## 4 Conclusions  
In the present work, the proteolytic activity of several starter cultures, isolated from meat and dairy products, sampled at different fermentation times and grown in different substrates, was evaluated. The fine optimization of the RP-HPLC method allowed screening of the proteolytic activity by following the chromatographic profile of selected peaks during the gradient elution. Of the investigated strains, _L. helveticus_ showed a higher likelihood, and more constant over the time, proteolytic activity and was, therefore, selected for further investigations. The results of the chromatographic analyses, coupled with in vitro antioxidant (ABTS and FRAP) and antihypertensive (ACE inhibitory) assays, evidenced a remarkable dependence of _L. helveticus_ activity on the starting substrate. This aspect could be gainfully exploited when a specific activity needs to be privileged to functionalize a food with pro-health peptides. Moreover, a nano-HPLC-HRMS system was implemented to discriminate those fractions which, differently from the non-fermented substrates, would be potentially responsible for the found activity.  
The preliminary results obtained in this study underline the necessity of considering a multiplicity of factors to exploit the potential bioactivity associated with peptides produced upon fermentation. Evidently, innovative separation and purification processes are mandatory to isolate the desirable peptide fractions following an initial screening, being aware, at the same time, that a significant variation in terms of bioactivities can be  
Figure 5: TIC (total ion current) zoomed-in the separative gradient interval 40–50 min of SM-Substrate, SM-TCA, and SM-Beads samples acquired in DDA mode. The light red squares highlight the peaks of interest.  
expected by the use of purified peptides over the non-purified hydrolyses. All these aspects become particularly crucial when large-scale production of healthful food or food ingredients is intended. In this context, more in-depth investigations addressing peptide sequence identification would enable the molecular characterization of such bioactive components. This, in turn, would also result in being helpful to reach a better knowledge of specific targets.  
The following are available online at [[https://www.mdpi.com/2076-341](https://www.mdpi.com/2076-341)]([https://www.mdpi.com/2076-341](https://www.mdpi.com/2076-341)) 7/11/2/811/s1, Table S1: Time sampling scheme for strain fermentation activity evaluation, Table S2: Comparison between L. acidophilus (LA 14) and L. helveticus (LH) activity upon 72 h fermentation, reported as mean area values of reference peaks (measured in mV-s) \(\pm\) standard deviation (SD), n = 3 for each selected peak., Figure S1: Full MS spectrum of the three samples a) SM-Substrate, b) SM-TCA, c) SM-Beads, related to the RT including the peak at 42.57 \(\pm\) 0.16 min. The m/z of the newly formed peptides are highlighted in red. Figure S2: Full MS spectrum of the three samples a) SM-Substrate, b) SM-TCA, c) SM-Beads, related to the RT including the peak at at 45.75 \(\pm\) 0.19 min. The m/z of the newly formed peptides are highlighted in red., Figure S3: Full MS spectrum of the three samples a) SM-Substrate, b) SM-TCA, c) SM-Beads, related to the RT including the peak at at 47.12 \(\pm\) 0.15 min. The m/z of the newly formed peptides are highlighted in red.  
Conceptualization, L.C. and B.T.C.-G.; investigation, F.I., A.A.A., and L.G.; writing--original draft preparation, F.I.; data curation, L.R. and F.B.; writing--review and editing, F.I., F.B., and L.C.; supervision, L.C. All authors have read and agreed to the published version of the manuscript.  
This research received no external funding.  
Not applicable.  
Data contained within the article or supplementary material are here presented for the first time.  
The authors declare no conflict of interest.