## Results and Discussion  
### Evaluation of the Fermentation Activity by RP-HPLC  
A selection of ten starter cultures (see Section 2.3 for details), isolated from meat and dairy products and identified in previous works [18; 19; 20; 21; 22], was tested for their ability to grow in aerobic and anaerobic conditions. The selected strains were inoculated in reconstituted SM and screened via RP-HPLC to assess their proteolytic activity. Thepreliminary outcomes highlighted a higher activity, chromatography evaluated, of _L. acidophilus_ LA 14 (ref. 1004) and _L. helveticus_ (ref. LH, field strain) for the production of peptide components in fermented milk. Therefore, such selected microorganisms were submitted to more detailed investigations by monitoring their activity at different times (between 12 h and 400 h upon fermentation). Based on data reported by other authors [27, 28], in order to get the separation of the major number of peaks belonging to the desirable peptide fraction, several chromatographic experimental conditions were properly selected. Accordingly, the gradient profile, along with column temperature and flow rate, was progressively adapted by relying upon the heuristic \"trial-and-error\" method [29]. The preliminary optimization step was carried out by using a common reversed phase stationary phase. The addition of 0.1% (v) TFA as lipophilic ion-pairing additive to both components of the mobile phase (water and ACN) revealed to be an optimal compromise to guarantee the reproducibility of chromatographic runs [30, 31, 32, 33, 34]. Additionally, the simultaneous optimization of both temperature (from 25 to 40 \({}^{\circ}\)C) and gradient steepness demonstrated being a powerful tool to control band spacing and separation of the complex mixtures under investigation [32, 33, 34]. The optimal identified experimental conditions were screened on several RP columns [29, 35] (See Section 2.2 for details). The Gemini NX column proved to be the best performing one in terms of separation and resolution factor and, therefore, was selected to carry out the chromatographic analyses applied to the real samples. The scanning of microorganism activity and successive selection were made by following the chromatographic trend of some reference peaks during the gradient elution. In Figure 1, the chromatographic profiles obtained from SM fermented with an _L. acidophilus_ strain and _L. helveticus_, in the timeframe 12-400 h, are shown. Based on these preliminary results, and in line with data reported by other authors [13, 36], _L. helveticus_ showed a higher likelihood, and more constant over the time, proteolytic activity than _L. acidophilus_. Figure 2 shows the comparison between the two strains' activities (upon 72 h fermentation), based on area values of reference peaks. Moreover, a comparison of antioxidant and ACEi activities between the two LAB strains, which confirms the higher properties of hydrolysate from _L. helveticus_ (upon 12 h fermentation), is exemplarily reported in Table 1.  
Detailed mean area values \(\pm\) SD (\(n\) = 3) are reported in Table S2 (Supplementary Material). Therefore, _L. helveticus_ stood out as a producer of BPs in fermented milk, further investigations were deepened to explore its use as potential candidate for BPs production. With the aim of maximizing _L. helveticus_ activity, the results obtained from fermented SM were compared with those produced by inoculating BHI and PW as substrates (Figure 3). In this phase, two extraction strategies were appraised: the more \"conventional\" extraction by centrifugation in the presence of TCA at 4 \({}^{\circ}\)C [37], and the extraction by using glass beads [38]. Comparing these two extraction methods, it was possible to determine the potential cell membrane disruption for the recovery of intracellular bioactive compounds. The results highlighted no significant differences between the two methods.