PubMed · 11798344
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Ruth A Etzel. Mycotoxins.. https://doi.org/10.1001/jama.287.4.425
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Aflatoxin (AF) contamination poses a severe global threat to food and medicinal material safety, yet existing research focuses on terminal AF metabolites while neglecting residual biosynthetic precursors, leading to potential underestimation of contamination risks. In this study, a UPLC-MS/MS method was established for the simultaneous quantification of six AFs and their five precursors in lotus seeds, with optimization of mass spectrum parameters, chromatographic separation conditions, and sample pretreatment. Method validation confirmed linearity (R2 > 0.99), LODs (0.03-0.36 μg/kg), and recoveries (76.53%-120.0%, RSD < 15%). Analysis of 41 natural lotus seed samples revealed a 63.4% AF contamination rate, dominated by B-group AFs, while O-methylsterigmatocystin (OMST) and versicolorin hemiacetal (VOH) were identified as the primary co-residual precursors with co-occurrence rates ≥ 50%. Notably, AFM1 was predominantly detected in natural samples with AFB1 concentrations exceeding 100 μg/kg. Artificial inoculation experiments further demonstrated that sterilization and sealing conditions modulated AF biosynthesis in lotus seeds, with non-sterilized and non-sealed groups showing delayed fungal metabolism and lower toxin accumulation. A significant linear correlation was observed between AFM1 and AFB1 levels (r = 0.94) in infected samples, demonstrating their accumulation levels are coupled with fungal overall metabolic flux. Given the high co-occurrence rate of OMST/VOH with AFB1 in natural samples, their individual and combined toxicities require in-depth investigation. This work deciphers matrix-specific AF dynamics in lotus seeds, supporting regulatory standard refinement (e.g., precursor inclusion) and targeted control (e.g., time-sensitive drying after harvest). Further studies will focus on exploring the molecular mechanisms of substrate-dependent AF synthesis.
Aspergillus sojae, which is believed to be a domesticated strain of Aspergillus parasiticus, contains all of the aflatoxin biosynthetic genes but is unable to produce aflatoxins and is generally recognized as safe (GRAS) for producing fermented foods. In A. parasiticus both aflR, the aflatoxin pathway-specific regulatory gene, and aflJ, a co-activator gene, are necessary for transcription of genes encoding the aflatoxin biosynthetic enzymes. A. sojae aflR differs from A. parasiticus aflR in that it encodes extra His and Ala, and has a pretermination defect that causes truncation of the carboxyl terminus of the predicted protein. A. sojae aflJ differs from A. parasiticus aflJ in that it encodes a predicted protein with Ser39 replaced by Ala and Ser283 replaced by Pro. Steady-state levels of aflatoxin biosynthetic gene transcripts of aflR, aflJ, pksA, nor1, ver1 and omtA in A. sojae as determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were much lower than those of A. parasiticus. Yeast two-hybrid assays showed that the truncated A. sojae AFLR did not interact with AFLJ of A. sojae and A. parasiticus but that an A. sojae AFLR reverted to the putative ancestral form interacted normally with AFLJ of A. sojae and A. parasiticus. Deletion analysis showed that both amino- and carboxy-terminal regions of the A. sojae AFLJ were important for the R-J interaction. The truncated A. sojae AFLR thus not only was impaired in its ability to activate transcription of aflatoxin biosynthetic genes, but also was unable to interact with AFLJ, in A. parasiticus both of which are required for normal expression of the aflatoxin biosynthetic genes. Consequently, the lack of aflatoxin-producing ability of A. sojae resulted primarily from two defects in the regulatory mechanism responsible for gene transcription.
The biosynthesis of aflatoxin in Aspergillus parasiticus is a complex process that involves the activities of at least 18 pathway enzymes. The distribution of these enzymes within fungal colonies and fungal cells is not clearly understood. The objective of this study was to investigate the distribution and subcellular location of Nor-1, Ver-1, and OmtA, which represent early, middle, and late enzymatic activities, respectively, in the aflatoxin biosynthetic pathway. The distribution of these three enzymes within A. parasiticus SU-1 was analyzed in time-fractionated, 72-h fungal colonies (fraction 1, 48-72 h; fraction 2, 24-48 h; fraction 3, 0-24 h). Western blot analysis and immunofluorescence microscopy demonstrated the highest abundance of Nor-1, Ver-1, and OmtA in colony fraction 2. Fungal tissues in this fraction were analyzed by immunoelectron microscopy. Nor-1 and Ver-1 were primarily localized to the cytoplasm, suggesting that they are cytosolic enzymes. OmtA was also detected in the cytoplasm. However, in cells located near the basal (substrate) surface of the colony, OmtA was predominantly detected in organelles tentatively identified as vacuoles. The role of this organelle in toxin biosynthesis is unclear. The relative distribution of OmtA to the cytoplasm or to vacuole-like organelles may depend on the age and/or physiological condition of the fungal cells.