JP2013173710A - Aflatoxin production inhibitor and method for producing the same, aflatoxin contamination control method, and aflatoxin production inhibitor-producing bacterium - Google Patents
Aflatoxin production inhibitor and method for producing the same, aflatoxin contamination control method, and aflatoxin production inhibitor-producing bacterium Download PDFInfo
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- JP2013173710A JP2013173710A JP2012040099A JP2012040099A JP2013173710A JP 2013173710 A JP2013173710 A JP 2013173710A JP 2012040099 A JP2012040099 A JP 2012040099A JP 2012040099 A JP2012040099 A JP 2012040099A JP 2013173710 A JP2013173710 A JP 2013173710A
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- aflatoxin
- production inhibitor
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- aflatoxin production
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Abstract
Description
本発明は、アフラトキシン産生阻害剤及びその製造方法、並びにアフラトキシン汚染防除方法、前記アフラトキシン産生阻害剤を産生する新規微生物であるアフラトキシン産生阻害剤産生菌に関する。 The present invention relates to an aflatoxin production inhibitor and a method for producing the same, an aflatoxin contamination control method, and an aflatoxin production inhibitor-producing bacterium that is a novel microorganism that produces the aflatoxin production inhibitor.
カビの二次代謝物には、有用な化合物が含まれる一方、マイコトキシンと呼ばれる毒性を示す化合物も多い。現在、マイコトキシンによる農作物の汚染は、世界的に深刻な問題となっており、安全な食糧を安定して得るために、マイコトキシン汚染防除の手段が求められている。 Mold secondary metabolites include useful compounds, while many toxic compounds called mycotoxins. Currently, contamination of crops with mycotoxins is a serious problem worldwide, and means for controlling mycotoxin contamination are required to stably obtain safe food.
マイコトキシンによる農作物の汚染のうち、最も深刻な問題となっているのが、アフラトキシンによる農作物の汚染である。アフラトキシンは、既知の天然物質中で最も強い発ガン性や急性毒性を有することが知られており、また、通常の調理方法等では分解されない化合物であることから、その健康被害は深刻である。アフラトキシンに汚染された農作物の摂取による死亡の報告は頻繁にあり、またアフラトキシンが原因と推定される肝臓ガンによる死者は、中国だけでも毎年数十万人にのぼっている。 Among the contamination of crops by mycotoxins, the most serious problem is the contamination of crops by aflatoxins. Aflatoxin is known to have the strongest carcinogenicity and acute toxicity among known natural substances, and it is a compound that is not decomposed by ordinary cooking methods, so that its health damage is serious. There are frequent reports of deaths from ingestion of aflatoxin-contaminated crops, and hundreds of thousands of deaths from liver cancer presumed to be caused by aflatoxins occur in China alone each year.
このような健康被害を防止するため、アフラトキシンの農作物汚染の規制値は、１０ｐｐｂ程度と低く設けられている。しかし、これにより、アフラトキシンで汚染された農作物を破棄することによる損害額も高額にのぼっている。例えば、米国でのアフラトキシン汚染による損失は、年間数百億円である。また、アジアにおけるアフラトキシン汚染による損失は更に大きく、年間１，０００億円以上にのぼると推定されている。 In order to prevent such health damage, the regulation value of aflatoxin contamination of agricultural crops is set as low as about 10 ppb. However, this also increases the amount of damage caused by destroying crops contaminated with aflatoxins. For example, the loss due to aflatoxin contamination in the United States is tens of billions of yen per year. Moreover, the loss due to aflatoxin contamination in Asia is even greater, and is estimated to be over 100 billion yen per year.
アフラトキシンの主な産生菌は、Ａｓｐｅｒｇｉｌｌｕｓ ｆｌａｖｕｓ及びＡｓｐｅｒｇｉｌｌｕｓ ｐａｒａｓｉｔｉｃｕｓであり、熱帯及び亜熱帯の環境下において、農作物の栽培や貯蔵の段階で、トウモロコシやピーナッツ等の農作物に感染してアフラトキシンを産生することが知られている（非特許文献１及び２参照）。熱帯及び亜熱帯以外の気候の地域においても、近年の地球温暖化現象による気候変動により、汚染地域の拡大が懸念されている。 The main producers of aflatoxins are Aspergillus flavus and Aspergillus parasiticus, which are known to produce aflatoxins by infecting crops such as corn and peanuts in the cultivation and storage stages of crops in tropical and subtropical environments. (See Non-Patent Documents 1 and 2). In regions other than the tropics and subtropics, there is a concern about the expansion of contaminated areas due to recent climate change caused by global warming.
従来から、アフラトキシン汚染防除の取り組みとして、アフラトキシン産生菌のゲノム解析やアフラトキシン産生に関与する遺伝子の同定等の基礎研究や、感染に抵抗性を有する品種の取得、アフラトキシン非産生菌との競合による汚染の軽減等の実用研究が行われている。しかしながら、アフラトキシン汚染防除の効果的かつ抜本的な方法は、未だ確立されていない。 Conventional efforts to control aflatoxin contamination include basic research such as genome analysis of aflatoxin-producing bacteria and identification of genes involved in aflatoxin production, acquisition of varieties that are resistant to infection, and contamination due to competition with non-aflatoxin-producing bacteria Practical research such as mitigation is being conducted. However, an effective and drastic method for controlling aflatoxin contamination has not yet been established.
アフラトキシン汚染防除方法としては、例えば、アフラトキシン産生菌の生育を阻害する抗カビ剤を使用する方法が考えられるが、強力な抗カビ剤は安全性に問題があり、また、該抗カビ剤の耐性菌の蔓延を引き起こす可能性がある。
一方、アフラトキシンは二次代謝物であるため、その産生を阻害しても産生菌の生育には影響を与えないと考えられることから、アフラトキシン産生のみを特異的に阻害する薬剤を利用することができれば、有効な汚染防除方法になりうると考えられる。
As a method for controlling aflatoxin contamination, for example, a method using an antifungal agent that inhibits the growth of aflatoxin-producing bacteria can be considered. However, a strong antifungal agent has a problem in safety, and the resistance of the antifungal agent is also considered. May cause fungal spread.
On the other hand, since aflatoxin is a secondary metabolite, even if its production is inhibited, it is thought that it does not affect the growth of the producing bacteria, so it is necessary to use a drug that specifically inhibits only aflatoxin production. If possible, it can be an effective pollution control method.
そこで、アフラトキシン産生を阻害する物質の探索が行なわれた結果、有機リン系の殺虫剤であるｄｉｃｈｌｏｒｖｏｓや、メラニン生合成系酵素を阻害するｔｒｉｃｙｃｌａｚｏｌｅに、アフラトキシン産生阻害活性が見出された（非特許文献３参照）。しかしながら、これらの化合物はアフラトキシン産生阻害活性が弱く、また該化合物自体の安全性に問題があることに加え、阻害活性の選択性に問題があることから実用化には至っていない。 Thus, as a result of searching for substances that inhibit aflatoxin production, aflatoxin production inhibitory activity was found in dichlorvos, an organophosphorus insecticide, and tricyclazole, which inhibits melanin biosynthesis enzymes (non-patented). Reference 3). However, these compounds have not been put into practical use because they have weak aflatoxin production inhibitory activity, and there are problems in the safety of the compounds themselves and in selectivity of inhibitory activity.
このように、耐性菌の蔓延を引き起こすことなく、アフラトキシン産生のみを特異的に阻害する作用を有する化合物が求められており、探索の結果いくつかの化合物も見出されている（特許文献１及び２参照）。しかし、安全性が高く、かつ実用性の高いアフラトキシン産生阻害剤としては、未だ満足なものが見出されていないのが現状である。 Thus, there has been a demand for a compound having an action of specifically inhibiting only aflatoxin production without causing the spread of resistant bacteria, and several compounds have been found as a result of the search (Patent Document 1 and 2). However, at present, no satisfactory aflatoxin production inhibitor has been found yet to be safe and highly practical.
本発明は、従来における前記諸問題を解決し、以下の目的を達成することを課題とする。即ち、本発明は、アフラトキシン産生を特異的かつ効果的に阻害し、安全性が高く、実用的なアフラトキシン産生阻害剤及びその効率的な製造方法、並びに前記アフラトキシン産生阻害剤を用いたアフラトキシン汚染防除方法、前記アフラトキシン産生阻害剤を産生するアフラトキシン産生阻害剤産生菌を提供することを目的とする。 An object of the present invention is to solve the above-described problems and achieve the following objects. That is, the present invention specifically and effectively inhibits aflatoxin production, is highly safe and practical aflatoxin production inhibitor and its efficient production method, and aflatoxin contamination control using the aflatoxin production inhibitor A method and an aflatoxin production inhibitor-producing bacterium that produces the aflatoxin production inhibitor.
前記課題を解決するため、本発明者らは鋭意検討した結果、以下のような知見を得た。即ち、新規な微生物として、受託番号ＦＥＲＭ ＡＢＰ−１１４６７のステノトロフォモナス スピーシーズ（Ｓｔｅｎｏｔｒｏｐｈｏｍｏｎａｓ ｓｐ．）Ｎｏ．２７株を分離することに成功し、この菌株の培養物が、優れたアフラトキシン産生阻害活性を有することを見出し、本発明の完成に至った。 In order to solve the above-mentioned problems, the present inventors have made extensive studies and as a result, obtained the following findings. That is, as a new microorganism, Stenotrophomonas sp. No. No. FERM ABP-11467 is available . 27 strains were successfully isolated, and the culture of this strain was found to have excellent aflatoxin production inhibitory activity, resulting in the completion of the present invention.
本発明は、本発明者らによる前記知見に基づくものであり、前記課題を解決するための手段としては、以下の通りである。即ち、
＜１＞ 受託番号ＦＥＲＭ ＡＢＰ−１１４６７のステノトロフォモナス スピーシーズ（Ｓｔｅｎｏｔｒｏｐｈｏｍｏｎａｓ ｓｐ．）Ｎｏ．２７株の培養物を少なくとも含むことを特徴とするアフラトキシン産生阻害剤である。
＜２＞ 受託番号ＦＥＲＭ ＡＢＰ−１１４６７のステノトロフォモナス スピーシーズ（Ｓｔｅｎｏｔｒｏｐｈｏｍｏｎａｓ ｓｐ．）Ｎｏ．２７株を培養する工程を少なくとも含むことを特徴とするアフラトキシン産生阻害剤の製造方法である。
＜３＞ 前記＜１＞に記載のアフラトキシン産生阻害剤を用い、アフラトキシン産生菌によるアフラトキシン産生を阻害することを特徴とするアフラトキシン汚染防除方法である。
＜４＞ アフラトキシン産生阻害剤を、農作物に施用し、該農作物に感染したアフラトキシン産生菌のアフラトキシン産生を阻害する前記＜３＞に記載のアフラトキシン汚染防除方法である。
＜５＞ 農作物が、穀類、ナッツ類、香辛料、豆類、ゴマ類、及び綿実から選択される少なくともいずれかである前記＜４＞に記載のアフラトキシン汚染防除方法である。
＜６＞ アフラトキシン産生阻害剤を、植物体に塗布し、該植物体に感染したアフラトキシン産生菌のアフラトキシン産生を抑制する前記＜３＞に記載のアフラトキシン汚染防除方法である。
＜７＞ 受託番号ＦＥＲＭ ＡＢＰ−１１４６７のステノトロフォモナス スピーシーズ（Ｓｔｅｎｏｔｒｏｐｈｏｍｏｎａｓ ｓｐ．）Ｎｏ．２７株であることを特徴とするアフラトキシン産生阻害剤産生菌である。
The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> accession number Stenotrophomonas species of FERM ABP-11467 (Stenotrophomonas sp. ) No. An aflatoxin production inhibitor comprising at least 27 cultures.
<2> accession number Stenotrophomonas species of FERM ABP-11467 (Stenotrophomonas sp. ) No. It is a method for producing an aflatoxin production inhibitor comprising at least a step of culturing 27 strains.
<3> A method for controlling contamination of aflatoxins, wherein the aflatoxin production inhibitor according to <1> is used to inhibit aflatoxin production by aflatoxin-producing bacteria.
<4> The method for controlling aflatoxin contamination according to <3>, wherein the aflatoxin production inhibitor is applied to a crop and the aflatoxin production of an aflatoxin-producing bacterium infected with the crop is inhibited.
<5> The method for controlling aflatoxin contamination according to <4>, wherein the crop is at least one selected from cereals, nuts, spices, beans, sesame seeds, and cottonseed.
<6> The method for controlling aflatoxin contamination according to the above <3>, wherein the aflatoxin production inhibitor is applied to a plant body to suppress aflatoxin production of an aflatoxin-producing bacterium infected with the plant body.
<7> accession number Stenotrophomonas species of FERM ABP-11467 (Stenotrophomonas sp. ) No. It is an aflatoxin production inhibitor producing bacterium characterized by being 27 strains.
本発明によれば、従来における前記諸問題を解決し、前記目的を達成することができ、アフラトキシン産生を特異的かつ効果的に阻害し、安全性が高く、実用的なアフラトキシン産生阻害剤及びその効率的な製造方法、並びに前記アフラトキシン産生阻害剤を用いたアフラトキシン汚染防除方法、前記アフラトキシン産生阻害剤を産生するアフラトキシン産生阻害剤産生菌を提供することができる。 According to the present invention, it is possible to solve the conventional problems and achieve the object, specifically and effectively inhibit aflatoxin production, and have high safety and practical aflatoxin production inhibitor and its An efficient production method, aflatoxin contamination control method using the aflatoxin production inhibitor, and an aflatoxin production inhibitor producing bacterium that produces the aflatoxin production inhibitor can be provided.
（アフラトキシン産生阻害剤）
本発明のアフラトキシン産生阻害剤は、受託番号ＦＥＲＭ ＡＢＰ−１１４６７のステノトロフォモナス スピーシーズ（Ｓｔｅｎｏｔｒｏｐｈｏｍｏｎａｓ ｓｐ．）Ｎｏ．２７株（以下、単に「Ｎｏ．２７株」と称することがある）の培養物を少なくとも含み、必要に応じて、更にその他の成分を含む。
前記アフラトキシン産生阻害剤の製造方法としては、特に制限はなく、目的に応じて適宜選択することができるが、後述する本発明のアフラトキシン産生阻害剤の製造方法により好適に製造される。
(Aflatoxin production inhibitor)
Aflatoxin production inhibitor of the present invention, Stenotrophomonas species accession number FERM ABP-11467 (Stenotrophomonas sp. ) No. It contains at least a culture of 27 strains (hereinafter sometimes simply referred to as “No. 27 strain”), and further contains other components as necessary.
There is no restriction | limiting in particular as a manufacturing method of the said aflatoxin production inhibitor, Although it can select suitably according to the objective, It manufactures suitably with the manufacturing method of the aflatoxin production inhibitor of this invention mentioned later.
＜培養物＞
前記培養物としては、前記Ｎｏ．２７株の培養物であれば、特に制限はなく、目的に応じて適宜選択することができ、例えば、培養液、培養上清、培養菌体、培養菌体の破砕物などが挙げられる。
また、前記培養物の抽出物であってもよい。前記培養物の抽出物とは、例えば、該培養物に適当な有機溶媒（例えば、エタノール、メタノール、アセトンなど）を加えて懸濁した後、遠心分離や濾過膜分離等を行い菌体と分離した抽出上清や、該抽出上清に対して、通常用いられる単離精製処理を行ったものなどが挙げられる。
これらは、１種単独で使用してもよく、２種以上を併用してもよい。これらの中でも、培養液、培養上清が好ましい。
なお、前記Ｎｏ．２７株は、放射線照射やその他の変異処理に供することにより、アフラトキシン産生阻害活性を高めるような処理を施されたものであってもよい。
<Culture>
As the culture, the above-mentioned No. If it is a culture of 27 strains, there is no restriction | limiting in particular, According to the objective, it can select suitably, For example, a culture solution, a culture supernatant, a culture | cultivation microbial cell, the crushed material of a culture | cultivation microbial cell etc. are mentioned.
Moreover, the extract of the said culture may be sufficient. The culture extract is, for example, added with a suitable organic solvent (for example, ethanol, methanol, acetone, etc.) and suspended in the culture, followed by centrifugation, filtration membrane separation, etc. Extracted supernatants, and those obtained by subjecting the extracted supernatants to commonly used isolation and purification treatments.
These may be used alone or in combination of two or more. Among these, a culture solution and a culture supernatant are preferable.
In addition, said No. The 27 strains may have been subjected to treatment that increases aflatoxin production inhibitory activity by subjecting it to irradiation or other mutation treatments.
前記アフラトキシン産生阻害剤中の前記培養物の含有量としては、特に制限はなく、目的に応じて適宜選択することができる。また、前記アフラトキシン産生阻害剤は、前記培養物そのものであってもよい。 There is no restriction | limiting in particular as content of the said culture in the said aflatoxin production inhibitor, According to the objective, it can select suitably. The aflatoxin production inhibitor may be the culture itself.
＜その他の成分＞
前記アフラトキシン産生阻害剤中の前記その他の成分としては、本発明の効果を損なわない限り、特に制限はなく、例えば、任意の農薬成分、農薬補助剤などが挙げられる。
前記アフラトキシン産生阻害剤中の、前記その他の成分の含有量としては、特に制限はなく、目的に応じて適宜選択することができる。
<Other ingredients>
The other components in the aflatoxin production inhibitor are not particularly limited as long as the effects of the present invention are not impaired, and examples thereof include arbitrary agricultural chemical components and agricultural chemical adjuvants.
There is no restriction | limiting in particular as content of the said other component in the said aflatoxin production inhibitor, According to the objective, it can select suitably.
＜＜農薬成分＞＞
前記農薬成分としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、以下の農薬の有効成分が挙げられる。
前記農薬としては、例えば、殺カビ剤、殺細菌剤、抗ウィルス剤、植物抵抗性誘導剤、殺虫剤、殺ダニ剤、殺線虫剤、昆虫生育調整剤、昆虫誘引剤、除草剤、植物生長調整剤、共力剤、薬害軽減剤、鳥類忌避剤、肥料、土壌改良剤などが挙げられる。これらは、１種単独で使用してもよく、２種以上を併用してもよい。
<< Agricultural chemical ingredients >>
There is no restriction | limiting in particular as said agrochemical component, According to the objective, it can select suitably, For example, the following active ingredients of an agrochemical are mentioned.
Examples of the pesticide include fungicides, bactericides, antiviral agents, plant resistance inducers, insecticides, acaricides, nematicides, insect growth regulators, insect attractants, herbicides, plants Examples include growth regulators, synergists, safeners, bird repellents, fertilizers, and soil conditioners. These may be used alone or in combination of two or more.
＜＜農薬補助剤＞＞
前記農薬補助剤は、担体と、界面活性剤と、その他の補助剤とを含有し、更に必要に応じてその他の成分を含有する。
<< Agrochemical adjuvant >>
The agrochemical adjuvant contains a carrier, a surfactant, and other adjuvants, and further contains other components as necessary.
−担体−
前記担体としては、農園芸用に用いることができるものであれば、特に制限はなく、目的に応じて適宜選択することができ、例えば、液体担体、固体担体などが挙げられる。これらは、１種単独で使用してもよいし、２種以上を併用してもよい。
-Carrier-
The carrier is not particularly limited as long as it can be used for agriculture and horticulture, and can be appropriately selected according to the purpose. Examples thereof include a liquid carrier and a solid carrier. These may be used individually by 1 type and may use 2 or more types together.
前記液体担体としては、例えば、水；イソプロピルアルコール、エチレングリコール等のアルコール類；シクロヘキサノン；メチルエチルケトン等のケトン類；プロピレングリコールモノメチルエーテル、ジエチレングリコールモノ−ｎ−ブチルエーテル等のエーテル類；ケロシン、軽油等の脂肪族炭化水素類；キシレン、トリメチルベンゼン、テトラメチルベンゼン、メチルナフタリン、ソルベントナフサ等の芳香族炭化水素類；Ｎ−メチル−２−ピロリドン等のアミド類；脂肪酸のグリセリンエステル等のエステル類；大豆油、ナタネ油等の植物油などが挙げられる。 Examples of the liquid carrier include water; alcohols such as isopropyl alcohol and ethylene glycol; cyclohexanone; ketones such as methyl ethyl ketone; ethers such as propylene glycol monomethyl ether and diethylene glycol mono-n-butyl ether; fats such as kerosene and light oil. Aromatic hydrocarbons such as xylene, trimethylbenzene, tetramethylbenzene, methylnaphthalene, and solvent naphtha; amides such as N-methyl-2-pyrrolidone; esters such as glycerin esters of fatty acids; soybean oil And vegetable oils such as rapeseed oil.
前記固体担体としては、例えば、澱粉、活性炭、大豆粉、小麦粉、木粉、魚粉、粉乳等の動植物性粉末；タルク、カオリン、ベントナイト、ゼオライト、珪藻土、ホワイトカーボン、クレー、アルミナ、炭酸カルシウム、塩化カリウム、硫安等の鉱物性粉末などが挙げられる。 Examples of the solid carrier include animal and vegetable powders such as starch, activated carbon, soybean flour, wheat flour, wood flour, fish flour, and milk powder; talc, kaolin, bentonite, zeolite, diatomaceous earth, white carbon, clay, alumina, calcium carbonate, and chloride. Examples thereof include mineral powders such as potassium and ammonium sulfate.
−界面活性剤−
前記界面活性剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、非イオン性界面活性剤、陰イオン性界面活性剤、陽イオン性界面活性剤、両性界面活性剤などが挙げられる。これらの界面活性剤は、１種単独で使用してもよいし、２種以上を併用してもよい。
-Surfactant-
There is no restriction | limiting in particular as said surfactant, According to the objective, it can select suitably, For example, a nonionic surfactant, anionic surfactant, a cationic surfactant, an amphoteric surfactant Etc. These surfactants may be used alone or in combination of two or more.
前記非イオン性界面活性剤としては、例えば、ポリオキシエチレンアルキルエーテル、ポリオキシエチレンアルキルアリールエーテル、ポリオキシエチレンスチリルフェニルエーテル、ポリオキシエチレンアルキルエステル、ポリオキシエチレンソルビタンアルキレート、ポリオキシエチレンフェニルエーテルポリマー、ポリオキシエチレンアルキレンアリールフェニルエーテル、ポリオキシエチレンアルキレングリコール、ポリオキシエチレンポリオキシプロピレンブロックポリマーなどが挙げられる。 Examples of the nonionic surfactant include polyoxyethylene alkyl ether, polyoxyethylene alkyl aryl ether, polyoxyethylene styryl phenyl ether, polyoxyethylene alkyl ester, polyoxyethylene sorbitan alkylate, polyoxyethylene phenyl ether. Examples thereof include polymers, polyoxyethylene alkylene aryl phenyl ethers, polyoxyethylene alkylene glycols, and polyoxyethylene polyoxypropylene block polymers.
前記陰イオン性界面活性剤としては、例えば、リグニンスルホン酸塩、アルキルアリールスルホン酸塩、ジアルキルスルホサクシネート、ポリオキシエチレンアルキルアリールエーテルサルフェート、アルキルナフタレンスルホン酸塩、ポリオキシエチレンスチリルフェニルエーテルサルフェートなどが挙げられる。 Examples of the anionic surfactant include lignin sulfonate, alkylaryl sulfonate, dialkyl sulfosuccinate, polyoxyethylene alkyl aryl ether sulfate, alkyl naphthalene sulfonate, polyoxyethylene styryl phenyl ether sulfate, and the like. Is mentioned.
前記陽イオン性界面活性剤としては、例えば、アルキルアミン塩などが挙げられる。
前記両性界面活性剤としては、例えば、第４級アンモニウム塩アルキルベタイン、アミンオキサイドなどが挙げられる。
Examples of the cationic surfactant include alkylamine salts.
Examples of the amphoteric surfactant include quaternary ammonium salt alkylbetaines and amine oxides.
−その他の補助剤−
前記その他の補助剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、粘結剤、増粘剤、固着剤、防腐防カビ剤、溶剤、農薬活性成分の安定化剤、酸化防止剤、紫外線防止剤、結晶析出防止剤、消泡剤、物性向上剤、着色剤などが挙げられる。
前記粘結剤、増粘剤、固着剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、デキストリン、セルロース、メチルセルロース、エチルセルロース、カルボキシメチルセルロース、ヒドロキシエチルセルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、カルボキシメチルデンプン、プルラン、アルギン酸ナトリウム、アルギン酸アンモニウム、アルギン酸プロピレングリコールエステル、グアーガム、ローカストビーンガム、アラビアゴム、キサンタンガム、ゼラチン、カゼイン、ポリビニルアルコール、ポリエチレンオキサイド、ポリエチレングリコール、エチレン・プロピレンブロックポリマー、ポリアクリル酸ナトリウム、ポリビニルピロリドンなどが挙げられる。
-Other adjuvants-
The other adjuvant is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a binder, a thickener, a sticking agent, an antiseptic and fungicide, a solvent, and stabilization of an agrochemical active ingredient. Agents, antioxidants, ultraviolet light inhibitors, crystal precipitation inhibitors, antifoaming agents, physical property improvers, colorants and the like.
The binder, thickener, and fixing agent are not particularly limited and may be appropriately selected depending on the intended purpose.For example, dextrin, cellulose, methylcellulose, ethylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, Hydroxypropyl methylcellulose, carboxymethyl starch, pullulan, sodium alginate, ammonium alginate, propylene glycol ester alginate, guar gum, locust bean gum, gum arabic, xanthan gum, gelatin, casein, polyvinyl alcohol, polyethylene oxide, polyethylene glycol, ethylene / propylene block polymer , Sodium polyacrylate, polyvinylpyrrolidone and the like.
＜剤型＞
前記アフラトキシン産生阻害剤の剤型としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、乳剤、懸濁剤、水和剤、水溶剤、液剤、ゾル剤（フロアブル剤）、顆粒水和剤、粉剤、細粒剤、粒剤、錠剤、油剤、噴霧剤、煙霧剤、エアゾール剤、ペースト剤などが挙げられる。これらの中でも、液剤が好ましい。
前記各剤の製造方法としては、特に制限はなく、公知の方法により製造することができる。
<Dosage form>
The dosage form of the aflatoxin production inhibitor is not particularly limited and may be appropriately selected depending on the intended purpose. For example, emulsion, suspension, wettable powder, aqueous solvent, liquid, sol (flowable) Granule wettable powder, powder, fine granules, granules, tablets, oils, sprays, fumes, aerosols, pastes and the like. Among these, a liquid agent is preferable.
There is no restriction | limiting in particular as a manufacturing method of each said agent, It can manufacture by a well-known method.
＜混用あるいは併用＞
前記アフラトキシン産生阻害剤は、他の殺菌剤（殺カビ剤、殺細菌剤、抗ウィルス剤、植物抵抗性誘導剤など）、殺虫剤、殺ダニ剤、殺線虫剤、昆虫生育調整剤、昆虫誘引剤、除草剤、植物生長調整剤、共力剤、薬害軽減剤、鳥類忌避剤、肥料、土壌改良剤等との混用あるいは併用することもできる。
<Mixed or combined>
The aflatoxin production inhibitors include other fungicides (fungicides, bactericides, antiviral agents, plant resistance inducers, etc.), insecticides, acaricides, nematicides, insect growth regulators, insects It can also be used in combination or in combination with attractants, herbicides, plant growth regulators, synergists, safeners, bird repellents, fertilizers, soil conditioners and the like.
（アフラトキシン産生阻害剤の製造方法）
本発明のアフラトキシン産生阻害剤の製造方法は、受託番号ＦＥＲＭ ＡＢＰ−１１４６７のステノトロフォモナス スピーシーズ（Ｓｔｅｎｏｔｒｏｐｈｏｍｏｎａｓ ｓｐ．）Ｎｏ．２７株を培養する工程を少なくとも含み、必要に応じて、更にその他の工程を含む。
(Method for producing aflatoxin production inhibitor)
The method for producing an aflatoxin production inhibitor according to the present invention has been described in Stenotrophomonas sp. No. No. FERRM ABP-11467. It includes at least a step of culturing 27 strains, and further includes other steps as necessary.
前記培養は、前記Ｎｏ．２７株を栄養培地（以下、単に「培地」と称することがある）中に接種し、前記アフラトキシン産生阻害剤の産生に良好な温度で培養することによって行われる。 The culture is performed as described in No. 1 above. 27 strains are inoculated into a nutrient medium (hereinafter sometimes simply referred to as “medium”) and cultured at a temperature suitable for the production of the aflatoxin production inhibitor.
前記栄養培地としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、従来ステノトロフォモナス スピーシーズの培養に利用されている公知のものを使用することができる。
前記栄養培地に添加する栄養源としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、市販されているペプトン、酵母エキス、肉エキス、コーン・スティープ・リカー、綿実粉、落花生粉、大豆粉、ＮＺ−アミン、カゼイン、硝酸ソーダ、硝酸アンモニウム、硫酸アンモニウム等の窒素源；トマトペースト、グリセリン、シュークロース、デンプン、グルコース、ガラクトース、マンノース、デキストリン、糖みつ、脂肪等の炭素源；食塩、リン酸塩、炭酸カルシウム、硫酸マグネシウム等の無機塩；などが挙げられる。
また、前記培地中の成分として、前記栄養源の他、例えば、微量の金属塩、消泡剤としての動物油、植物油、鉱物油などを添加することもできる。
これらの材料は、前記Ｎｏ．２７株が利用し、アフラトキシン産生阻害剤の産生に役立つものであればよく、公知の培養材料はすべて用いることができる。
There is no restriction | limiting in particular as said nutrient medium, According to the objective, it can select suitably, For example, the well-known thing conventionally utilized for the culture | cultivation of Stenotrophomonas species can be used.
There is no restriction | limiting in particular as a nutrient source added to the said nutrient medium, According to the objective, it can select suitably, For example, commercially available peptone, yeast extract, meat extract, corn steep liquor, cottonseed powder Nitrogen sources such as peanut flour, soybean flour, NZ-amine, casein, sodium nitrate, ammonium nitrate, ammonium sulfate; carbon sources such as tomato paste, glycerin, sucrose, starch, glucose, galactose, mannose, dextrin, sugarcane, fat Inorganic salts such as sodium chloride, phosphate, calcium carbonate, magnesium sulfate, and the like.
Moreover, as a component in the culture medium, in addition to the nutrient source, for example, a trace amount of metal salt, animal oil, vegetable oil, mineral oil, or the like as an antifoaming agent can be added.
These materials are the same as those described in No. 1 above. Any known culture material can be used as long as it is used by 27 strains and is useful for the production of an aflatoxin production inhibitor.
アフラトキシン産生阻害剤の産生のための種母としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、寒天培地上で培養した前記Ｎｏ．２７株の生育物などが挙げられる。 There is no restriction | limiting in particular as a seed mother for production of an aflatoxin production inhibitor, According to the objective, it can select suitably, For example, said No. cultivated on the agar medium. Examples include 27 strains.
前記培養の条件としては、特に制限はなく、目的に応じて適宜選択することができるが、好気的条件で培養することが好ましい。 The culture conditions are not particularly limited and may be appropriately selected depending on the intended purpose, but are preferably cultured under aerobic conditions.
前記培養の方法としては、斜面（スラント）培養、平板（プレート）培養などの固体（寒天）培養や、液体培養のいずれであってもよいが、前記アフラトキシン産生阻害剤を大量に産生させる観点から、液体培養が好ましい。前記液体培養としては、振とう培養、静置培養、攪拌培養のいずれであってもよいが、振とう培養が好ましく、回転振とう培養がより好ましい。なお、大量培養を行う場合には、発酵槽等を用いて培養を行ってもよい。 The culture method may be solid (agar) culture such as slant culture, flat plate culture, or liquid culture, but from the viewpoint of producing a large amount of the aflatoxin production inhibitor. Liquid culture is preferred. The liquid culture may be any of shaking culture, stationary culture, and stirring culture, but shaking culture is preferable, and rotary shaking culture is more preferable. In addition, when mass culture is performed, the culture may be performed using a fermenter or the like.
前記培養の温度としては、前記Ｎｏ．２７株の発育が実質的に阻害されずに、アフラトキシン産生阻害剤を産生しうる範囲であれば、特に制限はなく、使用する産生菌に応じて適宜選択することができるが、２０℃〜３７℃が好ましい。 As the culture temperature, the above-mentioned No. There is no particular limitation as long as the growth of 27 strains is not substantially inhibited and an aflatoxin production inhibitor can be produced, and it can be appropriately selected according to the producing bacteria to be used. ° C is preferred.
前記培養の期間としては、特に制限はなく、アフラトキシン産生阻害剤の蓄積に合わせて適宜選択することができる。通常、培養３日間〜７日間でアフラトキシン産生阻害剤の蓄積が最高となる。 There is no restriction | limiting in particular as said culture | cultivation period, According to accumulation | storage of an aflatoxin production inhibitor, it can select suitably. Usually, accumulation of an aflatoxin production inhibitor is highest in 3 to 7 days of culture.
（アフラトキシン汚染防除方法）
本発明のアフラトキシン汚染防除方法は、本発明の前記アフラトキシン産生阻害剤を用い、アフラトキシン産生菌によるアフラトキシン産生を阻害する方法であり、前記アフラトキシン産生菌が付着及び／又は感染した対象物、若しくは、前記アフラトキシン産生菌の付着及び／又は感染の発生を防止したい対象物に対し、前記アフラトキシン産生阻害剤を施用する方法であれば、特に制限はなく、目的に応じて適宜選択することができる。
(Aflatoxin contamination control method)
The aflatoxin contamination control method of the present invention is a method of inhibiting aflatoxin production by an aflatoxin-producing bacterium using the aflatoxin production inhibitor of the present invention, and the target to which the aflatoxin-producing bacterium adheres and / or is infected, or There is no particular limitation as long as it is a method in which the aflatoxin production inhibitor is applied to an object for which adhesion of aflatoxin producing bacteria and / or the occurrence of infection is to be prevented, and can be appropriately selected depending on the purpose.
前記対象物としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、植物体、農作物などが挙げられる。
前記農作物としては、例えば、トウモロコシ、コメ、ソバ、ハトムギ等の穀類；ピーナッツ、ピスタチオナッツ、ブラジルナッツ等のナッツ類；ナツメグ、唐辛子、パプリカ等の香辛料；コーヒー豆等の豆類；ゴマ類；綿実；などが挙げられる。
There is no restriction | limiting in particular as said target object, According to the objective, it can select suitably, For example, a plant body, agricultural products, etc. are mentioned.
Examples of the agricultural crops include grains such as corn, rice, buckwheat and pearl barley; nuts such as peanut, pistachio nut and brazil nut; spices such as nutmeg, chili and paprika; beans such as coffee beans; sesame seeds; And so on.
前記アフラトキシン産生阻害剤を施用する方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記アフラトキシン産生阻害剤をそのまま、又は水等で希釈した状態のアフラトキシン産生阻害剤を、散布（例えば、噴霧、ミスティング、アトマイジング、散粉、散粒、水面施用、箱施用等）する方法、土壌施用（例えば、混入、潅注等）する方法、表面施用（例えば、塗布、粉衣、被覆等）する方法、浸漬する方法などが挙げられる。 The method for applying the aflatoxin production inhibitor is not particularly limited and may be appropriately selected depending on the purpose. For example, the aflatoxin production inhibitor in a state where the aflatoxin production inhibitor is diluted as it is or with water or the like. , Spraying, misting, atomizing, dusting, dusting, water surface application, box application, etc.), soil application (eg, mixing, irrigation, etc.), surface application (eg, application, powdering) For example, clothing, covering, etc.) and dipping methods.
前記アフラトキシン産生阻害剤の施用量としては、特に制限はなく、アフラトキシン産生阻害剤中の有効成分の濃度、製剤の形態、対象病害や作物の種類、病害による被害の程度、施用場所、施用方法、施用時期、混用あるいは併用する薬剤や肥料等の種類や使用量、気象などの種々の条件に応じて、適宜選択することができる。 The application amount of the aflatoxin production inhibitor is not particularly limited, the concentration of the active ingredient in the aflatoxin production inhibitor, the form of the preparation, the target disease or crop type, the degree of damage caused by the disease, the application location, the application method, It can be appropriately selected according to various conditions such as application time, type and amount of drugs and fertilizers to be mixed or used together, weather, and the like.
前記アフラトキシン産生阻害剤の施用濃度としては、特に制限はなく、目的に応じて適宜選択することができる。 There is no restriction | limiting in particular as an application density | concentration of the said aflatoxin production inhibitor, According to the objective, it can select suitably.
（アフラトキシン産生阻害剤産生菌）
本発明のアフラトキシン産生阻害剤産生菌は、ステノトロフォモナス スピーシーズ（Ｓｔｅｎｏｔｒｏｐｈｏｍｏｎａｓ ｓｐ．）Ｎｏ．２７株（受託番号ＦＥＲＭ ＡＢＰ−１１４６７）であり、該アフラトキシン産生阻害剤産生菌の培養物は、アフラトキシン産生阻害活性を有する。そのため、前記アフラトキシン産生阻害剤の産生菌として使用され得る。前記Ｎｏ．２７株の菌学的性状は、以下の通りである。
(Aflatoxin production inhibitor producing bacteria)
Aflatoxin production inhibitor producing bacteria of the present invention, Stenotrophomonas species (Stenotrophomonas sp.) No. 27 strains (Accession No. FERM ABP-11467), and the culture of the aflatoxin production inhibitor-producing bacterium has aflatoxin production inhibitory activity. Therefore, it can be used as a producer of the aflatoxin production inhibitor. No. The mycological properties of the 27 strains are as follows.
１．形態
（１）細胞の形態
光学顕微鏡（ＢＸ５０Ｆ４、オリンパス株式会社製）で観察したＮｏ．２７株の形態は、運動性を有するグラム陰性の桿菌であり（図１参照）、胞子は形成せず、菌体の大きさは、約０．７μｍ〜０．８μｍ×１．５μｍ〜２．０μｍであった。
なお、グラム染色は、フェイバーＧ（日水製薬株式会社製）を用いて行った。
（２）コロニーの形態
普通寒天培地（ｎｕｔｒｉｅｎｔ ａｇａｒ；Ｏｘｆｏｒｄ， Ｈａｍｐｓｈｉｒｅ， Ｅｎｇｌａｎｄ）を用い、３０℃で４８時間、好気的条件下で培養した結果、コロニーの色は黄色で、周縁は全緑であり、コロニーの形状は円形で、その直径は約２．０ｍｍ〜３．０ｍｍであり、隆起状態はレンズ状であり、コロニーの表面の形状はスムーズであり、透明度は不透明であり、粘稠度はバター様であった（図２参照）。
1. Morphology (1) Cell Morphology No. observed with an optical microscope (BX50F4, manufactured by Olympus Corporation) The 27 strain form is a gram-negative gonococci having motility (see FIG. 1), no spores are formed, and the size of the bacterial cells is about 0.7 μm to 0.8 μm × 1.5 μm to 2. It was 0 μm.
In addition, Gram dyeing | staining was performed using Faber G (made by Nissui Pharmaceutical Co., Ltd.).
(2) Colony morphology Using normal agar (Oxford, Hampshire, England) and culturing at 30 ° C for 48 hours under aerobic conditions, the colony color is yellow and the periphery is all green. Yes, the shape of the colony is circular, the diameter is about 2.0 mm to 3.0 mm, the raised state is lenticular, the shape of the surface of the colony is smooth, the transparency is opaque, the consistency Was butter-like (see FIG. 2).
２．生理的性質
（１）生育温度範囲
普通寒天培地（ｎｕｔｒｉｅｎｔ ａｇａｒ；Ｏｘｆｏｒｄ， Ｈａｍｐｓｈｉｒｅ， Ｅｎｇｌａｎｄ）を用い、４℃、２０℃、３０℃、３７℃、４０℃、及び４５℃の各温度で４８時間、好気的条件下で培養した結果、４０℃及び４５℃では生育は認められず、４℃〜３７℃の範囲で生育した。
（２）培地
普通寒天培地（ｎｕｔｒｉｅｎｔ ａｇａｒ；Ｏｘｆｏｒｄ， Ｈａｍｐｓｈｉｒｅ， Ｅｎｇｌａｎｄ）に、２質量％塩化ナトリウム又は４質量％塩化ナトリウムを添加して、３０℃で４８時間、好気的条件下で培養した結果、４質量％塩化ナトリウムでは生育は認められず、２質量％塩化ナトリウムでは生育が認められた。
（３）酸素要求性
普通寒天培地（ｎｕｔｒｉｅｎｔ ａｇａｒ；Ｏｘｆｏｒｄ， Ｈａｍｐｓｈｉｒｅ， Ｅｎｇｌａｎｄ）を用い、嫌気条件下で、３０℃にて４８時間培養した結果、弱いながらも育成は認められた。
（４）カタラーゼ反応
カタラーゼ反応は、陽性であった。
（５）オキシダーゼ反応
オキシダーゼ反応は、陰性であった。
（６）グルコースからの酸／ガス産生（酸産生／ガス産生）
グルコースからの酸／ガス産生は、陰性／陰性であった。
（７）Ｏ／Ｆテスト（酸化／発酵）
Ｏ／Ｆテスト（酸化／発酵）は、陽性／陰性であった。
（８）ガラクトースの酸化
ガラクトースの酸化は、陰性であった。
なお、前記（４）〜（７）は、ＢＡＲＲＯＷ， （Ｇ．Ｉ．） ａｎｄ ＦＥＬＴＨＡＭ， （Ｒ．Ｋ．Ａ．）： Ｃｏｗａｎ ａｎｄ Ｓｔｅｅｌ’ｓ Ｍａｎｕａｌ ｆｏｒ ｔｈｅ Ｉｄｅｎｔｉｆｉｃａｔｉｏｎ ｏｆ Ｍｅｄｉｃａｌ Ｂａｃｔｅｒｉａ． ３ｒｄ ｅｄｉｔｉｏｎ． １９９３、Ｃａｍｂｒｉｄｇｅ Ｕｎｉｖｅｒｓｉｔｙ Ｐｒｅｓｓ．に記載の方法に基づき試験した。
2. Physiological properties (1) Growth temperature range Using normal agar (Oxford, Hampshire, England) for 48 hours at 4 ° C, 20 ° C, 30 ° C, 37 ° C, 40 ° C and 45 ° C, As a result of culturing under aerobic conditions, no growth was observed at 40 ° C. and 45 ° C., and it grew in the range of 4 ° C. to 37 ° C.
(2) Medium As a result of adding 2% by mass sodium chloride or 4% by mass sodium chloride to normal agar medium (Oxford, Hampshire, England) and culturing at 30 ° C. for 48 hours under aerobic conditions Growth was not observed with 4% by mass sodium chloride, but growth was observed with 2% by mass sodium chloride.
(3) Oxygen requirement As a result of culturing at 30 ° C. for 48 hours under anaerobic conditions using a normal agar medium (Oxford, Hampshire, England), growth was observed although it was weak.
(4) Catalase reaction The catalase reaction was positive.
(5) Oxidase reaction The oxidase reaction was negative.
(6) Acid / gas production from glucose (acid production / gas production)
Acid / gas production from glucose was negative / negative.
(7) O / F test (oxidation / fermentation)
The O / F test (oxidation / fermentation) was positive / negative.
(8) Oxidation of galactose The oxidation of galactose was negative.
In addition, said (4)-(7) is BARROW, (GI) and FELTHAM, (RKA): Cowan and Steel's Manual for the Identification of Medical Bacteria. 3rd edition. 1993, Cambridge University Press. The test was conducted based on the method described in 1.
３．生化学試験、資性化試験
ＡＰＩ２０ＮＥ（ｂｉｏＭｅｒｉｅｕｘ社製、 Ｌｙｏｎ， Ｆｒａｎｃｅ）を用いた生化学試験及び資性化試験の結果は、下記表１に示すとおりであった。
3. Biochemical test and validation test The results of the biochemical test and validation test using API 20NE (manufactured by bioMerieux, Lyon, France) were as shown in Table 1 below.
４．１６Ｓ ｒＤＮＡ遺伝子解析
１６Ｓ ｒＤＮＡ遺伝子の部分塩基配列（１，４７２ｂｐ）を決定し、ＤＮＡデータベース（アポロンＤＢ−ＢＡ ＢＬＡＳＴ）に登録された公知菌株のデータと比較した。その結果、Ｎｏ．２７株の塩基配列は、以下に示すように、ステノトロフォモナス スピーシーズ（Ｓｔｅｎｏｔｒｏｐｈｏｍｏｎａｓ ｓｐ．）の１６ＳｒＤＮＡ遺伝子と高い相同性を示した。特に、ステノトロフォモナス リゾフィラ（Ｓｔｅｎｏｔｒｏｐｈｏｍｏｎａｓ ｒｈｉｚｏｐｈｉｌａ）ｅ−ｐ１０株（ＷＯＬＦ（Ａ．） ｅｔ ａｌ．， Ｉｎｔ． Ｊ． Ｓｙｓｔ． Ｅｖｏｌ． Ｍｉｃｒｏｂｉｏｌ．， ２００２， ５２， １９３７−１９４４参照）との相同性は、９９．５％であった。
なお、１６Ｓ ｒＤＮＡ遺伝子解析は、以下の条件で行った。
・サイクルシークエンス：ＢｉｇＤｙｅ Ｔｅｒｍｉｎａｔｏｒ ｖ３．１ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇ Ｋｉｔ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｍｓ，ＣＡ，ＵＳＡ）
・プライマー：９Ｆ、７８５Ｆ、８０２Ｒ、及び１５１０Ｒ
・装置：ＡＢＩ ＰＲＩＳＭ ３１３０ ×１ Ｇｅｎｅｔｉｃ Ａｎａｌｙｚｅｒ Ｓｙｓｔｅｍ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｍｓ，ＣＡ，ＵＳＡ）
・塩基配列決定：ＣｈｒｏｍａｓＰｒｏ １．４（Ｔｅｃｈｎｅｌｙｓｉｕｍ Ｐｔｙ Ｌｔｄ．， Ｔｅｗａｎｔｉｎ， ＡＵＳ）
・相同性検索及び簡易分子系統解析：
ソフトウェア：アポロン２．０（テクノスルガ・ラボ、静岡）
データベース：アポロンＤＢ−ＢＡ７．０（テクノスルガ・ラボ、静岡）
国際塩基配列データベース：ＧｅｎＢａｎｋ／ＤＤＢＪ／ＥＭＢＬ
4. 16S rDNA gene analysis The partial base sequence (1,472 bp) of the 16S rDNA gene was determined and compared with data of known strains registered in the DNA database (Apollon DB-BA BLAST). As a result, no. As shown below, the base sequence of 27 strains showed high homology with the 16S rDNA gene of Stenotrophomonas sp. In particular, Stenotrophomonas Rizofira (Stenotrophomonas rhizophila) e-p10 strain (WOLF (A.) Et al ., Int. J. Syst. Evol. Microbiol., 2002, 52, 1937-1944 refer) homology with the 99.5%.
The 16S rDNA gene analysis was performed under the following conditions.
Cycle sequence: BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA)
Primers: 9F, 785F, 802R, and 1510R
Apparatus: ABI PRISM 3130 × 1 Genetic Analyzer System (Applied Biosystems, CA, USA)
-Sequence determination: ChromaPro 1.4 (Technyllium Pty Ltd., Thewantin, AUS)
・ Homology search and simple molecular phylogenetic analysis:
Software: Apollon 2.0 (Techno Suruga Lab, Shizuoka)
Database: Apollon DB-BA 7.0 (Techno Suruga Lab, Shizuoka)
International nucleotide sequence database: GenBank / DDBJ / EMBL
下記表２及び表３に、アポロンＤＢ−ＢＡ及び国際塩基配列データベースをそれぞれ用いてＢＬＡＳＴ検索を行った結果得られた、前記Ｎｏ．２７株の塩基配列と、他の菌株との上位３０塩基配列の相同率を示す。
また、図３に、前記Ｎｏ．２７株の１６Ｓ ｒＤＮＡ塩基配列に基づく簡易分子系統樹を示す。図３において、左下の下線はスケールバーを表し、系統枝の分岐に位置する数字はブートストラップ値を表し、株名の末尾のＴはその種の基準株（Ｔｙｐｅ ｓｔｒａｉｎ）を表す。
Tables 2 and 3 below show the results of BLAST searches using the Apollon DB-BA and the international nucleotide sequence database, respectively. It shows the homology rate of the base sequence of 27 strains and the top 30 base sequences of other strains.
In addition, FIG. The simplified molecular phylogenetic tree based on the 16S rDNA base sequence of 27 strains is shown. In FIG. 3, the lower left underline represents a scale bar, the number located at the branch of the system branch represents the bootstrap value, and T at the end of the strain name represents that type of strain (Type strain).
以上の結果より、前記Ｎｏ．２７株は、ステノトロフォモナス スピーシーズ（Ｓｔｅｎｏｔｒｏｐｈｏｍｏｎａｓ ｓｐ．）に属するものと考えられた。これらの中でもステノトロフォモナス リゾフィラ（Ｓ．ｒｈｉｚｏｐｈｉｌａ）と最も近縁であったが、硝酸塩を還元せず、エスクリンを加水分解し（β−グルコシダーゼ活性）、レシチナーゼ活性を示し、キシロースを資化しない点などは、ステノトロフォモナス リゾフィラ（Ｓ．ｒｈｉｚｏｐｈｉｌａ）の性状と異なっていた。そこで、前記Ｎｏ．２７株をステノトロフォモナス スピーシーズ（Ｓｔｅｎｏｔｒｏｐｈｏｍｏｎａｓ ｓｐ．）Ｎｏ．２７株とした。
なお、前記Ｎｏ．２７株は、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（日本国茨城県つくば市東１丁目１番地１中央第６）に国際寄託申請し、平成２４年２月１５日に、受託番号ＦＥＲＭ ＡＢＰ−１１４６７として受託された。前記Ｎｏ．２７株は、東京大学農学部弥生キャンパスの土壌より分離されたものであり、毒性がなく安全な細菌である。
From the above results, the above-mentioned No. Twenty-seven strains were considered to belong to Stenotrophomonas sp. Among these, S. rhizophila was most closely related, but nitrate was not reduced, esculin was hydrolyzed (β-glucosidase activity), lecithinase activity was exhibited, and xylose was not assimilated. The points differed from the properties of Stenotrophomonas Rhizophila ( S. rhizophila ). Therefore, the above-mentioned No. 27 shares Stenotrophomonas species (Stenotrophomonas sp.) No. There were 27 strains.
In addition, said No. 27 shares were applied for an international deposit at the Patent Organism Depositary (National Institute of Advanced Industrial Science and Technology) (1st, 1st, 1st, 1st East, Tsukuba, Ibaraki, Japan). On February 15, 2012, the deposit number FERM Commissioned as ABP-11467. No. 27 strains were isolated from the soil of Yayoi Campus of the University of Tokyo, and are safe and non-toxic bacteria.
なお、他の菌にも見られるように、前記Ｎｏ．２７株は、性状が変化することがあるが、例えば、前記Ｎｏ．２７株に由来する突然変異株（自然発生又は誘発性）、形質接合体、遺伝子組換体などであっても、アフラトキシン産生阻害活性を有するものは、本発明のアフラトキシン産生阻害剤産生菌に含まれる。 In addition, as seen in other bacteria, the above-mentioned No. Although the 27 strains may have different properties, for example, the No. 27 strain. Among mutant strains derived from 27 strains (naturally occurring or inducible), zygotes, recombinants, etc., those having aflatoxin production inhibitory activity are included in the aflatoxin production inhibitor-producing bacteria of the present invention. .
以下に本発明の実施例を挙げて本発明を具体的に説明するが、本発明はこれらの実施例に何ら限定されるものではない。 EXAMPLES The present invention will be specifically described below with reference to examples of the present invention, but the present invention is not limited to these examples.
（実施例１）
＜アフラトキシン産生阻害剤の製造＞
ベネット寒天培地（１質量％グルコース、０．２質量％ポリペプトン（日本製薬株式会社製、名古屋）、０．１質量％肉エキス（極東製薬工業株式会社製、東京）、０．１質量％酵母エキス（Ｄｉｆｃｏ社製、ＭＤ、ＵＳＡ）、残部水；ｐＨ７．２）でＮｏ．２７株を２６．５℃で１週間培養後、このＮｏ．２７株を三角フラスコ（５００ｍＬ容量）に入ったベネット液体培地１００ｍＬに植菌し、２６．５℃で２日間静置培養することにより前培養を行った。
この前培養液５ｍＬを三角フラスコ（５００ｍＬ容量）に入ったベネット液体培地１００ｍＬに植菌し、２６．５℃で５日間静置培養することにより培養液を得た。この培養液を以下の試験例１に用いた。更にこの培養液を０．２５μｍの無菌フィルターに通し、菌体を除去して得られた培養上清を以下の試験例２に用いた。
Example 1
<Manufacture of an aflatoxin production inhibitor>
Bennett agar (1% glucose, 0.2% polypeptone (Nippon Pharmaceutical Co., Ltd., Nagoya), 0.1% meat extract (Kyokuto Pharmaceutical Co., Ltd., Tokyo), 0.1% yeast extract (Made by Difco, MD, USA), remaining water; After culturing 27 strains at 26.5 ° C. for 1 week, Twenty-seven strains were inoculated into 100 mL of Bennett liquid medium in an Erlenmeyer flask (500 mL capacity), and precultured by standing at 26.5 ° C. for 2 days.
5 mL of this preculture solution was inoculated into 100 mL of Bennett liquid medium contained in an Erlenmeyer flask (500 mL capacity), and a culture solution was obtained by stationary culture at 26.5 ° C. for 5 days. This culture solution was used in Test Example 1 below. Further, this culture solution was passed through a 0.25 μm sterile filter, and the culture supernatant obtained by removing the cells was used in Test Example 2 below.
（試験例１）
＜胞子懸濁液１の調製＞
アフラトキシンＢ１及びＧ１の産生菌として、Ａ．ｐａｒａｓｉｔｉｃｕｓ ＮＲＲＬ ２９９９（ＵＳＤＡ、Ａｇｒｉｃｕｌｔｕｒａｌ Ｒｅｓｅａｒｃｈ Ｓｅｒｖｉｃｅ、ＵＳＡより入手）をポテトデキストロース寒天培地（ＰＤＡ培地；Ｄｉｆｃｏ社製、ＭＤ，ＵＳＡ）の斜面培地上で、２７℃で１４日間培養後、その菌叢より胞子を白金耳で掻きとり、２．５×１０３ＣＦＵ／μＬになるようＴｗｅｅｎ２０を０．１質量％含む蒸留水に懸濁して胞子懸濁液を調製した。
(Test Example 1)
<Preparation of spore suspension 1>
As production bacteria aflatoxin B 1 and G 1, A. parasiticus NRRL 2999 (USDA, Agricultural Research Service, USA) was cultured on a potato dextrose agar medium (PDA medium; manufactured by Difco, MD, USA) for 14 days at 27 ° C. Was scraped with a platinum loop and suspended in distilled water containing 0.1% by mass of Tween 20 so as to be 2.5 × 10 3 CFU / μL to prepare a spore suspension.
＜胞子懸濁液２の調製＞
アフラトキシンＢ１の産生菌としてＡ．ｆｌａｖｕｓ ＩＭＦ４７７９８（Ｒｅｆｅｒｅｎｃｅｓｔｒａｉｎ ｏｆ ｔｈｅ Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｕｎｉｏｎ ｏｆ Ｍｉｃｒｏｂｉｏｌｏｇｉｃａｌ Ｓｏｃｉｅｔｉｅｓより入手）をポテトデキストロース寒天培地（ＰＤＡ培地；Ｄｉｆｃｏ社製、ＭＤ，ＵＳＡ）の斜面培地上で、２７℃で１４日間培養後、その菌叢より胞子を白金耳で掻きとり、２．５×１０３ＣＦＵ／μＬになるようＴｗｅｅｎ ２０を０．１質量％含む蒸留水に懸濁して胞子懸濁液を調製した。
<Preparation of spore suspension 2>
A as production strains aflatoxin B 1. flavus IMF47798 (Referencestrain of the International Union of Microbiological Societies than get) a potato dextrose agar medium (PDA medium; Difco Co., Ltd., MD, USA) on a slant medium of, after 14 days of culture at 27 ℃, the spores from the lawn Was then scraped with a platinum loop and suspended in distilled water containing 0.1% by mass of Tween 20 to 2.5 × 10 3 CFU / μL to prepare a spore suspension.
＜＜アフラトキシン産生阻害活性の評価＞＞
生ピーナッツ（約１．５ｇ／個）が２０個入った三角フラスコ（１００ｍＬ容量）に、蒸留水１０ｍＬを加え、１２０℃で１５分間オートクレーブを行った。オートクレーブしたピーナッツ（６個）を実施例１の培養液に浸漬した後、２４ウェルのマイクロプレートに１個／ウェルずつ移した。
コントロールとして、実施例１の培養液の代わりにベネット液体培地にピーナッツ（６個）を浸漬した後、２４ウェルのマイクロプレートに１個／ウェルずつ移した。
これらのウェルに入ったピーナッツに、前記調製した胞子懸濁液１（Ａ．ｐａｒａｓｉｔｉｃｕｓ ＮＲＲＬ ２９９９）又は胞子懸濁液２（Ａ．ｆｌａｖｕｓ ＩＭＦ ４７７９８）を１０μＬ／ウェルずつ植菌し、２７℃で３日間静置培養した。
<< Evaluation of aflatoxin production inhibitory activity >>
To an Erlenmeyer flask (100 mL capacity) containing 20 raw peanuts (about 1.5 g / piece), 10 mL of distilled water was added and autoclaved at 120 ° C. for 15 minutes. The autoclaved peanuts (6 pieces) were immersed in the culture medium of Example 1, and then transferred to a 24-well microplate one by one / well.
As a control, peanuts (6 pieces) were immersed in a Bennett liquid medium instead of the culture solution of Example 1, and then transferred to a 24-well microplate one by one / well.
Peanuts in these wells were inoculated with 10 μL / well of the prepared spore suspension 1 ( A. parasiticus NRRL 2999) or spore suspension 2 ( A. flavus IMF 47798) at 27 ° C. The culture was stationary for 1 day.
３日間培養後、ピーナッツの表面はいずれもカビの菌糸で覆われたが、カビの生育は、実施例１の培養液に浸したもの、コントロール共に良好であった。
カビの生育したピーナッツを１個ずつバイアル（直径１６．５ｍｍ、高さ４０ｍｍ）に移し、クロロホルム２．０ｍＬを加えてスパーテルで破砕し抽出した。得られた破砕抽出液０．１ｍＬを１．５ｍＬのマイクロチューブに入れ、風乾によりクロロホルムを留去後、得られた残渣を０．１ｍＬの９０容量％アセトニトリル水溶液に溶解した。この溶液を０．２５μｍの無菌フィルターに通した後、下記測定条件にて高速液体クロマトグラフ質量分析（ＬＣ／ＭＳ）によりアフラトキシンの質量を測定した。
また、下記測定条件にてアフラトキシンＢ１の標準品（Ａｆｌａｔｏｘｉｎ Ｂ１；Ｓｉｇｍａ社製）及びアフラトキシンＧ１の標準品（Ａｆｌａｔｏｘｉｎ Ｇ１；Ｓｉｇｍａ社製）を用いて検量線を作成し、実施例１の培養液に浸したピーナッツ及びコントロールのピーナッツ１個あたり（約１．５ｇ／個）のアフラトキシン質量（μｇ）を定量し、アフラトキシンＢ１及びアフラトキシンＧ１の合計質量（μｇ）及び該合計質量のピーナッツ６個の平均値を算出した。結果を下記表４及び図４に示す。
［ＬＣ／ＭＳの測定条件］
−ＨＰＬＣ−
・装置：２６９５ ＨＰＬＣ ｓｙｓｔｅｍ（Ｗａｔｅｒｓ社製、Ｍｉｌｆｏｒｄ，ＭＡ）
・カラム：Ｃａｐｃｅｌｌ−ｐａｋ Ｃ１８ ｃｏｌｕｍｎ ＵＧ１２０（直径２．０ｍｍ、長さ１５０ｍｍ、資生堂株式会社製）
・溶出液：Ａ １０ｍＭ酢酸アンモニウム水溶液／Ｂ アセトニトリル
・溶出条件：Ａ：Ｂ＝９０：１０からＡ：Ｂ＝０：１００で１０分間の直線グラジエント溶出
・流量：０．２ｍＬ／分間
・保持時間：アフラトキシンＢ１（１１．４５分間）
アフラトキシンＧ１（１１．７９分間）
−ＭＳ−
・装置：ｍｉｃｒｏ ｍａｓｓ ＺＱ（Ｗａｔｅｒｓ社製）
・イオン化：ＥＳＩ、ポジティブモード
・ソース温度：１２０℃
・脱溶媒温度：３５０℃
・コーン電圧：３０Ｖ
・脱溶媒ガス量：６００Ｌ／時間
・コーンガス量：５０Ｌ／時間
・キャピラリー電圧：２，８００Ｖ
・検出：アフラトキシンＢ１ ｍ／ｚ３１３（Ｍ＋Ｈ）＋
アフラトキシンＧ１ ｍ／ｚ３２９（Ｍ＋Ｈ）＋
After culturing for 3 days, the surface of the peanut was covered with mold mycelium, but the growth of the mold was good in both the control and the control immersed in the culture solution of Example 1.
The mold-grown peanuts were transferred one by one to a vial (diameter 16.5 mm, height 40 mm), added with 2.0 mL of chloroform, crushed and extracted with a spatula. 0.1 mL of the obtained crushed extract was put into a 1.5 mL microtube, chloroform was distilled off by air drying, and the resulting residue was dissolved in 0.1 mL of 90 vol% acetonitrile aqueous solution. After passing this solution through a 0.25 μm sterile filter, the mass of aflatoxin was measured by high performance liquid chromatography / mass spectrometry (LC / MS) under the following measurement conditions.
A calibration curve was prepared using a standard product of Aflatoxin B 1 (Aflatoxin B 1 ; manufactured by Sigma) and a standard product of Aflatoxin G 1 (Aflatoxin G 1 ; manufactured by Sigma) under the following measurement conditions. The amount of aflatoxin (μg) per peanut and control peanut soaked in the culture medium (about 1.5 g / piece) was quantified, and the total mass (μg) of aflatoxin B 1 and aflatoxin G 1 and the total mass The average value of 6 peanuts was calculated. The results are shown in Table 4 below and FIG.
[Measurement conditions for LC / MS]
-HPLC-
Apparatus: 2695 HPLC system (manufactured by Waters, Milford, MA)
Column: Capcell-pak C18 column UG120 (diameter 2.0 mm, length 150 mm, manufactured by Shiseido Co., Ltd.)
Eluent: A 10 mM ammonium acetate aqueous solution / B acetonitrile Elution conditions: A: B = 90: 10 to A: B = 0: 100, 10 minutes linear gradient elution Flow rate: 0.2 mL / min Retention time: Aflatoxin B 1 (11.45 minutes)
Aflatoxin G 1 (11.79 minutes)
-MS-
・ Device: micro mass ZQ (manufactured by Waters)
-Ionization: ESI, positive mode-Source temperature: 120 ° C
-Desolvation temperature: 350 ° C
・ Cone voltage: 30V
-Desolvation gas amount: 600 L / hour-Cone gas amount: 50 L / hour-Capillary voltage: 2,800 V
Detection: aflatoxin B 1 m / z 313 (M + H) +
Aflatoxin G 1 m / z 329 (M + H) +
表１の結果より、実施例１の培養液に浸漬することにより、アフラトキシン産生菌によるピーナッツへのアフラトキシン蓄積が明白に減少した。特にＡ．ｆｌａｖｕｓのピーナッツへのアフラトキシン蓄積を強く阻害した。 From the results shown in Table 1, accumulation of aflatoxins in peanuts by aflatoxin-producing bacteria was clearly reduced by immersion in the culture solution of Example 1. In particular A. It strongly inhibited aflatoxin accumulation in flavus peanuts.
（試験例２）
実施例１の培養上清を、２４ウェルのマイクロプレートに、０．１ｍＬ／ウェル、０．３ｍＬ／ウェル、０．５ｍＬ／ウェル、０．７ｍＬ／ウェル、又は０．９ｍＬ／ウェル入れ、各ウェルの液体合計量が１ｍＬとなるようにポテトデキストロース液体培地を加えた。また、コントロールとして、実施例１の培養上清の代わりにポテトデキストロース液体培地を１ｍＬ／ウェル添加した。
それぞれのウェルに、試験例１で調製した胞子懸濁液１（Ａ．ｐａｒａｓｉｔｉｃｕｓ ＮＲＲＬ ２９９９）又は胞子懸濁液２（Ａ．ｆｌａｖｕｓ ＩＭＦ ４７７９８）を１０μＬ／ウェルずつ植菌し、２７℃で３日間静置培養した。
(Test Example 2)
Put the culture supernatant of Example 1 in a 24-well microplate at 0.1 mL / well, 0.3 mL / well, 0.5 mL / well, 0.7 mL / well, or 0.9 mL / well. Potato dextrose liquid medium was added so that the total amount of liquid was 1 mL. As a control, potato dextrose liquid medium was added at 1 mL / well instead of the culture supernatant of Example 1.
To each well, spore suspension 1 was prepared in Test Example 1 (A. Parasiticus NRRL 2999) or spore suspension 2 (A. Flavus IMF 47798) was inoculated by 10 [mu] L / well, 3 days at 27 ° C. The culture was stationary.
３日間培養後、それぞれのウェルの培養液をミラクロス（Ｃａｌｂｉｏｃｈｅｍ社製、 Ｌａ Ｊｏｌｌａ、 Ｃａｌｉｆｏｒｎｉａ、 ＵＳＡ）で濾過し、菌体と濾液（培養上清）とに分離した。
前記菌体は、５ｍＬの蒸留水で洗浄後１．５ｍＬのマイクロチューブに移し、１００℃で３時間乾燥させた。乾燥後、菌体入マイクロチューブの質量を測定し、空のマイクロチューブの質量を差し引いた質量を菌体質量とした。
前記濾液は、該濾液１ｍＬをクロロホルム０．２ｍＬで抽出し、風乾によりクロロホルムを留去後、０．１ｍＬの９０容量％アセトニトリル水溶液に溶解した。この溶液を０．２５μｍの無菌フィルターに通した後、試験例１と同様の方法で、高速液体クロマトグラフ質量分析（ＬＣ／ＭＳ）によりアフラトキシンの質量を定量した。結果を表５に示す。また、胞子懸濁液１（Ａ．ｐａｒａｓｉｔｉｃｕｓ ＮＲＲＬ ２９９９）を植菌した結果を図５Ａに、胞子懸濁液２（Ａ．ｆｌａｖｕｓ ＩＭＦ ４７７９８）を植菌した結果を図５Ｂに示す。
After culturing for 3 days, the culture solution in each well was filtered with Miracloth (Calbiochem, La Jolla, California, USA), and separated into cells and filtrate (culture supernatant).
The cells were washed with 5 mL of distilled water, transferred to a 1.5 mL microtube, and dried at 100 ° C. for 3 hours. After drying, the mass of the microbial cell-containing microtube was measured, and the mass obtained by subtracting the mass of the empty microtube was defined as the microbial cell mass.
For the filtrate, 1 mL of the filtrate was extracted with 0.2 mL of chloroform, and the chloroform was distilled off by air drying, and then dissolved in 0.1 mL of 90 vol% acetonitrile aqueous solution. After passing this solution through a 0.25 μm sterile filter, the mass of aflatoxin was quantified by high performance liquid chromatography / mass spectrometry (LC / MS) in the same manner as in Test Example 1. The results are shown in Table 5. Moreover, the result of inoculating spore suspension 1 ( A. parasiticus NRRL 2999) is shown in FIG. 5A, and the result of inoculating spore suspension 2 ( A. flavus IMF 47798) is shown in FIG. 5B.
表２の結果より、実施例１の培養上清を０．５ｍＬ／ウェルあるいは０．７ｍＬ／ウェルの割合で添加した場合、Ａ．ｐａｒａｓｉｔｉｃｕｓあるいはＡ．ｆｌａｖｕｓのアフラトキシン産生がほぼ完全に阻害された。しかし、その際、カビの菌体質量には大きな変化はなく、実施例１の培養上清は、カビの生育にはほとんど影響を及ぼさなかった。 From the results in Table 2, when the culture supernatant of Example 1 was added at a rate of 0.5 mL / well or 0.7 mL / well, A. parasiticus or A. Flavus aflatoxin production was almost completely inhibited. However, at that time, there was no significant change in the fungal mass, and the culture supernatant of Example 1 had little effect on the growth of the fungus.
試験例２の結果より、Ｎｏ．２７株は、その培養上清にカビの生育を阻害せず、かつアフラトキシンの産生を特異的に阻害する物質を産生していることがわかった。そのアフラトキシン産生阻害物質が試験例１の培養液を用いた試験において、ピーナッツにおけるアフラトキシン産生を阻害していると推察される。
この結果より、Ｎｏ．２７株の培養液及びＮｏ．２７株の産生するアフラトキシン産生阻害物質は、共にアフラトキシン汚染防除に有効であることが示唆された。
From the results of Test Example 2, no. It was found that 27 strains produced substances that did not inhibit mold growth and specifically inhibited aflatoxin production in the culture supernatant. It is speculated that the aflatoxin production inhibitory substance inhibits aflatoxin production in peanuts in the test using the culture solution of Test Example 1.
From this result, no. No. 27 culture medium and It was suggested that both aflatoxin production inhibitors produced by 27 strains are effective in controlling aflatoxin contamination.
ＦＥＲＭ ＡＢＰ−１１４６７ FERM ABP-11467
本発明の新規微生物である受託番号ＦＥＲＭ ＡＢＰ−１１４６７のステノトロフォモナス スピーシーズ（Ｓｔｅｎｏｔｒｏｐｈｏｍｏｎａｓ ｓｐ．）Ｎｏ．２７株は、アフラトキシン産生阻害剤を産生するアフラトキシン産生阻害剤産生菌であり、前記Ｎｏ．２７株の培養物は、優れたアフラトキシン産生阻害活性を有することから、アフラトキシン産生阻害剤及びアフラトキシン汚染防除方法に好適に利用可能である。 Stenotrophomonas species of accession number FERM ABP-11467 is a novel microorganism of the present invention (Stenotrophomonas sp.) No. Strain 27 is an aflatoxin production inhibitor-producing bacterium that produces an aflatoxin production inhibitor. Since the culture of 27 strains has excellent aflatoxin production inhibitory activity, it can be suitably used for an aflatoxin production inhibitor and aflatoxin contamination control method.
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| CN109762761B (en) * | 2019-01-10 | 2022-01-07 | 河南农业大学 | Stenotrophomonas acidiphila capable of efficiently degrading aflatoxin B1 and application thereof |
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