及びグラム陰性細菌等の菌類に対して、広く抗菌活性を有することが分かった。 From the results in Table 3, isoamyl acetate inhibited spore germination in all 15 filamentous fungi strains listed in Table 3, and 5 yeast strains, 7 Gram-positive bacteria strains, and 2 Gram-negative bacterial strains listed in Table 3. In all, its growth was inhibited. Therefore, it was found that the antibacterial substance of the present invention has a broad antibacterial activity against fungi such as mold, yeast, gram positive bacteria, and gram negative bacteria.
[実施例７]
酢酸イソアミルによる真菌胞子の発芽阻害に関して、その形態の経時変化を観察した。
上記実施例４と同様に、アスペルギルス・ニガーの胞子混釈アガープラグを作製し、
１６０μＬの酢酸イソアミル標準品が添加されたマイクロカップとともにガラスシャーレ内に静置した。２８℃で４８時間培養した後、一方のサンプルでは酢酸イソアミルをガラスシャーレ内から取り除き、他方のサンプルでは酢酸イソアミルをそのままシャーレ内に残し、いずれのサンプルもその後さらに４８時間培養し、培養開始から０時間後（培養開始時）、５時間後、７時間後、及び４８時間後にアガープラグ表面の胞子および菌糸の状態を電子顕微鏡で観察した。また、コントロールとして、酢酸イソアミルを用いずに培養した菌についても同様に観察を行った。
図９に、（ａ）酢酸イソアミルに曝露せずに通常培養したアスペルギルス・ニガー胞子、（ｂ）酢酸イソアミルに曝露した状態で培養したアスペルギルス・ニガー胞子、（ｃ）酢酸イソアミルに曝露した後、酢酸イソアミルを除去して培養したアスペルギルス・ニガーの、培養開始から０時間後、５時間後、７時間後、及び４８時間後の観察結果を示す。
[Example 7]
Regarding the inhibition of fungal spore germination by isoamyl acetate, the time course of its morphology was observed.
As in Example 4 above, an Aspergillus niger spore mixed agar plug was prepared,
The sample was left in a glass petri dish together with a microcup to which 160 μL of isoamyl acetate standard was added. After culturing at 28 ° C. for 48 hours, in one sample, isoamyl acetate was removed from the glass petri dish, and in the other sample, isoamyl acetate was left in the petri dish, and both samples were further cultured for 48 hours thereafter. After time (at the start of culture), after 5 hours, 7 hours, and 48 hours, the state of spores and hyphae on the surface of the agar plug was observed with an electron microscope. In addition, as a control, the same observation was performed on bacteria cultured without using isoamyl acetate.
FIG. 9 shows (a) Aspergillus niger spores normally cultured without exposure to isoamyl acetate, (b) Aspergillus niger spores cultured in a state exposed to isoamyl acetate, (c) After exposure to isoamyl acetate, acetic acid The observation results of Aspergillus niger cultured after removing isoamyl after 0 hours, 5 hours, 7 hours and 48 hours from the start of the culture are shown.
図９の結果から、通常培養の（ａ）では胞子の発芽が認められたが、酢酸イソアミルに曝露した状態で培養を行った（ｂ）では、４８時間後においても胞子の発芽が見られなかった。
また、酢酸イソアミルに曝露した後、酢酸イソアミルを除去した（ｃ）では、酢酸イソアミル除去後に胞子の発芽が認められた。この結果からから、酢酸イソアミルはアスペルギルス・ニガーの殺菌効果ではなく、胞子の発芽を阻害する静菌効果を有することが分かった。
From the results shown in FIG. 9, spore germination was observed in the normal culture (a), but no spore germination was observed after 48 hours in the culture (b) exposed to isoamyl acetate. It was.
In addition, in (c) where isoamyl acetate was removed after exposure to isoamyl acetate, spore germination was observed after isoamyl acetate removal. From this result, it was found that isoamyl acetate has not the bactericidal effect of Aspergillus niger but the bacteriostatic effect of inhibiting spore germination.
[実施例８]
酢酸イソアミルによる細菌及び酵母の生育阻害関して、その形態の経時変化を観察した。
具体的には、まず、エスケリキア・コリを、ＮＢアガー平板培地に塗布して２８℃で２４時間培養して、エスケリキア・コリが生育しているＮＢアガー平板培地を作製した。また、サッカロマイセス・セレビシエを、ＹＭアガー平板培地に塗布して２８℃で２４時間培養して、サッカロマイセス・セレビシエが生育しているＹＭアガー平板培地を作製した。
得られたそれぞれの培地を滅菌済みの型を用いてくりぬき、細菌又は酵母が混釈されたアガープラグを作製し、実施例４と同様にガラスシャーレ内にＮＢまたはＹＭ平板培地上に、該アガープラグと、１６０μＬ酢酸イソアミル入りのマイクロカップとを設置し、２８℃で４８時間培養した後、アガープラグ及びアガープラグ周囲のエスケリキア・コリ又はサッカロマイセス・セレビシエの生育を電子顕微鏡で観察した。
図１０に、エスケリキア・コリ（Ｅ．ｃｏｌｉ）又はサッカロマイセス・セレビシエ（Ｓ．ｃｅｒｅｖｉｓｉａｅ）の、（ａ）通常培養開始時点（０時間後）、（ｂ）４８時間通常培養を行った後、及び（ｃ）酢酸イソアミルに曝露した状態で４８時間培養を行った後の、観察結果を示す。
[Example 8]
With respect to inhibition of bacterial and yeast growth by isoamyl acetate, changes in the morphology over time were observed.
Specifically, first, Escherichia coli was applied to an NB agar plate medium and cultured at 28 ° C. for 24 hours to prepare an NB agar plate medium on which Escherichia coli was grown. Further, Saccharomyces cerevisiae was applied to a YM agar plate medium and cultured at 28 ° C. for 24 hours to prepare a YM agar plate medium on which Saccharomyces cerevisiae was grown.
Each of the obtained media is hollowed out using a sterilized mold to prepare an agar plug mixed with bacteria or yeast, and in the same manner as in Example 4, the agar plug is placed on the NB or YM plate medium. A plug and a microcup containing 160 μL of isoamyl acetate were placed and cultured at 28 ° C. for 48 hours, and then growth of Escherichia coli or Saccharomyces cerevisiae around the agar plug and agar plug was observed with an electron microscope.
FIG. 10 shows (a) the start of normal culture (after 0 hours), (b) after 48 hours of normal culture of E. coli or Saccharomyces cerevisiae, and (b) c) The observation results after culturing for 48 hours in a state exposed to isoamyl acetate are shown.
図１０の結果から、酢酸イソアミルに曝露したエスケリキア・コリでは、細胞内構造に変化が認められた。また、酢酸イソアミルに曝露したサッカロマイセス・セレビシエでは、細胞内小器官の崩壊が認められた。
これらの結果から、酢酸イソアミルは細菌や酵母の細胞内に影響を及ぼすことにより、その生育を阻害していることが推察された。
From the results shown in FIG. 10, changes in the intracellular structure were observed in Escherichia coli exposed to isoamyl acetate. In Saccharomyces cerevisiae exposed to isoamyl acetate, disruption of intracellular organelles was observed.
From these results, it was inferred that isoamyl acetate inhibited the growth of cells by affecting bacteria and yeast cells.
本発明の抗菌性物質は、特にカビ、酵母、細菌等の菌類の増殖や生育が問題となる食品分野等で利用が可能である。 The antibacterial substance of the present invention can be used particularly in the food field where the growth and growth of fungi such as molds, yeasts and bacteria is a problem.
ＮＩＴＥ ＢＰ−２９７
ＮＩＴＥ ＢＰ−４７３
ＮＩＴＥ ＢＰ−４７４
NITE BP-297
NITE BP-473
NITE BP-474
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| JP2016524471A (en) * | 2013-06-18 | 2016-08-18 | アンハイザー−ブッシュ インベブ ソシエテ アノニムＡｎｈｅｕｓｅｒ−Ｂｕｓｃｈ ＩｎＢｅｖ ＳＡ | Method for preparing fermented beverages and beverages thus produced |
| EP3622819B1 (en) * | 2017-05-11 | 2022-01-05 | Universitat Politècnica de València | Use of a compound for protecting plants by means of stomatal closure and method for protecting plants by means of stomatal closure which comprises applying said compound to the plants |
| CN114456962A (en) * | 2022-03-17 | 2022-05-10 | 贵州大学 | Preparation method for fermenting grapefruit rice wine by using compound microorganisms |
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| JP2016524471A (en) * | 2013-06-18 | 2016-08-18 | アンハイザー−ブッシュ インベブ ソシエテ アノニムＡｎｈｅｕｓｅｒ−Ｂｕｓｃｈ ＩｎＢｅｖ ＳＡ | Method for preparing fermented beverages and beverages thus produced |
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