Penicillium citrinum isolated from environments inhibited aflatoxin production by Aspergillus parasiticus and the inhibitory substance was confirmed to be citrinin (CTN). CTN was converted to a new substance in the culture medium of A. parasiticus and the substance “CTN-KA adduct” was (1R,3S,4R)-3,4-dihydro-6,8-dihydroxy-1-(3-hydroxy-6-(hydroxymethyl)-4-oxo-4H-pyran-2-yl)-3,4,5-trimethyl-1H-isochromene-7-carboxylic acid. Since the CTN-KA adduct did not inhibit aflatoxin production by A. parasiticus, CTN’s toxicity was supposed to be alleviated with KA by converting CTN to the CTN-KA adduct.

アフラトキシン生合成に係る25以上の酵素遺伝子の中で、4遺伝子についてそれぞれ遺伝子破壊株を作製し、性状検討を行った。3種類の破壊株が同じ中間体を蓄積したのに対し、verA遺伝子破壊株は新規の黄色蛍光性中間体を蓄積し、それが、分子量374で、二つのコンフォーマーから校正されることが確認された。最終的に、verA遺伝子がDemethylsterigmatocystinのキサントン骨格形成に関与することが明らかとなった。

我々が開発したDV-AM法を用いて、輸入ナッツ中のアフラトキシン生産菌を検索したところ、ケニア産マカデミアナッツと南アフリカ産ピーナッツからアフラトキシン生産菌が検出された。かびはそれぞれ、Aspergillus flavus及びAspergillus parasiticusであることが確認された。以上の結果から、DV-AM法は食品安全の重要管理点の解明に有用であると示唆された。

The CTN-KA adduct does not show antimicrobial activity or any cytotoxic effect on HeLa cells, suggesting that CTN was detoxified by KA by the formation of the CTN-KA adduct. When the A. parasiticus mutants with impaired KA production were incubated with CTN, they were more susceptible to CTN than the wild KA-producing strain. When KA was added to the CTN-containing agar medium, fungal growth inhibition with CTN was remarkably mitigated, suggesting that the production of KA protected the fungal growth from CTN’s toxicity. CTN enhanced the production of KA by A. parasiticus, whereas the enhancement of KA production with CTN was repressed by the addition of α-tocopherol, suggesting that KA production is enhanced by oxidative stress via the formation of reactive oxygen species caused by CTN. Regulation mechanism of CTN on KA production, AF production, and fungal growth was proposed.

沖縄のサトウキビ畑の土壌からアフラトキシン生産菌をスクリーニングした。ジクロルボスーアンモニア法（DV-AM法）でまずアフラトキシン生産カビを単離して、得られたカビを通常の培地で培養した後、アンモニア処理（AM法）を行った。その結果、日本で初めてAspergillus novoparasiticusを単離することに成功した。以上のことから、DV-AM法とAM法を組み合わせてスクリーニングに用いることで、異なるアフラトキシン生産性を示す多様なカビを区別して検出できることが明らかとなった。

Citrinin (CTN) is a nephrotoxic and hepatotoxic substance to animals and contamination with CTN has been widely detected in cereals, feed, and supplements. We found that CTN produced by Penicillium citrinum inhibited aflatoxin (AF) production by Aspergillus parasiticus. Interestingly, when A. parasiticus was cultured in YES liquid medium supplemented with CTN, CTN was disappeared together with recovery of AF productivity with time. We confirmed that kojic acid (KA) caused CTN disappearance, and incubation of CTN and KA caused a novel substance, that was (1R,3S,4R)-3,4-dihydro-6,8-dihydroxy-1-(3-hydroxy-6-(hydroxymethyl)-4-oxo-4H-pyran-2-yl)-3,4,5-trimethyl-1H-isochromene-7-carboxylic acid (named as the “CTN-KA adduct”). The CTN-KA adduct was confirmed that unlike CTN, CTN-KA adduct is a non-toxic substance to microorganisms and HeLa cells. These results indicated that CTN can be detoxified with KA through formation of the CTN-KA adduct. We also found that A. parasiticus as well as A. oryzae with impaired KA production were more susceptible to CTN than the wild KA-producing strain. In contrast, KA addition of the mutants mitigated fungal growth inhibition with CTN. These results suggested that the production of KA protected the fungal growth from CTN’s toxicity. Also, we found that addition of CTN to the culture medium remarkably enhanced KA production by either A. parasiticus or A. oryzae. RT-PCR experiments showed that CTN enhanced the expression of KA biosynthetic genes (kojA, kojR, and kojT) of A. parasiticus. In contrast, enhancement of KA production with CTN was repressed in the presence of antioxidants, α-tocopherol or butylated hydroxy anisole. These results suggested that KA production in A. parasiticus is enhanced by oxidative stress via formation of reactive oxygen species caused by CTN. In this work, we proposed a regulation scheme of CTN for each of KA production, AF production, and fungal growth in A. parasiticus.

We obtained 10 kinds of raw nuts and seeds from foreign countries via internets to screen for aflatoxigenic fungi and investigated whether these nuts contained aflatoxigenic fungi by using dichlorvos-ammonia method (DV-AM method). Peanuts from South Africa and the macadamia nuts from Australia were found to contain aflatoxigenic A. parasiticus. These results showed that some aflatoxigenic fungi are coming from foreign countries to Japan by attaching to the imported raw nuts.

We tried to isolate aflatoxigenic species of Aspergilli from two different sugarcane fields during cultivation of sugarcane in Okinawa, Japan, by using a newly developed dichlorvos-ammonia (DV-AM) method. Aspergillus pseudonomius isolates and A. novoparasiticus were finally detected. These results demonstrated that the DV-AM method is practically useful for isolation of the minor species of aflatoxigenic Aspergilli.