2.4. Cheese production and quality analysis

Eight cheeses were produced following the Vacherin Fribourgeois PDO technology (Figure 2). The trials were replicated in two consequent days in the pilot plant facility at Agroscope (Liebefeld, Switzerland). Two controls, one with raw milk and the other with thermized milk, were produced using only the starter culture. A starter culture commonly used in Vacherin Fribourgeois PDO production (Lactococcus lactis and Leuconostoc mesenteroides; Liebefeld Kulturen AG) was also added at the proportion of 0.12% to the vat milk. The natural adjunct cultures were added at the proportion of 0.5% to the vat milk. The pH-value of the cheeses was determined using a pH electrode.

(A) Vacherin Fribourgeois PDO cheese making flow chart. (B) Acidification curve during the cheese making; the adjunct culture added in each trial is reported in the legend. Refer to Figure 1 for samples’ abbreviation.

Volatile carboxylic acids were analyzed in the 120 d ripened cheeses using a Hewlett Packard HP 6890 gas chromatograph (Agilent Technologies, Basel, Switzerland) as described by Fröhlich-Wyder et al. (2013).

Biogenic amines were analyzed in the 120 d ripened cheeses as described by Ascone et al. (2017) using a UPLC system (UltiMate 3000 RS; Thermo Fisher Scientific) equipped with a C18 column (Accucore C18: 2.6 mm, 150 _ 4.6 mm; Thermo Fisher Scientific). All measurements were carried out in duplicate.

Total free amino acids and di- and tripeptides were analyzed in the 120 d ripened cheeses with the ophthaldialdehyde (OPA) method (Egger et al., 2019). Briefly, the samples were diluted 10-fold, prior to precipitation with perchloric acid (0.5 mol/L), and then derivatized with OPA in the presence of 2-mercapto-ethansulfonic acid. The produced 1-alkylthio-2-alkylisoindol compound was measured at 340 nm. To calculate the results, a standard curve based on glutamic acid was used.

The extent of proteolysis in the 120 d ripened cheese was measured by analyzing the following compounds: total nitrogen (TN), water-soluble nitrogen (WSN) and non-protein nitrogen (NPN) according to Kjeldahl (Collomb et al., 1990).

Cheese samples at 1 d and 120 d of ripening were analyzed for moisture, dry matter (IDF, 1982), fat (IDF, 1987), and fat in dry matter (FDM) using common standard methods.

Cheese samples at 1 d and 120 d of ripening were analyzed for lactic acid and citric acid concentration and L-leucine aminopeptidase (LAP) activity. For the determination of lactate, 1.25 g of cheese were homogenized in 50 mL of water using an OmniPrep Multi-Sample Homogenizer (Omni International, Kennesaw, United States). For the determination of citrate, 5 g of cheese were used. The homogenates were then incubated at 2°C for 20 min. Particles and fat were removed by filtration. The concentration of D- and L-lactate, and citrate in the filtrates was determined using commercial enzymatic assay kits (R-Biopharm AG, Murten, Switzerland). L-leucine-aminopeptidase (LAP) activity was determined using a colorimetric assay with L-leucine-4-nitroanilide as substrate. For the assays, 60 μL cheese filtrate (1.25 g cheese sample homogenized in 50 mL water and filtered) and 250 μL of L-leucine-4-nitroanilide (final concentration: 0.995 mmol/L) in phosphate buffer containing 2 mmol/L Mg²⁺ (pH = 7.4) were mixed in a microtiter plate. Enzyme activity was calculated based on the micromolar extinction coefficient of 4-nitroaniline measured by a SpectraMax ABS plus plate reader (Molecular Devices) after 2 h of incubation using the SoftMax Pro software (Molecular Devices).