Aflatoxins are secondary metabolites produced by some fungi such as Aspergillus flavus and Aspergillus parasiticus. Aflatoxins are detected in various food commodities in the world and are the most dangerous contaminants in food and feed due to their very strong toxicity and carcinogenicity. Besides their effects on human and animal health, aflatoxins have a serious impact on the agricultural economy. Aflatoxin contamination can occur during growing of crop in field, harvest and storage. Susceptible crops of aflatoxin contamination include corn, peanut, cottonseed and various cereals. Several decontamination and detoxification strategies for aflatoxins have been attempted to resolve the aflatoxin contamination problem. However, most of the strategies are limited in effectiveness. Therefore, effective methods for preventing food and feed from aflatoxin contamination are strongly required. Antifungal agents may be useful for resolving aflatoxin contamination problems. However, there are few fungicides effective against aflatoxingenic fungi in field and inhibition of fungal growth may lead to spread of resistant strains. On the other hand, the method using specific aflatoxin production inhibitors may have a high potential to prevent aflatoxin contamination. However, there are few practically useful inhibitors at present.

In this study, I searched for specific aflatoxin production inhibitors among plant essential oils and microbial metabolites, and studied the mode of action of the obtained inhibitors. I also evaluated the effectiveness of the inhibitor and a bacterium for preventing aflatoxin contamination in peanut during storage at a tropical area where aflatoxin contamination occurs naturally.

Chapter I An aflatoxin production inhibitor from the essential oil of Betula alba

By the screening search for new aflatoxin production inhibitors among essential oil, the essential oil of B. alba was found to inhibit aflatoxin production. One of the active components involved in the oil was identified as methyl syringate. Methyl syringate inhibited aflatoxin production of A. parasiticus and A. flavus without affecting the fungal growth. Methyl syringate reduced mRNA levels of genes (aflR, pksA, and omtA) encoding proteins required for aflatoxin biosynthesis and inhibited production of norsolorinic acid, a biosynthetic intermediate involved in the early step of aflatoxin biosynthetic pathway, suggesting that methyl syringate inhibited an early regulatory step leading to expression of AflR, a key regulatory protein necessary for expression of aflatoxin biosynthetic enzymes.

Although methyl syringate showed high selectivity for afaltoxin production inhibition, methyl gallate, methyl 3,4,5-trimethoxybenzoate, and methyl 3-O-methylgallate inhibited both aflatoxin production and fungal growth of A. parasiticus and A. flavus. Methyl syringate showed a much weaker DPPH radical scavenging activity than methyl gallate, methyl 3-O-methylgallate, syringic acid, gallic acid or 3-O-methylgallic acid. This suggests that the aflatoxin production inhibitory activity of methyl syringate does not correlate to DPPH radical scavenging activity.

Chapter II Aflatoxin production inhibitors from bacterial strain no. 27

During the screening of bacteria producing aflatoxin production inhibitors, the strain no. 27 was found to produce selective aflatoxin production inhibitors. Two diketopiperazines were isolated from 25 % and 50% ethanol fractions of charcoal column chromatography of the culture filtrate. Each diketopiperazine inhibited aflatoxins B1 and G1 production of A. parasiticus and aflatoxin B1 production of A. flavus without affecting the fungal growth. They inhibited norsorolinic acid production and reduced the mRNA levels of aflR gene, suggesting that their targets are present in an early regulatory step leading to expression of AflR. Among stereoisomers, only an L- L isomer showed strong aflatoxin production inhibitory activity.

The results of analysis of the 16S rDNA sequence indicated that the strain no. 27 is a member of the genus Stenotrophomonas and most closely related to Stenotrophomonas rhizophila. Stenotrophomonas sp. is known to have good properties suitable for agricultural use as a biocontrol agent. Especially, S. rhizophila is most favorable due to its non-pathogenic property. Strong aflatoxin production inhibition by co-culturing of the strain no. 27 with aflatoxigenic fungus was found in a liquid medium. This co-culture assay may be useful for screening microbes with aflatoxin production inhibitory activity.

Chapter III Effects of methyl syringate and strain no. 27 on aflatoxin contamination in peanuts during storage

The effectiveness of methyl syringate for preventing aflatoxin contamination in peanuts during storage was evaluated. The experimental trials were performed at both laboratory and farmer's warehouse in Thailand. In the laboratory experiment, when peanut was dipped in 80 mM of ethanolic methyl syringate solution, the amount of aflatoxin produced by A. flavus in peanut was significantly reduced. In the farmer's warehouse experiment, methyl syringate weakly reduced the amount of aflatoxin in peanuts with shell during three weeks of storage at the same concentration as used in the laboratory experiment, but no effect was observed after six weeks of storage.

The effects of strain no. 27 on aflatoxin contamination in peanuts during storage were also evaluated at laboratory and farmer's warehouse. The results of laboratory experiment showed that the culture broth of strain no. 27 was effective to reduce aflatoxin in peanuts. The experimental trials at farmer's warehouse were performed in both rainy and dry seasons in Thailand. The culture broth of strain no. 27 was very effective to reduce aflatoxin contamination in peanuts with shell during the storage at the first and second trials. The activity of the culture broth of strain no. 27 was not reduced after keeping at 4 ℃ for one month. Furthermore, the culture broth of strain no. 27 was still effective at 39 ℃ in the second trial experiments. These properties may be suitable for practical use of the bacterium as a biocontrol agent. This is the first report that practical effectiveness of a bacterium which produces aflatoxin production inhibitors is shown. We are now planning experiments to test the effectiveness of strain no. 27 for preventing various crops from aflatoxin contamination.

Aspergillus flavusやAspergillus parasiticusは農作物に感染し、強い毒性と発ガン性を有するアフラトキシンを農作物中に生産する。農作物のアフラトキシン汚染は、人や家畜の健康に害をなし、また甚大な経済的損失を引き起こしている。しかし現在、実用的なアフラトキシン汚染防除法はほとんどなく、より効果的な方法の開発が強く求められている。本論文は、天然物を対象とした新しいアフラトキシン生産阻害物質の探索とそれらのアフラトキシン汚染防除への応用について述べたものであり、3章より構成される。

序論に続き、第1章では、精油を対象にA. parasiticusのアフラトキシン生産を阻害する物質を探索した結果と得られた阻害物質およびその類縁化合物の活性について述べている。阻害活性が見出されたシラカバの精油より活性物質の精製が行われ、シリング酸メチルが活性物質として単離・同定された。シリング酸メチルはA. parasiticusおよびA. flavusのアフラトキシン生産をIC50値が1 mM程度の強さで阻害した。その際、カビの成育には影響を与えない選択性を持つことが示された。シリング酸、没食子酸、3-O-メチル没食子酸、3,4,5-トリメトキシ安息香酸およびそれらのメチルエステルの8化合物中、A. parasiticusのアフラトキシン生産を選択的に阻害する化合物はシリング酸メチルだけであることが示された。またそれら8化合物のDPPH(1,1-diphenyl-2-picrylhydrazyl)ラジカル捕捉活性が調べられ、ラジカル捕捉活性はアフラトキシン生産阻害活性と相関しないことが示された。シリング酸メチルはアフラトキシン生合成経路初期段階の生合成中間体であるノルソロリン酸の生産を阻害した。また、アフラトキシン生合成酵素の発現を調節するタンパク質AflRをコードする遺伝子aflRおよびAflRの調節を受ける生合成酵素遺伝子pksA、omtAの転写を抑制することより、シリング酸メチルの作用点はAflRの発現に至るまでの制御過程にあることが示唆された。

第2章では、土壌より分離した細菌の代謝産物を対象に、アフラトキシン生産阻害物質を探索した結果得られたNo. 27株の生産する阻害物質に関して述べている。No. 27株はStenotrophomonas rhizophilaに最も近縁のStenotrophomonas sp.と同定された。No. 27株の培養液上清よりアフラトキシン生産阻害物質が精製され、活性炭カラムクロマトグラフィー、逆相高速液体クロマトグラフィーにより2種の活性物質が単離された。それらはcyclo(L-Ala-L-Pro)およびcyclo(L-Val-L-Pro)と同定された。両者のA. parasiticusおよびA. flavusのアフラトキシン生産阻害活性は同等であり、IC50値が0.7 mM程度の強さで阻害することが示された。その際、カビの成育には影響を与えなかった。cyclo(L-Ala-L-Pro)、cyclo(D-Ala-D-Pro)、cyclo(L-Ala-D-Pro)、cyclo(D-Ala-L-Pro)が調製されそれらのアフラトキシン生産阻害活性が調べられたところ、活性を示すのはcyclo(L-Ala-L-Pro)だけであることが示された。cyclo(L-Ala-L-Pro)およびcyclo(L-Val-L-Pro)はノルソロリン酸の生産を阻害し、aflR、pksA、omtAの転写を抑制したことよりその作用点はAflRの発現に至るまでの制御過程にあることが示唆された。No. 27株とA. flavusを共培養し、アフラトキシン生産とカビの成育に与える影響を調べたところ、アフラトキシン生産は共培養に用いたNo. 27株の菌体数に応じて阻害され、カビの成育には影響がないことが示された。

第3章では、第1章および第2章で見出されたシリング酸メチルおよびNo. 27株の効果を、自然状態でアフラトキシン汚染が見られるタイのピーナッツ貯蔵所において試験した結果について述べている。ピーナッツ貯蔵所での実験を行う前に、生ピーナッツをサンプル溶液に浸して効果を見る実験室でのモデル実験が行われ、アフラトキシン生産阻害効果が見られるシリング酸メチル溶液の濃度およびNo. 27株菌体懸濁液の菌体数が調べられ、ピーナッツ貯蔵所で実験を行う際の条件が決定された。タイではピーナッツを収穫、乾燥後、殻付きの状態で袋に入れ、出荷までの数週間程度貯蔵所で保管する。その貯蔵段階でのアフラトキシン汚染が問題となっている。そこで、貯蔵開始時にシリング酸メチル溶液あるいはNo. 27株菌体懸濁液でピーナッツを浸漬処理し、3週間貯蔵した後でのアフラトキシン汚染に対する効果を調べた。シリング酸メチル80 mM溶液で処理した場合、有意に貯蔵後のピーナッツに含まれるアフラトキシン量の減少が見られたが効果は弱いものであった。No. 27株の菌体懸濁液で処理した場合は菌体数に応じたアフラトキシン量の大幅な減少が見られ、No. 27株はアフラトキシン汚染防除に有効であることが示された。

以上、本研究はアフラトキシン生産阻害活性を示す物質を新たに見出し、阻害物質あるいは阻害物質生産菌がアフラトキシン汚染防除に有効であることを示したものであり、学術上、応用上貢献するところが少なくない。よって、審査委員一同は、本論文が博士(農学)の学位論文として価値あるものと認めた。