Sausage Microbiome Analysis Protocol

The samples were taken from randomly chosen parts of sausages using the dynamics described within Section 2.1, by making small cuts in several places with a sterile scalpel. All parts collected from three sausages were pooled and mixed into one sample. The DNA extraction was performed using the Zymo BIOMICS DNA Mini Kit (Zymo Research, Irvine, CA, USA) from a minimum of 200 mg/pooled sample from each time point, according to the manufacturer’s protocol. The concentration of isolated DNA was measured using a Qubit Fluorometric Quantitation device (Qubit 4 Fluorometer, Invitrogen, Carlsbad, CA, USA). Amplicon sequencing was performed using a 2 × 300 bp paired-end sequencer on a MiSeq sequencer according to the manufacturer’s instructions (Illumina, San Diego, CA, USA) at a commercial sequencing service (FISABIO, Valencia, Spain). To identify bacterial communities, the V3-V4 region of the 16S rRNA gene was amplified with defined forward (5′-CCTAGCGGGNGGCWGCAG-3′) and reverse (5′-GACTACHVGGGTATCTAATCC-3′) primers [20 (link)].