# Chemical-Sensory Traits of Fresh Cheese Made by Enzymatic Coagulation of Donkey Milk
## 2 Materials and Methods
### Chemical and Sensory Analyses  
The moisture content in cheese was assessed by the method IDF 4:1986, fat by the Soxhlet method, total protein by the Kjeldhal method, ash by muffle furnace incineration at 530 \({}^{\circ}\)C, and pH by the HI99165 pH Meter equipped with penetration electrode (Hanna Instruments, Woonsocket, RI, USA). Total fatty acids (TFA) analyses were performed on milk, whey and cheese by gas chromatography (GC), as reported by [PERSON] et al. [15], with some modifications. Briefly, the fatty acids methyl esters were obtained by transesterification of about 25 mg lipid sample obtained by Soxhlet extraction (for cheese) and centrifugation at 2.360 RCF at 4 \({}^{\circ}\)C (for milk and whey), previously dissolved in 2 mL of petroleum ether. Samples were injected into a Fisons model MFC 800 gas chromatograph (Fisons, Milan, Italy) equipped with a 60 m \(\times\) 0.32 mm i.d. and 0.5 \(\upmu\)m film thickness fused-silica capillary column (Stabilwax, Restek, Bellefonte, PA, USA). The operating conditions were: (a) oven temperature 70 \({}^{\circ}\)C for 5 min, then to 220 \({}^{\circ}\)C at 1 \({}^{\circ}\)C/min, and held at 220 \({}^{\circ}\)C for 30 min; (b) carrier gas helium 20 cm/s at 170 \({}^{\circ}\)C; (c) injector at 250 \({}^{\circ}\)C, 1 \(\upmu\)L; split 40:1; (d) flame-ionisation detector, 250 \({}^{\circ}\)C. Volatile compounds were extracted by the HS-SPME technique using a DVB-CAR-PDMS fiber (Supelco, Bellefonte, Pa., USA), as reported by [PERSON] et al. [16], with some modifications. The sample (1.0 mL milk or 1 g cheese), 0.2 g sodium chloride and 10 L of 3-pentanone (internal standard, 81.9 ng/L in water) were placed in 20 mL screw-cap vial, tightly capped with a PTFE-silicon septum, and conditioned for 10 min at 40 \({}^{\circ}\)C. A TriPlus RSH\({}^{\upmu\text{m}}\) Autosampler (Thermo Fisher Scientific, Rodano, Italy) was used to optimise and standardise the entire extraction procedure. The fiber then was introduced into the headspace of the vial for 40 min at 40 \({}^{\circ}\)C. Then, desorption of volatiles from fiber took place in a splitless mode for 3 min at 220 \({}^{\circ}\)C. The separation of volatile compounds was performed by a Trace 1300 gas chromatograph (Thermo Fisher Scientific) equipped with a VF-WAXms capillary column (Agilent, Santa Clara, CA, USA), 60 m length \(\times\) 0.25 mm I.D. \(\times\) 0.25 \(\upmu\)m film, and coupled with an ISQ single quadrupole mass spectrometer (Thermo Fisher Scientific). The chromatographic conditions were: oven, 45 \({}^{\circ}\)C (5 min) to 210 \({}^{\circ}\)C at 4 \({}^{\circ}\)C/min, held for 3 min; detector, source temperature 250 \({}^{\circ}\)C; transfer line temperature 250 \({}^{\circ}\)C; carrier gas, helium at constant flow of 0.4 mL/min. The impact energy was 70 eV. Data were acquired using the full-scan mode in the range of 35 to 150 m/z at an acquisition rate of 7.2 Hz. Volatile compounds were tentatively identified by comparing the experimental spectra with those reported in the National Institute of Standards and Technology (NIST) Library, and with those obtained by the available pure standard compounds. Semi-quantitation was carried out by considering the relative areas of the compounds related to that of the internal standard, and assuming arbitrarily that their response factors were the same. Figure 1: Technological scheme used for donkey milk cheesemaking. Acquisition and processing of peaks was carried out using Xcalibur v 4.1 software (Thermo Fisher Scientific). All analyses were done in triplicate, and means and standard deviations were calculated. Two different electrophoresis (EF) techniques were used for investigating the protein fraction in milk, whey and cheese. A sample of isoelectric whey, used as a standard, was also prepared. The milk and cheese samples were analysed by urea polyacrylamide gel electrophoresis (PAGE) as indicated by [PERSON] [17], whereas sodium dodecyl sulphate (SDS) PAGE as reported by [PERSON] et al. [18] was applied to the whey samples.