Application of aqueous ozone for controlling Diaporthe toxica, a mycotoxin producing fungal species

Interest in plant-based proteins, particularly those obtained from lupin beans, has rapidly increased in last years. On the other side, concerns for potential risks associated with this food matrix have been raised, since lupin is the primary source of a mycotoxigenic fungal species, Diaporthe toxica. Phomopsin-A (PHO-A), the main mycotoxin produced by this fungus, showed deleterious effects on livestock, and has been classified as possibly harmful for humans. Unfortunately, only limited information on this species is available in the scientific literature. Moreover, no treatments are documented for the reduction of PHO-A in lupin intended for food consumption. Ozone, a strongly oxidizing molecule, showed a great potential in the inactivation of both microorganisms and related toxins, but no research has been conducted on PHO-A. This study aims to characterize D. toxica, including its growth and metabolites production. Moreover, ozone treatments with different durations have been conducted on inoculated lupin beans, to evaluate its effectiveness and applicability in reducing PHO-A. Different agar media (Oat Flake Medium (OFM), Potato Dextrose Agar (PDA), Yeast extract, Peptone, Dextrose (YPD), Malt Extract Agar (MEA), Water Agar (WA) and WA with lupin beans) have been inoculated by using an 8 mm mycelial plug of a 5-day-old D. toxica culture, and then incubated at 25.0 °C for 21 days. Fungal growth was evaluated by mens of diameter growth measurements, and the spore production was confirmed by optical microscopy. In addition, lupin beans were inoculated following 3 different protocols, and PHO-A was quantified over time. Finally, inoculated lupins were subjected to a 7.00 ppm aqueous ozone treatment for 4, 6 and 8 hours; then, fungal load and PHO-A reduction were evaluated by microbiological sampling and SPE extraction followed by UHPLC-MS/MS analysis, respectively. Moreover, alkaloid variation was investigated by HPLC-MS/MS. Results from fungal growth evidenced the ability of D. toxica to develop mycelium in different media, particularly on OFM and PDA. With respect to the spores production, it was achieved only by using lupin beans as growth substrate. Successively, PHO-A production data in different conditions showed a diverse fungal adaptation to the environment: on rehydrated lupin beans, D. toxica produced PHO-A values up to 1082.17 ppm, while in commercial lupins PHOA only reached 138.81 ppm. Moreover, the evaluation of alkaloids revealed that this strain can produce alkaloids similar to those of lupin, if grown on this legume. Concerning the effectiveness of aqueous ozone in the reduction of the microbial load and PHO-A, the treatments could not reduce D. Toxica count significantly in most cases, but nearly 20% PHO-A reduction was observed for all the samples. In conclusion, results highlighted some critical aspects of D. toxica, introducing new information to better understand its metabolism. The knowledge gained in this study might help reduce the risk of PHO-A formation in lupin beans. Moreover, aqueous ozone treatment demonstrated a promising potential for the reduction of PHO-A.