## Results and Discussion
### Antioxidant and ACE-Inhibitory Activity of Peptide Hydrolysates  
Several food-derived peptides have been found to exhibit antioxidant properties generally without noticeable side effects and with high activity and easy absorption [39; 40]. Several properties such as structure, hydrophobicity, amino acids composition and their specific positioning and configuration in the peptide sequence, seem to significantly influ  
Figure 1: Chromatographic profiles obtained from SM substrate fermented with (**a–c**) _L. acidophilus_ strain (strain LA 14) and (**d–f**) _L. helveticus_ (LH field strain), at time points of 12 h, 72 h, and 400 h, respectively. The hydrolyses were extracted by TCA at 4 \({}^{\circ}\)C (see Section 2.4 for details). An enlarged portion of each chromatogram in the time-window 15–30 min is reported to evidence the richness of the chromatographic profile relatively to minor intensity detected peaks. The interference peak detected at 12 min corresponds to the TCA additive. ence the antioxidant activity of peptides [41; 42]. Furthermore, hydrolysate concentration and molecular weight were also found to affect antioxidant properties [43]. In the present study, the antioxidant activity of the hydrolysates from _L. helveticus_ fermented substrates was evaluated and compared by relying upon the free radical scavenging activity (measured through the ABTS assay) and the reducing capacity (measured through the FRAP assay). As shown in Table 2, the hydrolysates obtained by using BHI as starting fermentation substrates showed the highest values of antioxidant activity determined by FRAP and ABTS assays. Instead, a controversial behavior was observed for the hydrolysates from SM and PW substrates; indeed, while in the former case the lowest antioxidant activity was measured by ABTS, in the latter the lowest FRAP activity was obtained. Figure 2: Graphical representation of the comparison between _L. acidophilus_ (LA 14) and _L. helveticus_ (LH) activity upon 72 h fermentation, based on area values of reference peaks (measured in mV-s). Data are the mean \(\pm\) standard deviation (SD) of three replicates. Data were analyzed by a Student’s unpaired _t_-test. *******_p_ \(<\) 0.0001, ***_p_ \(<\) 0.001, ***_p_ \(<\) 0.01, *_p_ \(<\) 0.05. Concerning the ACEi activity, _L. heleticus_ strains have been designated among the preferential sources to achieve a hypotensive effect, thanks to the excellent microbial \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Hydrolysate** & **ABTS (\(\mathbf{\mu}\)mol TE/mL)** & **FRAP (\(\mathbf{\mu}\)mol TE/mL)** & **ACEi Activity\% \({}^{\star}\)** \\ \hline SM-TCA & 17.00 \(\pm\) 0.00 & 23.02 \(\pm\) 0.00 & 81.77 \(\pm\) 1.37 \\ BHI-TCA & 245.40 \(\pm\) 0.00 & 29.55 \(\pm\) 0.00 & 53.65 \(\pm\) 1.82 \\ PW-TCA & 55.67 \(\pm\) 0.00 & 9.58 \(\pm\) 0.00 & 87.30 \(\pm\) 1.59 \\ \hline SM-Beads & 26.27 \(\pm\) 0.00 & 20.84 \(\pm\) 0.00 & 70.12 \(\pm\) 3.81 \\ BHI-Beads & 261.40 \(\pm\) 0.00 & 25.12 \(\pm\) 0.00 & 49.59 \(\pm\) 0.49 \\ PW-Beads & 64.87 \(\pm\) 0.00 & 10.53 \(\pm\) 0.00 & 97.75 \(\pm\) 1.87 \\ \hline \hline \end{tabular} \({}^{\star}\)Measured by reversed phase-high performance liquid chromatography (RP-HPLC).  
\end{table} 
Table 2: Summary of antioxidant (by ABTS and FRAP assays) and ACEi activities of hydrolysates. Values are expressed as mean values \(\pm\) standard deviation (SD), \(n\) = 2. Figure 3: Chromatographic analysis of _L. heleticus_ hydrolysates obtained with the three substrates (SM, BHI, and PW) and extracted by TCA at 4 \({}^{\circ}\)C or by using glass beads. The interference peak detected at 12 min corresponds to the TCA additive. proteolytic system involved in conversion of milk proteins into oligopeptides [16; 43]. Accordingly, we tested the ACEi activity of _L. helveticus_ hydrolyses produced in the presence of the three selected substrates. The results highlighted an in vitro activity close or over 50%, comparable with data reported by other authors [12; 13; 43].