*2.10. Caco-2 Cell Culture*

Caco-2 cells were obtained from DISCOG (University of Padova, Padova, Italy). The cells cultured in DMEM (high glucose) supplemented with 10% FBS, were used between 35 and 60 passages. In order to perform transepithelial transport, 8 × 10<sup>4</sup> cells were seeded on Transwell © cell culture inserts (0.4 μm pore sizes, 12 mm diameter, 1.12 cm<sup>2</sup> grown surface; Corning Life Sciences, Tewksbury, MA, USA). Cells were grown, di fferentiated for 21 days and the monolayer integrity was estimated by transepithelial electrical resistance (TEER) (Millicell ® ERS-2 volt-ohmmeter, EDM Millipore, Darmstadt, Germany) showing values higher than 1100 Ω × cm2.

#### *2.11. Transepithelial Transport of Fractions or Peptides Through Caco-2 Cell Monolayers*

The intestinal barrier crossing capacity of the fractions or synthetic peptides through Caco-2 cells monolayer was evaluated using Transwell ® insert model according to Tonolo et al. [26]. Briefly, after 21 days of culturing, Caco-2 cells di fferentiated forming a monolayer that delimited an upper part (apical compartment) and lower part (basolateral compartment). The monolayer was rinsed three times with Hank's balanced salt solution (HBSS) and 10 mM D-glucose and equilibrated for 30 min at 37 ◦C. After, cells were incubated in the presence of 0.75 mL HBSS containing 150 μg fraction or 75 μg peptide in the apical chamber at 37 ◦C for 120 min. Samples collected from both compartments (apical and basolateral) at di fferent times were centrifuged at 11,600× *g*, filtered with 0.45 μm filter, frozen and lyophilized. Subsequently, the obtained samples were dissolved in 100 μL of 0.1% TFA aqueous solution and evaluated by RP-HPLC and Matrix assisted laser desorption/ionization- time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS).
