*2.1. Determination of Proteolytic Activity of Lactobacillus Strains*

Both the initial and the overall proteolytic activity of all cultures were determined using the method of Church et al. [15]. The proteolytic activity of each *Lactobacillus* strain was determined in reconstituted skim milk (RSM) after 6 and 24 h of fermentation at a temperature of 37 ◦C for determination of the initial and the overall proteolytic activity, respectively. All the organisms were activated from their frozen forms by one transfer into MRS broth (*Lactobacillus* Broth acc. to DE MAN, ROGOSA and SHARPE, Merck, Poland). The obtained cultures were passaged twice by a transfer 1 mL of inoculum into 100 mL of sterile supplemented (with glucose and yeast extract) reconstituted skim milk (SRSM). Then, 1 mL of the inoculum from the SRSM was transferred into 100 mL of RSM and after 24 h of incubation at 37 ◦C, and 1 mL of culture was transferred to 100 mL of RSM and incubated for either 6 or 24 h.

For determinations of the proteolytic activity of the analyzed strains, trichloroacetic acid (TCA) filtrates of the samples were prepared by mixing 5 mL of the sample with 1 mL of distilled water and 10 mL of 0.75 N TCA (Avantor Performance Materials, Poland), followed by centrifugation (MPW-352R centrifuge, Poland) at 4000 g and 4 ◦C for 30 min. The supernatants were filtered through a 0.45 μm syringe membrane filter (MILLEX HV, Milipore, Poland). The proteolytic activity of all cultures was determined by the reaction of 150 μL of the TCA filtrate with 3 mL of o-phthaldialdehyde reagent (OPA, Sigma-Aldrich, Poland). Absorbance was measured after vortexing and 2 min incubation at room temperature at 340 nm (Genesis UV-VIS Spectrophotometer, Thermo Scientific).
