## 2 Materials and Methods
### Analytical Methods
Filtered aliquots of the neutralized supernatant were analyzed by a Waters Alliance 2695 apparatus at a 30.0 cm \(\times\) 7.8 mm column Aminex HPX-87H (Bio-Rad Laboratories, California, USA), coupled to a differential refractometer (Refractive Index Detector, Waters 2410). The mobile phase used was H\({}_{2}\)SO\({}_{4}\) (0.005 M) with a flow rate of 0.5 mL/min (isocratic elution) and a column temperature of \(T\) = 60 \({}^{\circ}\)C. The samples were diluted to appropriate concentration and filtered through a 0.2 \(\upmu\)m membrane filter before injection (injection volume = 20 \(\upmu\)L). The sugars were detected by RI at 880 nm and the results were quantified, corresponding to standards. Total cellular lipids (L, g/L) were extracted from TDW, using the blend of solvents chloroform/methanol (C/M) [47] in a proportion 2:1 \(v/v\), respectively, for 72-96 h, as described in [PERSON] et al. [48]. The solvents were recycled by evaporation (Flash Evaporator--Rotavapor R114) and the cellular lipids were gravimetrically determined. A sample (c. 30 mg) of the total cellular lipids was dissolved in C/M 2/1 \(v/v\) (1 mL) for Thin Layer Chromatography (TLC) analysis, and the rest was converted to the respective fatty acid methyl esters (FAME) in a two-step reaction, as described in [PERSON] et al. [44]. TLC was conducted as described by [PERSON] et al. [49]; aluminum sheets silica-gel 60 (20 cm \(\times\) 20 cm \(\times\) 0.25 mm dimensions--Merck, Darmstadt, Germany) was used for the development of neutral lipids. The plate was activated before use by heating for 2 h at \(T\) = 80 \({}^{\circ}\)C, then the lipid standards (cholesterol, oleic acid, and triolein at a concentration of 30 mg/mL) and samples were manually injected, and the plate was placed in an all-glass chromatography chamber at room temperature, containing a mixture of solvents (\(n\)-hexane/diethyl ether/glacial acetic acid--70:30:1, \(v/v/v\)), as mobile phase. The chromatographic separation (c. 45 min) was followed by drying in a fume hood for 30 min. The visualization was conducted through exposure of the developed TLC plate to iodine vapor in an iodine-saturated chamber. Gas chromatography analysis of FAME was carried out in a GC apparatus (Fisons GC 8000 Series), equipped with a capillary CPWAX 52 CB column (dimensions 60 m \(\times\) 0.32 mm, film thickness 1.20 \(\upmu\)m) and a flame ionization detector (FID, Fisons). Helium (He) is used as the carrier gas, with a gas flow of 2 mL/min; the analysis was isothermally run to \(T\) = 240 \({}^{\circ}\)C, the temperatures of injector and detector were \(T\) = 240 \({}^{\circ}\)C and \(T\) = 250 \({}^{\circ}\)C, respectively, and the FAME were identified and quantified by comparing the retention times and areas of their peaks to those observed for their respective standards (Supelco 37 Component FAME Mix, Sigma-Aldrich, Taufkirchen, Germany). The individual FA content was expressed as weight percentage (g/100 g of total FA). Residual lactose (Lac, g/L) determination was carried out in Shimadzu Type High Performance Liquid Chromatography (HPLC, Waters Alliance 2695) device, equipped with UV and RI (2414 Refractive Index) detectors, as described above. The determination of free amino nitrogen (FAN, mg/L) of \(\alpha\)-amino acids or N-terminals of peptides and proteins was conducted by the ninhydrin photometric method [50]. Total Kjeldahl Nitrogen (TKN, g/L) of second cheese whey cultures were assayed according to Kjeldahl method [51] in a Kjeltek(tm) 8100 Distillation Unit (Foss A/S, Hillerad, Denmark). Residual protein (RP, g/L) of culture broth was rapidly determined according to the colorimetric method suggested by [PERSON] [52].