Diversity of Mycotoxigenic Penicillium and Associated Mycobiota in Dry-Cured Meat (Cecina, León, Spain) Revealed by a Polyphasic Approach

<i>Cecina de León</i> is a traditional Spanish dry-cured beef product whose surface, as in other similar meat products, becomes heavily colonised by fungi during ripening, raising concerns related to possible mycotoxin contamination. This study aimed to characterise the mycobiota associated with <i>cecina</i> and its production environment, with particular emphasis on mycotoxigenic <i>Penicillium</i> species. Seventy-eight <i>cecina</i> samples and 26 air samples were collected from meat-processing plants and local markets in the province of León (Spain) and analysed for fungal counts, water activity and pH. A total of 101 mould isolates and 16 yeasts were recovered, with <i>Penicillium</i> accounting for 88% of all moulds. Sixteen <i>Penicillium</i> species were identified using a polyphasic approach integrating macro- and micromorphological analysis, extrolite production, molecular markers (<i>BenA</i>, <i>CaM</i> and ITS), and MALDI-TOF MS. Mycotoxin screening by HPTLC and HPLC-PDA targeted cyclopiazonic acid, ochratoxin A, patulin, citrinin, griseofulvin and mycophenolic acid, revealing that 51% of the <i>Penicillium</i> isolates were mycotoxin producers, mainly <i>P. commune</i>. The proposed polyphasic strategy, including MALDI-TOF MS as a rapid complementary tool, offers a practical framework for the surveillance of fungal communities and mycotoxin risk in meat-processing environments.