Aflatoxins have frequently occurred in nuts, spices, and cereals. Accurate determination of aflatoxins is required to assess the risk of human intoxication. Liquid chromatography mass spectrometry (LCMS) is a powerful tool for the confirmation of aflatoxins, but it is not sufficient for routine analysis yet. We described here about an application for the determination of aflatoxins by LCMS electrospray ionization with a selected ion monitoring mode. Our analytical method provided the quantitative linearity at concentrations of 0.5-10 ng/ml. The relative standards deviation of aflatoxins was less than 4.34 % (10 ng/ml) after six times repeated injection. The present method was applied to the determination of aflatoxins in 6 food samples such as peanuts, pistachio nuts, almond, maize, nutmeg and red pepper harvested from markets. Aflatoxins B1, B2, G1, and G2 could be detected at the concentrations of more than 1 ng/g.
A confirmatory analytical method for aflatoxins in a complicated sample such as spices utilizing a column switching technique was developed. Using both DIOL and ODS columns as a pre-column and a main column, respectively, three applications of two-dimensional liquid chromatography for aflatoxins in sample extracts not fully refined were achieved. First, a fraction containing all aflatoxins B1, B2, G1 and G2 was separated from sample matrices within pre-column elution and reinjected into the main column followed by gradient elution. Second, a fraction containing only aflatoxin B1 was separated and analyzed in the same manner, that is to say, single peak heartcutting. The quantitative detection limits were 10μg/kg for G1 and 5μg/kg for the others in eight spices. Third, a large-volume sample injection was successfully accomplished. As a result, we could improve tenfold the quantitative detection limits. Over and above that, consideration of the reliability of identification in chromatographic analysis was carried out.
Oil palm is an important plant for edible oil production. Prior to harvesting the palm fruit bunches from which the oil is extracted, the fronds beneath the palm fruit are removed. Research has shown that the waste palm fronds can be used as cattle feed. However, mycotoxin contamination of these fronds, especially aflatoxin contamination, is of concern. Therefore, we developed an analytical method for determining aflatoxins in dried oil palm fronds (OPF) and compound feed containing OPF. Aflatoxins were extracted with 80 % acetonitrile and passed through a multi-functional column (MycoSep #228) as a clean-up step prior to analyses. Aflatoxins were analyzed by both TLC and HPLC. The recovery of aflatoxins from samples spiked at 5 to 20 μg/kg (aflatoxin B1, B2, G1 and G2 individually) was about 75 % and RSDs were less than 10 %. The detection limit of aflatoxin B1 in OPF by TLC was 5 μg/kg. No aflatoxins were detected among ten of the OPF samples analyzed, however, aflatoxins were produced and detected in samples that were inoculated with potent aflatoxin producing strains.
All fungal polyketide synthase (PKS) genes so far cloned code for iterative type I PKSs that are different from bacterial modular type I PKSs and type II PKSs. In order to clarify how fungal PKSs control their reactions to produce specific product compounds, fungal PKSs such as Aspergillus terreus ATX, Aspergillus nidulans WA, Colletotrichum lagenarium PKS1, Aspergillus fumagatus Alb1p and their derivatives, were expressed under α-amylase promoter in heterologous host Aspergillus oryzae. Chemical identification of products confirmed their functions and gave some insights into how their reactions are controlled.
Aflatoxins are toxic, carcinogenic secondary metabolites that are produced by some strains of Aspergillus flavus, A. parasiticus, A. nomius, and A. tamarii. Biosynthetic pathway of aflatoxins consists of more than 18 enzyme steps from acetyl coenzyme A. Many of the enzyme genes involved in aflatoxin biosynthesis have been isolated, and most of them are clustered. A regulatory gene product, AflR, regulates expression of these enzyme genes. This review summarizes the enzyme steps involved in aflatoxin biosynthesis and introduces the features of this pathway and regulation mechanism of aflatoxin production.
Aflastatin A (AsA) is an inhibitor of aflatoxin production by Aspergillus parasiticus and it also inhibits production of other polyketide metabolites by fungi such as melanin produced by Colletotrichum lagenarium. To elucidate the mode of action of AsA, the effect of AsA on the biosynthetic pathway of each aflatoxin and melanin was examined. AsA inhibited production of norsolorinic acid, an early biosynthetic intermediate of aflatoxin. The transcription of genes encoding some aflatoxin biosynthetic enzymes and a gene encoding a regulatory protein for expression of the biosynthetic enzymes (aflR) were significantly reduced by the addition of AsA. These results suggest that AsA inhibits a very early step in aflatoxin biosynthesis prior to the transcription of aflR. On the other hand, AsA inhibited the production of scytalone, an intermediate of melanin biosynthesis, and it severely impaired the expression of PKS1 encoding a melanin biosynthetic enzyme. This suggests that AsA inhibits an early step in melanin production similarly to the case in aflatoxin production. We also found that AsA elevated the glucose consumption and ethanol accumulation by A. parasiticus, which suggested that AsA can influence glucose metabolism in the fungus.