## 2 Materials and Methods
### Hydrolysis of SPC  
SPC hydrolysis was carried out according to the protocol provided by Penta-Ramos and [PERSON] [15] with slight modifications. Firstly, five separate water solutions of SPC containing 3% protein (w/v) each were prepared. All of them had the non-adjusted pH of 7.0\(\pm\)0.1. SPC solutions were continuously and gently stirred, as well as pre-heated for 5 min using a Heidolph Unimax Modular Incubator 1010 (Heidolph Instruments GmbH & CO. KG, Schwabach, Germany). The preincubation temperatures were typical of the activity of enzymes used, according to their specifications provided by the manufacturer, and were as follows: bromelain and ficin (50 \({}^{\circ}\)C), papain (65 \({}^{\circ}\)C), and proteinase K (37 \({}^{\circ}\)C). Finally, 3 hour hydrolysis of four SPC samples was conducted under continuous stirring and the enzyme-to-substrate ratio (protein) of 1:100 (w/w) [15; 16]. The pH values of SPC solutions were optimal for enzyme activity as provided by the manufacturer, i.e. : 7.0 for bromelain, papain, and proteinase K; and 6.5 for ficin. In the case of the last sample, its pH was adjusted using 0.1 M HCl. Afterwards, all hydrolysates were heated at 90 \({}^{\circ}\)C for 15 min to inactivate the enzymes and then freeze-dried. Finally, four SPC hydrolysates were produced, namely B-SPC, F-SPC, P-SPC, and PK-SPC, the abbreviations referring to bromelain-, ficin-, papain-, and proteinase-K-SPC hydrolysates, respectively, according to the convention used in the previous paper [9]. The fifth sample, 0-SPC, was a reference sample being the non-hydrolyzed SPC solution. All hydrolysates were prepared in duplicate. RP-HPLC for Monitoring the Process of SPC Hydrolysis and RP-HPLC-MS/MS for the Identification of SPC-Originating Peptides  
RP-HPLC (reversed-phase high performance liquid chromatography) was applied to observe the progress in the hydrolysis of SPC using the above-mentioned enzymes. The preparation of SPC/SPC hydrolysate samples taken for RP-HPLC and RP-HPLC-MS/MS (reversed-phase high performance liquid chromatography online with tandem mass spectrometry), as well as the parameters of devices for chromatography were exactly the same as in the protocol by [PERSON] et al. [9]. Firstly, two milligrams of freeze-dried SPC/SPC hydrolysate sample were dissolved in 300 \(\upmu\)L of a buffer containing 0.1 M Bis-Tris and 4 M urea. Then, two microliters of 2-mercaptoethanol were added, and the mixture was vortexed and finally incubated at room temperature (1 hour). Then, six-hundred-eighty microliters of 6 M urea solution in a mixture of ACN and H\({}_{2}\)O (v/v; 100: 900; pH 2.2 was adjusted by the addition of TFA) were added to the sample and stirred. Finally, the samples were centrifuged (10 min; 10,000\(\times g\)) [9]. The RP-HPLC analysis was carried out on Shimadzu(r) devices comprised of two LC-20 AD pumps, an SIL-20 AC HT autosampler, a CBM-20A controller, a CTO-10 AS VP thermostat, an SPD-M20A photodiode detector, a DGU-20A5 degasser, and an FRC-10A fraction collector. The Jupiter Proteo Phenomener(r) column (Torrance, CA, USA; 250 \(\times\) 2 mm; particle diameter: 4\(\upmu\)m; pore diameter: 90A) was applied for RP-HPLC analysis of the samples. According to the protocol by [PERSON] et al. [9], Solvent A was 0.01 % TFA water solution (v/v), whereas Solvent B was 0.01 % (v/v) TFA dissolved in acetonitrile (ACN). The gradient of Solvent B increased from 0 to 40 % during 60 min. The column was washed with Solvent B (40-100 %, 60-65 min; 100%, 65-70 min, 100 -0 %, 70-71 min; 0 %, 71-80 min). Data registration was between the 0 th and 80 th minute of the RP-HPLC analysis. The injection volume was 30 \(\upmu\)L; the flow rate was 0.2 mL \(\times\) min\({}^{-1}\); and the column temperature was 30 \({}^{\circ}\)C. All chromatograms were acquired at 220 nm [9]. Finally, the chromatographic profiles of the samples taken before the 60 th min of the RP-HPLC analysis were the subject of discussion. Similarly, the exact procedure as that described by [PERSON] et al.