Sample preparation and genomic DNA extraction

Inoculated milk samples (100 mL) were first subjected to centrifugation at 6000×g (30 min, 10 °C) using a Heraeus Multifuge (Thermo Fisher Scientific, Waltham, USA; Rotor 75002005), after which the supernatant was removed. The resulting cell pellets were kept at − 20 °C. Curd and cheese samples were prepared for DNA isolation by blending 10 g of sample with 90 mL of pre-warmed 2% (w/v) sodium citrate buffer (Merck, Darmstadt Germany) in a BagMixer^® lab blender (Interscience for Microbiology, Saint Nom, France) for 2 min at maximum speed using a BagFilter^® 400 P lab blender bag (< 250 μm lateral filter). The filtered supernatant was again centrifuged at 6000×g (30 min, 10 °C). After removal of the supernatant, the pellets were kept at − 20 °C until further processing.

For isolation of genomic DNA, the method published by Luzzi et al. (2020) was modified to suit DNA isolation from inoculated milk, curd and cheese samples. For this, the pelleted samples were defrosted at ambient temperature and resuspended in 1 mL of the lysis buffer used by Luzzi et al. (2020). Next, to each sample 0.4 g of sterile 0.1 mm Zirconium/glass-beads^® (Carl Roth, Karlsruhe, Germany) were added and samples were homogenised for 5–10 min at 80 rpm using an Intelli-Mixer RM-2 M (ELMI, Calabasas, CA, USA) bead-beater set at the U2-mode until homogenous. The samples were incubated at 70 °C with shaking at 400 rpm (Eppendorf Thermomixer, Hamburg, Germany) for 15 min. Next, the samples were centrifuged (9600×g for 5 min at 4 °C) using a Heraeus Fresco21 (Thermo Fisher Scientific, Waltham, MA, USA) centrifuge. The supernatant was centrifuged repeatedly under the same conditions until a clear lysate was obtained. To increase the final genomic DNA yield, 300 μL of fresh lysis buffer was added after the clear lysate had been harvested, and the homogenisation steps using Zirconium/glass-beads^®, heat treatment and centrifugation were repeated once as described above.

A 10% volume of 10 M ammonium acetate (relative to the absolute sample volume; Merck, Darmstadt, Germany) was given to the respective clear lysate samples. These were then placed on ice for 10 min and subsequently centrifuged at 16,200×g (10 min, 4 °C). To precipitate the DNA, one volume of 2-propanol (4 °C; Carl Roth, Karlsruhe, Germany), was then added to each sample. After mixing thoroughly, the samples were placed on ice for 45 min. This was followed by centrifugation at 16,200×g (15 min, 4 °C). The supernatant was discarded and the nucleic acid-containing pellets were washed with 1 mL of 70% ethanol (Carl Roth, Karlsruhe, Germany). Subsequently, the pellets were dried and resuspended in 100 μL of 10 mM Tris-HCl pH 8.0.

Samples were then further processed using the QIAamp DNA Stool Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the protocol outlined by Luzzi et al. (2020). Briefly, the RNA was first digested using 4 μL of a 10 mg/mL DNAse-free RNase solution (VWR International GmbH, Darmstadt, Germany), after which the proteins were degraded with 30 μL of 20 mg/mL proteinase K solution (AppliChem GmbH, Darmstadt, Germany). The QIAGEN kit’s ‘AL Buffer’ was then added to each sample prior to usage of the QIAamp spin columns to capture and wash the DNA as documented by Luzzi et al. (2020). The concentration of genomic DNA from samples was determined with a Qubit^® 3.0 Fluorometer and the Qubit™ dsDNA BR Assay Kit following the kit specifications (Thermo Fisher Scientific, Darmstadt, Germany).