Buffered peptone water (bpw)

Samples were pre-enriched in buffered peptone water (BioMaxima S.A., Lublin, Poland, 1:10 v/w; 18 ± 2 h at 37 ± 1 °C) followed by selective isolation and differentiation on BEAA medium (Bile Esculin Azide Agar ISO 15788:2009) or CHROMagar Orientation (BioMaxima S.A., Lublin, Poland) differentiating a wide spectrum of microorganisms. After overnight incubation at 37 ± 1 °C, 3 colonies from each plate with a characteristic morphology (BEAA: small gray colonies on the darked of the medium around the growth, CHROMagar Orientation: turquoise colonies) were selected and transferred to nutrient agar (Oxoid, Hampshire, United Kingdom) which was incubated overnight at 37 ± 1 °C.
The identification of microorganisms was performed using the MALDI TOF MS technique (Matrix Assisted Laser Desorption and Ionisation Time of Flight Mass Spectrometry) and the MALDI Biotyper (MBP COMPASS 4.1). All samples were prepared in accordance with the Standard operating procedure formic acid extraction (EX) method (Brucker Daltonic GmbH, Revision 3, August 2013). Obtaining a log score (log(score)) in the range of 2000–3000 was considered to give a high degree of confidence of the best match to the reference species in the database. Strains belonging to the genus Enterococcus were preserved using LB medium with 15% glycerol (POCh, Gliwice, Poland) and stored at −20 °C until further analysis.

For microbiological testing, ten grams of cheese were homogenized with 90 mL of sterile buffered peptone water and consecutive decimal dilutions were prepared by mixing 1 mL of preceding dilution with 9 mL of buffered peptone water (BioMaxima, Warsaw, Poland) in test tubes [48 ]. Total aerobic bacteria count (TABC) [49 ], number of mesophilic lactococci [50 ], yeast and molds [51 ], and coliform [52 ] were determined according to the respective standard procedures on the respective media, i.e., plate count agar (PCA), M17 agar, chloramphenicol agar and VRBL agar (BioMaxima, Poland). The plates were incubated under aerobic conditions at 30 °C for 72 h (TABC and Lactococcus sp.), 25 °C for 120 h (yeasts and molds) and 30 °C for 24 h (coliforms). After incubation, microorganisms were counted, and results calculated and expressed in log cfu/g.
Emulsions of cheese samples prepared in water (1:1) were subjected to pH measurement using a CP-411 pH-meter (Elmetron, Zabrze, Poland). The water activity of freshly grated cheese samples was measured using LabMaster-aw (Novasina AG, Lachen, Switzerland) [53 ].