Microbial communities and volatile profile of Queijo de Azeitão PDO cheese, a traditional Mediterranean thistle-curdled cheese from Portugal

The production of ovine or caprine milk cheeses with thistle rennet is a common practice in the Mediterraneanbasin. The aim of the present study was to obtain information on bacteria and yeast communities harboured byQueijo de Azeit˜ao PDO cheese through viable counting and, for the first time, via metataxonomic analysis.Moreover, solid phase microextraction (SPME) technique was applied to characterize Queijo de Azeit˜ao PDOcheese volatile compounds. Nine cheese samples were collected from three different artisan producers located inPortugal. The results of physico-chemical analyses showed significant differences between producers, with meanvalues ranging from 5.40 ± 0.25 (Producer 1) to 6.00 ± 0.22 (Producer 2). As for TTA, Producer 1 showed thehighest mean value attesting at 18.04 ± 6.57 mL of 0.1 M NaOH used to reach pH 8.3. Regarding lactic acidconcentration, Producer 1 showed the highest mean value attesting at 0.488 ± 0.106 g 100 g 1, whereas, foracetic acid, no significant differences were evidenced among producers with values comprised between0.141 ± 0.021 g 100 g 1 and 0.245 ± 0.016 g 100 g 1. No significant differences were observed between overallmean values of the three producers for viable counts of presumptive lactococci, thermophilic cocci, presumptivelactobacilli, thermophilic lactobacilli and total mesophilic aerobes with values in the order of 7–8 log cfu g 1.Moreover, no significant differences were evidenced for viable counts of coagulase-negative cocci, enterococci,Enterobacteriaceae and Pseudomonadaceae. As for eumycetes, cheeses from Producer 1 showed the lowest meanvalue (2.78 ± 2.42 log cfu g 1) in respect with values detected in cheeses from Producer 2 and 3. Concerningmicrobiota and mycobiota of the analyzed cheeses, the alpha diversity index did not show any significant differencebetween the three producers in terms of composition and complexity of the microbial population. Asimple composition was apparently shared by the three producers, whose cheese manufactures were dominatedby the presence of Leuconostoc mesenteroides (37% of the relative frequency in average), Lactococcus lactis (29%),Lacticaseibacillus zeae (4.7%), Lentilactobacillus kefiri (4.4%), Serratia spp. (3.5%), Lactiplantibacillus plantarum(2.7%), and Latilactobacillus sakei (2.5%). The mycobiota composition showed the neat dominance of Yarrowialipolytica (46.7% of the relative frequency in average), followed by Candida ethanolica (13.6%), Kurtzmaniellazeylanoides (9.4%), Geotrichum candidum (8.8%), Galactomyces geotrichum (8.7%), Kluyveromyces lactis (3.5%),and Geotrichum silvicola (2.7%). The volatile profile analysis allowed 24 different compounds to be identified: 7acids, 7 esters, 4 alcohols, 3 ketones, 2 aromatic hydrocarbons, and 1 aldehyde. The most represented volatileorganic compounds (VOCs) were 2-butanone, butanoic acid and hexanoic acid. A positive correlation betweenLen. kefiri and hexanoic acid and isopentyl isobutyrate was observed (P < 0.05), whereas Y. lipolytica displayedthe highest number of positive correlations with 3-methyl-butanal, 2-pentanone and 2-pentanol (P < 0.05). Tothe authors’ knowledge, this is the very first detection of Len. kefiri in a raw ewe’s milk cheese coagulated withvegetable rennet.