Sterile ringer solution

Ten grams of grated cheese (Biraghi s.p.a., Cuneo, Italy) were contaminated with 10 mL of ten-fold serial dilutions of an overnight pure culture of L. lactis subsp. lactis 39FL4 and added of 30 mL of sterile Ringer solution (Oxoid). The samples were homogenized in a Stomacher (Interscience, Rockland, MA, USA) for 30 sec and 1 mL of each sample was subjected to RNA extraction by using the MasterPureTM Complete DNA and RNA Purification Kit (Epicentre). Ribonucleic acid samples were treated as reported above (paragraph 2.1) and each cDNA sample was submitted to qPCR. Standard curve was constructed by plotting the Ct values against CFU/g, evaluated by traditional plating on M17 agar supplemented with lactose (5 g/L), of the grated cheese inoculated with the overnight L. lactis subsp. lactis 39FL4 culture. The reactions were carried out in triplicate and mean values were considered for standard curve construction. The R² and E of the amplifications were calculated according to Rutledge and Cote [13] (link).
The limit of detection of L. lactis was evaluated by RT-qPCR in grated cheese specifically inoculated with 5, 10, 50, 100, 1000 CFU/g of the microorganism. Commercial grated cheese was checked for the absence of lactococci before use.

An aliquot of 50 ml of bulk tank milk and the disposable in-line milk filter were collected from a dairy farm located in Piedmont, North-West Italy; the selected farm had 200 lactating cows and a mean daily milk production volume of 5,000 L. Samples were collected directly from the tank in sterile conditions, inserted in sterile plastic containers, and transferred to the Laboratory of Food Inspection at the Department of Veterinary Science – University of Turin. Upon arrival, 90 ml of sterile ringer solution (Oxoid Ltd., Basingstoke, Hampshire, UK) was added to a 10 g of aseptically weighed milk filter in a sterile stomacher bag and homogenized with a Stomacher® 400 circular (Seward Ltd., Worthing, UK) at 230 rpm for 2 min. The bulk milk (sample M) and the obtained filter homogenate (sample F) were divided into three aliquots of 15 ml each (M1, M2, M3; F1, F2, F3), which were subjected to different treatment and sequencing depth; aliquots were processed as shown in Figure 1.