*2.7. Gel Permeation Chromatography*

The freeze-dried scotta hydrolysates and control samples were reconstituted in water at 6 mg mL−<sup>1</sup> and filtered on a 0.2 μm filter, then analyzed by Gel Permeation high performance liquid Chromatography (GPC) using an Agilent 1260 HPLC system equipped with a DAD detector. The separation was performed at 25 ◦C in isocratic mode with a mobile phase composed of 70:30 ACN:H2O with 0.1% TFA, flowing continuously at 0.5 mL min−<sup>1</sup> through a Phenomenex Yarra SEC-2000 column (300 × 7.8 mm; pore size 3 μm). Samples were filtered through 0.2 μm nylon filter and 20 μL were injected. The analytical signal was acquired for 30 min at 214 nm. A calibration of molecular weights (MW) was obtained by acquiring the retention volume of the following pure standards covering a MW range from 181.19 to 66,500 Da: Tyr, Asp-Glu, Leu-Trp-Met-Arg, bacitracin, aprotinin, α-La, β-Lg, BSA. The obtained linear model was adopted to determine the MW distribution of the hydrolysates. The results were expressed as relative abundance by summing the areas of the peaks detected at different molecular weight (obtained by the calibration curve) ranges (1 kDa, 1–5 kDa, 5–10 kDa and >10 kDa), as previously reported by [41]. Data were analyzed as described in Section 2.4.

A semi-preparative GPC step was further performed on the above-described samples by injecting 100 μL in the same conditions above described. The fraction corresponding to a calculated MW between 5000 and 330 Da was collected for further LC-MS/MS analysis.
