Metagenomic pipeline

The bacterial community was analyzed by amplifying the V3–V4 hypervariable region of the 16S rRNA gene using primers and procedure described by Klindworth et al. (2013) (link). The fungal population was studied by amplification of the D1 domain of the 26S rRNA gene using primers and conditions described by Mota-Gutierrez et al. (2019) (link). 26S rRNA target was chosen because it showed greater taxonomic resolution and robustness compared to the ITS region. PCR amplicons were purified following the Illumina metagenomic pipeline (Illumina Inc., San Diego, CA, United States). Sequencing was performed with a MiSeq platform (Illumina), generating 250 bp paired-end reads.

For DNA extraction from milk and NMC, 1 mL of samples were firstly centrifuged at 4 °C at 12,000× g for 30 min, and supernatant was removed together with fats by using a sterile cotton swab. Pellet was washed with 500 µL of 1X phosphate buffered saline (PBS) and resuspended in 200 µL of ATL buffer (Qiagen, Hilden, Germany) and proteinase K (Qiagen, Hilden, Germany) following the manufacturer’s instruction of the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany). DNA was then extracted with the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) following the manufacturer’s instruction. For DNA extraction from the cheese, 10 g of sample were homogenized in a sterile stomacher bag with 90 mL of sterile Ringer solution (Oxoid, Milan, Italy) and mixed in a Stomacher 400 Circulator (Seward Ltd, Worthing, UK) at 300 rpm for 3 min. One mL of the first decimal dilution was transferred into 1.5 mL micro-tube and DNA was extracted by using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany).
Mycobiota was studied though the amplification of the D1 domain of the 26S rRNA gene using primers and condition described by Mota-Gutierrez et al. [17 (link)]. PCR amplicons were purified following the Illumina metagenomic pipeline (Illumina Inc. San Diego, CA, USA). Sequencing was performed with a MiSeq platform (Illumina), generating 250 bp paired-end reads.