Exploring Fungal Biodiversity During Artisanal Cheese Ripening In Natural Mine Environment

The CheeseMine project explored an innovative approach to cheese aging in the Dossena’s mine (Italy), enhancing traditional dairies while safeguarding cultural and environmental authenticity. This study investigated the autochthonous fungal biodiversity of the artisanal Italian cheese Ol Minadùr during mine-aging, focusing on community composition and potential health risks associated with toxigenic fungi. DNA-metabarcoding of 23 mine-aging cheeses, from 4 batches, aged 90 and 180 days, and produced by 5 dairy farms, revealed dominance of Debaryomyces hansenii and Mucor lanceolatus. In addition, non-EU regulated mycotoxin-producing fungi were detected at low relative abundance. Shannon index showed that the mycobiota richness increases over time, while beta diversity analysis revealed significant differences in mycobiota composition of cheese produced by different dairies and ripening. A case study, carried out on a single dairy, compared both fungal biodiversity and volatile organic compounds (VOCs) in cheeses aged in refrigerated cells vs mine. Cell-aged cheeses showed that Penicillium solitum and Candida parapsilosis were the most occurring species at 90 days, and Torulaspora delbrueckii and Saccharomyces cerevisiae at 180 days. Mine-aged ones exhibited higher fungal diversity, dominated by M. lanceolatus, alongside Arachnotheca glomerata and Fusarium domesticum. Sequencing of specific DNA markers was performed to overcome the limited taxonomic resolution of ITS2-based DNA metabarcoding and accurately identify fungal species [1]. VOCs determined by SPME-GC-MS revealed that cell environment promoted higher levels of hexanoic and 3-methylbutanoic acids, ethyl-hexanoate, and ethanol, developing more cheesy, pungent, and slightly fruity notes, associated with Aspergillus, Penicillium, Candida, and Kluyveromyces species. In contrast, mine-aged cheeses were characterized by higher amounts of 2-butanol, 2-heptanone, and 2-phenylethanol, exhibiting fruitier, floral, and sweeter aromas associated with Fusarium and Mucor species. Results showed that uncontrolled natural environments can harbor non-toxigenic mycobiota, which act as secondary starters, shaping distinctive cheese sensory profiles. On the other hand, continuous monitoring of fungal biodiversity is essential also for safety, in order to eventually detect toxigenic species and provide a better food risk assessment.