# Evaluation of a New Bulk Packaging Container for the Ripening of Feta Cheese
## 2 Materials and Methods
### Physico-Chemical Parameter Analyses  
The pH of the cheeses was determined using a model WTW (SenTix SP-DIN, 2010) pH meter (Wissenschaftlich-Technische Werkstatten GmbH, Weilheim, Germany). The moisture of the cheees was determined according to the oven-drying method [7]. Cheese salt content was determined using the Volhard method. The fat content of the cheeses was determined using the Gerber van Gulik method. Cheese protein content, total nitrogen (TN), and soluble nitrogen fractions (water-soluble nitrogen (% WSN), nitrogen soluble in 12% trichloroacetic acid (% TCA-SN), and nitrogen soluble in 5% phosphotungistic acid (% PTA-SN)) were determined according to the Kjeldahl method using Inkje1 1210 M apparatus (Behr Labor-Technik GmbH., Dusseldorf, Germany). Lactose was determined according to Anifantakis [8] as follows: 2 g of cheese were weighed in a 250 mL Erlenmeyer flask; then, 10 mL 0.5 M NaOH and 100 mL deionized water were added, and the mixture was homogenized. The mixture was then transferred to a 200 mL volumetric flask and 10 mL of 10% ZnSO4 \(\cdot\) 7 H\({}_{2}\)O solution was added to the flask; the flask was filled to the mark. The contents of the flask were then filtered. Then, 1 mL of the filtrate along with 1 mL of 5% phenol were transferred to a new test tube and 5 mL of H\({}_{2}\)SO\({}_{4}\) (d = 1.84 g/mL) was added dropwise. The test tube was then placed in a water bath at T = 100 \({}^{\circ}\)C for 10 min, after which the test tube contents were cooled to 15-20 \({}^{\circ}\)C and the absorption of the solution was measured at \(\lambda\) = 490 nm. In parallel, standard solutions of lactose in the range of 0-200 ug/mL were prepared, and their absorption was measured at the same wavelength following the above procedure. The lactose content of cheese was calculated based on the standard curve prepared.  
Semi-Quantitative Determination of Volatile Compounds Using Solid Phase Microextraction (SPME)--Gas Chromatography/Mass Spectroscopy (GC/MS)  
A 3 g sample of grated cheese, along with 10 \(\upmu\)L of an internal standard solution (0.134 mg/mL 4-methyl-2-pentanone), was placed in a 20 mL glass serum vial and sealed with a Teflon-coated septum and an aluminum crimp cap. SPME was performed with a DVB/CAR/PDMS 30/50 \(\upmu\)m fiber (Supelco, Bellefonte, PA, USA).  
The vial was then placed in a 50 \({}^{\circ}\)C water bath and, after allowing 15 min for equilibration, the fiber was exposed to the vial headspace for 15 min. GC analysis of the volatile compounds adsorbed onto the SPME fiber was carried out on a DB-5 MS 60 m \(\times\) 320 \(\upmu\)m i.d. \(\times\) 1 \(\upmu\)m column (Agilent, Santa Clara, CA, USA). An Agilent Technologies 7890A GC System equipped with an Agilent Technologies 5975C MS System detector was used (Wilmington, DE, USA) for the analysis of volatiles. The injector was operated in the split mode (2:1 split ratio) at a temperature of 250 \({}^{\circ}\)C. The column was initially maintained at 40 \({}^{\circ}\)C for 2 min; it was then heated to 170 \({}^{\circ}\)C at a rate of 5 \({}^{\circ}\)C/min, then heated to 260 \({}^{\circ}\)C at a rate of 10 \({}^{\circ}\)C/min and held at 260 \({}^{\circ}\)C for 2 min. The flow rate of the helium carrier gas was 1.5 mL/min. The temperature of the transfer line was held constant at 250 \({}^{\circ}\)C. The MS conditions were as follows: MS Quadrapole temperature 150 \({}^{\circ}\)C; source temperature 230 \({}^{\circ}\)C. Acquisition was performed in electron impact (70 eV) mode and the mass/charge was (m/z): 29-300. Peak identification was performed by comparison of the mass spectra of the eluting compounds to those of the [PERSON] library (Wiley 275, J. Wiley & Sons Ltd., Chichester, England). Retention indices (RI) of volatile compounds were calculated using n-alkane (C8-C20) standards (Fluka, Buchs, Switzerland). All above SPME conditions were the result of optimization carried out in preliminary experiments.