This study describes the fungal microbiota of Pelardon cheese, a french goat PDO cheese during cheese making and ripening and microbiota of environmental species.
Metabarcoding analysis was performed on 34 samples (3 raw milks, 3 whey, 3 curds, 6 cheese core and 6 cheese rind samples and 13 environmental samples). The same raw material and cheese samples, diluted in citrate buffer, were used as described previously for culture-dependent analyses (see 2.2). Aliquots of 1 mL were centrifuged at 9000 g for 15 min at 4°C, then supernatants were removed and the cell pellets immediately stored at -20°C until total DNA extractions. Regarding environmental samples, pellets were obtained from liquid samples by centrifuging 1 mL (9000 g, 15 min, 4°C), whereas for surface samples, swabs were placed in 1 mL of TS diluent prior to centrifugation (9000 g, 15 min, 4°C) to obtain cell pellets.  Total DNA extractions were performed using the DNeasy Blood and tissue kit (Qiagen, Germany) with a supplementary initial enzymatic lysis. First, cell pellets were thawed at room temperature then resuspended in 400 µL of lysis buffer (Tris-HCl 20 mM at pH 8.0, EDTA 2 mM, Triton X-100 1.2 %) supplemented with lysozyme (20 mg mL-1) and mutanolysin (5 U µL-1), then Rnase (25 µg mL-1; Qiagen, Germany) and lyticase (0.5 U µL-1; Sigma-Aldrich, Germany) were added. DNA extracts). ITS3f/ITS4_Kyo1 primers (5'-GCATCGATGAAGAACGCAGC-3'/5'-TCCTCCGCTTWTTGWTWTGC-3') (Toju et al. 2012) were used, targeting the ITS2 region. ITS2 amplifications and sequencing steps were performed in the same run at Genome Quebec sequencing platform (MacGill University, Canada) using Illumina Miseq PE300 technology generating 2x300 bp reads.