*2.4. Sample Preparation for* α*-Lac and* β*-Lg Determination*

Defatted whey supernatant was prepared from both HTST- and HPP-treated samples by centrifuging them at 9000 g for 15 min at 4 ◦C. Then, the whey portion was obtained by adjusting the pH to 4.6 using 8 M acetic acid, followed by centrifugation at the same conditions. The pH of the supernatant was readjusted to 6.8 using 3 M NaOH.

#### *2.5. Determination of* α*-Lac and* β*-Lg Content*

An enzyme-linked immunosorbent assay (ELISA) method was followed to measure the concentration of α-Lac and β-Lg. The sandwich ELISA was performed using bovine α-Lac and β-Lg Quantitation Kits (catalogue E10-128 and E10-125, respectively) and an ELISA Starter Accessory Kit (catalogue E103), purchased from Bethyl Laboratories, TX, USA. The ELISA plates were coated, and supplied bovine α-Lac and β-Lg were diluted to obtain standard curves, following the manufacturer's protocols. A multimode plate reader with associated software (Perkin Elmer's EnSpire Multimode Plate reader, Waltham, Massachusetts, USA) was used to read the absorbance of ELISA plates at 450 nm. The unknown concentration of α-Lac and β-Lg of treated samples was obtained in duplicate from the standard curves. Retention of α-Lac and β-Lg was calculated as a percentage of α-Lac and β-Lg in untreated samples, respectively, as given in Equation (2).

$$\text{Retention} = \frac{c\_t}{c\_o} \times 100,\tag{2}$$

where *c*<sup>t</sup> and *c*<sup>o</sup> represent the concentrations of α-Lac or β-Lg after and before treatment, respectively.
