## 2 Materials and Methods  
### Bacterial Strains and Culture Conditions  
_Enterococcus faecalis_ ATCC 47077, NCTC 13379 (_vanB+_), EFS2 ATCC 202014, ATCC 29212, _E. ficeium_ ATCC 700221, ATCC 19434, ATCC 51559 (_vanA+_) and C68 CRA-FLC, _E. gallinarum_ ATCC 49573 (_vanC1+_), _E. casselfluxus_ ATCC 25788, _Lactococcus lactis_ subsp. _lactis_ NCTC 6681, ATCC 11454, _L. lactis_ subsp. _cremoris_ ATCC 19257, DSM 20069, _Lacticasiebacillus casei_ ATCC 393, _L. paracasei_ DSM 5622, _L. rhamnus_ NCTC 12953, _Lactiplantiobacillus plantarum_ ATCC 55883, _Lactobacillus delbrueckii_ subsp. _lactis_ NCTC 13361, _L. delbrueckii_ subsp. _bulgaricus_ DSM 2008, _Lactobacillus_ spp. 30a ATCC 33222, _Levilactobacillus brevis_ NCTC 13386 and ATCC 367, _Limosilactobacillus fermentutum_ ATCC 55884, _Listeria monocytogenes_ ATCC7644 and EU-RL 12M0B098, _Escherichia coli_ O157 VTEC ISS, ATCC 25952 and EURL C07, _Salmonella_ Typhimurium ATCC 14028 and _Staphylococcus aureus_ ATCC 33862 were used as positive control strains in specific PCR reactions, antimicrobial production tests and cheese making trial. All culture media were provided by Biolife Italiana, S.r.l. (Milan, Italy). Enterococci and rod shaped LAB were propagated in De Man, Rogosa and Sharpe (MRS) broth (20.0 g/L glucose, 10.0 g/L peptone, 8.0 g/L meat extract, 4 g/L yeast extract, 3 g/L sodium acetate, 2 g/L triammonium citrate, 2 g/L K\({}_{2}\)HPO\({}_{4}\), 0.2 g/L MgSO\({}_{4}\), 0.05 g/L MnSO\({}_{4}\), 1 mL sorbitan monooleate and pH 6.5) and incubated for 24-48 h at 37 \({}^{\circ}\)C in anaerobic jars containing Oxoid(tm) AnaerobicGen(tm) sachets (Thermo Fisher Scientific, Monza, Italy). Lactococci were subcultured on M17 broth or agar in anaerobic conditions at 30 \({}^{\circ}\)C for 24-48 h. Coagulase positive cocci, _E. coli_, _L. monocytogenes_ and _Salmonella_ Typhimurium were propagated in Brain Heart Infusion (BHI) broth at 37 \({}^{\circ}\)C in aerobiosis for 24-48 h. Cryopreservation of cultures was performed in Microbank tubes (Biolife Italiana) at \(-80\) \({}^{\circ}\)C.  
Sampling was carried out according to the ISO 707:2008 norm [12]. Samples were collected from nine batches of cheese produced in three dairy factories belonging to the producer association \"Consorzio di Tutela del Pecorino di Farindola\" and comprised milk, curd and cheese at days 7, 14, 21, 35, 49, 63, 77, 91, 121 and 150 of ripening during the period March to September 2018. Samples were transferred into sterile containers, in the manufacturing plant or in the ageing rooms, transported to the laboratory and stored in refrigeration conditions for a maximum of 48 h until analysis. The collected amounts were 1 L of milk, 500 g of curd and a whole piece of cheese. The a\({}_{\text{w}}\) determination was carried out with the Aqualab 4 TE device (Meter Group, Pullman, WA, USA) according to the norm ISO 18787:2017 [13]. The determination of pH was carried out using a Seven Easy pH Meter (Mettler-Toledo S.P.A., Milan, Italy) on a suspension of 10 g of sample homogenized in 90 mL of deionized water for 1 min in a Stomacher 400 (Biosigma, Cona, VE, Italy). LAB counts were carried out as described by [PERSON] et al. [14]. Namely, LAB were counted and isolated on MRS agar medium and M17 agar medium (19.0 Di-sodium-glycerophosphate, 11.0 g/L agar, 5 g/L lactose, 5 g/L meat digest, 5 g/L tryptone, 5 g/L soya peptone, 2.5 g/L yeast extract, 0.5 g/L ascorbic acid, 0.25 g/L MgSO\({}_{4}\) and pH 6.9), incubated at 37 \({}^{\circ}\)C for 72 h in anaerobic jars and Slanetz and Bartley (SB) medium (20.0 g/L tryptose, 13 g/L agar, 5 g/L yeast extract, 2.0 g/L glucose, 4.0 g/L K\({}_{2}\)HPO\({}_{4}\), 0.4 g/L sodium azide, 0.2 g/L 2,3,5-Triphenyltetrazololium Chloride and pH 7.2), selective for enterococci, and incubated at 37 \({}^{\circ}\)C for 24 h in aerobiosis. Presumptive LAB were tested for catalase activity by transferring an individual colony onto a glass slide and adding a drop of 3% (\(v/v\)) H\({}_{2}\)O\({}_{2}\). Gram staining was also carried out.