Development and application of a competitive enzyme immunoassay for the detection of penitrem A in fungal mycelium: Evidence for frequent occurrence of multiple indole-containing mycotoxins in mouldy foods

Abstract

The present study aimed to develop a novel enzyme immunoassay (EIA) for the tremorgenic mycotoxin, penitrem A (PTA). This competitive EIA, based on rabbit anti-PTA polyclonal antibodies and a PTA-bovine serum albumin conjugate, yielded a mean standard curve detection limit of 220 ng/mL. The PTA-EIA, together with two EIAs for paxilline (PAX) and ergoline alkaloids, were used to study the toxin profiles in culture of fungi isolated from some naturally infected food sample materials, including dairy products, citrus fruits, and walnuts. PTA was produced by eight out of 27 mycelium extracts from malt extract agar and Sabouraud glucose chloramphenicol selective agar (2–200 μg/mL). Ten isolates from all types of food were also positive in the PAX-EIA (0.004–4 μg/mL). The EIA for ergoline alkaloids yielded positive results (0.0002–0.4 μg/mL) in isolates from dairy products and from walnuts, but not from citrus fruits. Control analyses of selected fungal extracts by HPLC-MS/MS for PTA and PAX qualitatively confirmed the EIA results, poor quantitative agreement could be attributed to the presence of penitrems other than PTA, and to PAX analogues, respectively. Selected PTA-positive fungal isolates were subjected to sequencing of the internal transcribed spacer region and the β-tubulin gene, and were identified as Penicillium polonicum and P. crustosum. In conclusion, Penicillium spp. capable to produce PTA, PAX, and ergoline alkaloids, either alone or in combination, appear to be quite common as contaminants on foods at the retail or production level. Some are capable to produce PTA in culture at high levels, which would have to be considered as toxic if present in food.