The native β-Lg concentration after heat treatment was determined by HPLC using the supernatant recovered after centrifugation (9000 rpm for 30 min at 4 °C) of the samples adjusted to pH 4.6. The chromatographic system was an Alliance e2695 Separation Module (Waters, Milford, MA, USA), composed by a XBridge Protein BEH C4 300 Å, 3.5 µm, 4.6 mm × 250 mm separation column associated with a guard column (Sentry Guard Cartridge, Waters). The two mobile phases used were 0.1% (v/v) of trifluoroacetic acid (99%, Acros Organics) in Milli-Q water as solvent A and 0.1% (v/v) trifluoroacetic acid, 80% acetonitrile (HPLC grade, Thermo Fisher Scientific) and 20% Milli-Q as solvent B. 20 µL of each sample was injected in the column maintained at 40 °C. The separation was performed at a flow rate of 1 mL/min with gradient elution and a detection wavelength of 215 nm. Analyses were repeated two times for each sample and three times for each standard. Calibration standards in the range from 0.25 to 3 g/L were prepared by dissolving a β-lg powder (BioPure industrial powder, Davisco, Foods International, Inc., Minnesota: β-lg at 88.85% purity) in Milli-Q water.
The native β-lg concentrations were calculated by averaging the measured chromatographic areas and converting each area into concentration by means of the calibration curve.
The native β-lg concentrations were calculated by averaging the measured chromatographic areas and converting each area into concentration by means of the calibration curve.