2.3. In Vitro Digestion of Lf

In vitro digestions were performed in two steps, representing the gastric and duodenal processes [14]. In the first gastric digestion step, the pH of protein samples was adjusted to 2.5 with 0.5 M HCl. Pepsin was dissolved in Simulated Gastric Fluid (SGF) (0.15 M NaCl, pH 2.5), and was added to the protein samples to give a final concentration of 165 U of pepsin per mg of protein. Half of the gastric digested sample was further hydrolyzed in duodenal digestion. The other half of the gastric digested sample was centrifuged at 15,000× g for 30 min at 4 °C and the supernatant was collected and stored at −20 °C for antibacterial activity assay.

In stage two duodenal digestion, the pH of the digesta from the gastric digestion was firstly adjusted to 6.5 with 0.1 M NaOH. Trypsin 34.5 U per mg of whey protein and chymotrypsin 0.4 U per mg of whey protein were added into the digesta. The digestion was performed in a shaking incubator (170 rpm) at 37 °C, 60 min for stage one and 30 min for stage two. Aliquots were withdrawn from the mixture at the beginning and the end of gastric and duodenal digestions to evaluate protein hydrolysis profile using SDS-PAGE. Hydrolysis was terminated by heating at 80 °C for 15 min. All the digested protein samples were centrifuged at 15,000× g for 30 min at 4 °C and the supernatant was collected and stored at −20 °C for antibacterial activity assay.