*2.2. Scotta Concentrate Preparation*

Ovine scotta was freshly collected from a dairy plant located in north Sardinia (Italy) after the manufacturing of ovine Ricotta cheese, and immediately refrigerated to 4 ◦C, delivered to the lab and stored at −20 ◦C. Scotta was thawed to 20 ◦C immediately before the concentration step. The mean chemical composition, determined according to the literature [37] was as follows: pH 6.19 ± 0.11; total solids, 6.73 ± 0.28% ( *w*/*w*); fat, 0.05 ± 0.02% (*w*/*w*); total nitrogen (TN), 0.14 ± 0.01% ( *w*/*w*); nitrogen soluble in water (NS), 0.08 ± 0.05% (*w*/*w*); non-protein nitrogen (NPN), 0.07 ± 0.05% ( *w*/*w*); ash, 0.34 ± 0.23% ( *w*/*w*).

Scotta (5 L) underwent a preliminary continuous skimming at 15,000× *g* using a lab-scale cream separator (TLE 100, Tecnolatte, Lodi, Italy), then was consecutively filtered through 5, 1.2, and 0.65 μm on conventional cartridge filters (Sartopure PP3 Midicap, Sartorius, Goettingen, Germany) with a surface area of 0.21, 0.15, and 0.15 m<sup>2</sup> respectively, fed by a SartoJet Membrane Pump (Sartorius, Goettingen, Germany). The 0.65 μm filtered skimmed scotta underwent tangential filtration at 20 ◦C through a 10 kDa Hydrosart membrane (Sartocon Slice Cassette, Sartorius, Goettingen, Germany), keeping a constant transmembrane pressure of 0.5 bar, and monitoring the removed permeate weight, which was precalculated in order to obtain a nitrogen concentration factor of 4×. The retentate was then reconstituted to the original weight by adding ultrapure (UP) water and subsequently underwent a diafiltration step using the same membrane, in order to reduce the mineral fraction and lactose. The collected retentate was sampled at the end of the process for total nitrogen (NT) determination and then stored at −20 ◦C until the subsequent hydrolysis steps.

### *2.3. Enzymatic Hydrolysis of Retentate Scotta Samples*

The retentate obtained in Section 2.2, with a total nitrogen of 0.50 ± 0.01% ( *w*/*w*), was split between two experiments to be hydrolyzed with bromelain (BSPH) and pancreatin (PSPH), respectively. For each experiment, three hydrolyses on 50 g (*n* = 3) of retentate were performed. Lab-scale enzymatic reactions were performed for 4 h. Enzymatic hydrolysis were performed, as recommended by the manufacturer, at 50 ◦C for bromelain and at 40 ◦C for pancreatin. The enzyme-substrate (E:S) ratio was fixed at 4% (enzyme weight to protein weight) for both experimental groups. Further three 50 g retentate control samples (CTRL) underwent the same procedure without enzyme addition, setting the temperature and duration to 50 ◦C and 4 h, respectively. The enzymatic reactions were performed in a water bath at constant temperature ( ±0.05 ◦C) and continuous magnetic stirring at 500 rpm using an AREX-6 Digital PRO Hot Plate Stirrer (Velp Scientific, Bohemia, NY, USA) equipped with a VTF EVO digital thermoregulator. All the reactions were stopped by heating the mixtures at 90 ◦C for 10 min to inactivate the proteases. Afterward, the mixtures were centrifuged twice at 14,000× *g* (Neya 16 R, Remi Elektrotechnki LTD, Vasai, India) for 15 min at 4 ◦C, and the precipitate was discarded. The obtained supernatants of bromelain scotta protein hydrolysate (BSPH), pancreatin scotta protein hydrolysate (PSPH) and of the control were freeze-dried (Labconco, Kansas City, MO, USA) and stored at −20 ◦C until further analysis, such as DPP-IV inhibition, antioxidant capacity and an antibacterial assay, as well as gel permeation chromatography (GPC) and LC-MS/MS characterization.
