## 2 Materials and Methods
### Bacterial Strain Selection and Propagation  
Several strains, which showed growth in the range 10-37 \({}^{\circ}\)C, both in aerobic and anaerobic conditions without gas production from dextrose, were tested for acidifying activity in skim milk (SM, BD Difco, 232,100). Then, based on the instantaneous acidification rate and its maximum value [18; 19; 20; 21; 22], a selection of ten strains was used for the production of BPs.  
The strains tested in this work were: _Lactococcus lactis_ ssp. lactis (internal reference (ref. 79, field strain), _Lactococcus lactis_ ssp. lactis (ref. 81, field strain), _Lactobacillus casei_ ssp. casei (ref. 80, filed strain), _Lactobacillus acidophilus_ ATCC 4356 (ref. 618), _Lactobacillus acidophilus_ LA 14 (ref. 1004), _Lactobacillus acidophilus_ (ref. 80/3, field strain), _Lactobacillus helicticus_ (ref. LH, field strain), _Enterococcus facicum_ UBEF-41 (ref. 1003), and _Saccharomyces cerevisiae_ var boulardii MTCC-5375 (ref. 1005). The morphological, biochemical and physiological characterization, the growth curves at several temperatures, including refrigeration conditions, the acidifying activity and their ability to improve palatability of certain food, along with safety considerations, have been reported by the authors in previous papers [18; 19; 20; 21; 22]. Before the test, freeze-dried strains of the starter cultures were grown aerobically in nutrient broth (NB, Oxoid CM0001, Basingstoke, UK) at 37 \({}^{\circ}\)C for 24 h. Each strain was then sub-cultured in skim milk (BD Difco, Franklin Lakes, NJ, USA, 232,100) at 37 \({}^{\circ}\)C for 24 h. The total viable cell (TVC) count (on nutrient agar, NA, Oxoid CM0003, incubated at 37 \({}^{\circ}\)C in aerobiosis for 24 h) after 24 h of incubation (Sanyo MIR-153 incubator, Moriguchi-City Osaka, Japan) was approximately 1 \(\times\) 10\({}^{9}\) cfu/mL. For tests, the strains were inoculated into skim milk (BD Difco) to get an initial concentration of approximately 1 \(\times\) 10\({}^{7}\) cfu/g, which mimics the initial starter concentration in dairy products fabrication. The fermentation activity was monitored at different time points (Table S1, Supplementary Material).  
Evaluation of Different Substrates and Comparison of Two Extraction Protocols for Milk Fermented with L. heleticus  
_L. heleticus_ (Ref. LH) was inoculated in three different culture media, skim milk (SM, BD Difco), brain heart infusion (BHI, Oxoid) and peptone water (PW, Oxoid) and fermentation was carried out for 12 h and 110 h at 37 \({}^{\circ}\)C in aerobiosis. The conditions of bacterial growth and inoculation described in Section 2.3 were applied when the three substrates (SM, BHI, and PW) were used as culture media.  
Two extraction protocols were used for peptide recovery within the fermented products, as described below:  
(_i_) an aliquot of fermented substrate (1.0 mL) was treated with an aqueous TCA solution (1.0% \(w/v\), 500 uL), vortexed (ZX3 VELP Scientific, Usmate, Italy) for 1 min and centrifuged (ALC 4239R high speed refrigerated centrifuge, International PBI, Milan, Italy) at 10,000 \(\times\)\(g\) for 10 min at 4 \({}^{\circ}\)C. For each sample the supernatant was separated from the precipitated fraction and stored at \(-\)20 \({}^{\circ}\)C until further investigations;  
(_ii_) an aliquot of fermented substrate (1.0 mL) was treated with glass beads (100 mg) and an aqueous solution of urea/TRIS-HCl (urea 2.0 M + Tris-HCl 50 mM, pH 7.4, 500 uL). The mixture was sonicated for 10 min at 25 \({}^{\circ}\)C, and then centrifuged at 10,000\(\times\)\(g\) for 10 min at 20 \({}^{\circ}\)C. For each sample the supernatant was separated from the precipitated fraction and stored at \(-\)20 \({}^{\circ}\)C until further investigations.