*2.3. Aqueous Extract of Samples*

150 mL of fermented milk were mixed with 150 mL of distilled water in an orbital shaker at room temperature (RT) for 5 min. Then the solution was centrifuged at 16,800× *g* for 25 min at 15 ◦C. The supernatants were filtered through Whatman Chr 1 (GE Healthcare, Chicago, IL, USA) and the obtained aqueous extracts were stored at –20 ◦C until use [22].

#### *2.4. Solid Phase Extraction of Bioactive Fractions from Aqueous Preparations*

For the extraction of peptide fractions, a solid phase STRATA C18 E column (Phenomenex, Torrance, CA, USA) was employed. The column was initially conditioned with 50 mL of 100% acetonitrile (ACN) and rinsed with 125 mL of 0.1% trifluoroacetic acid (TFA) aqueous solution. Aliquots of 50 mL of aqueous extract, obtained as described above, were loaded onto the column. After washing with 125 mL of 0.1% TFA, a discontinuous gradient step of ACN was applied in order to obtain peptide enriched fractions [22]. Briefly, the column was eluted with 50 mL of 5%, 30% and 50% ACN solutions and the fractions 5–30% ACN and 30–50% ACN were collected, frozen at –80 ◦C and lyophilized (Freeze Drier, Edwards, Burgess Hill, west Sussex, UK), then stored at –20 ◦C until further analysis.
