## 4 Conclusions  
Based on the _in vitro_ tests conducted, the strains _P. acidilactici_ CNTA 1059, _L. plantarum_ CNTA 600, _L. brevis_ CNTA 1374, and _B. subtilis_ CNTA 517 seem to fulfil the necessary requirements to be used as protective cultures. These strains are properly identified to the species level, are listed in the Qualified Presumption of Safety (QPS) list, do not exhibit acquired resistance to target antibiotics, and do not display toxicgenicity upon contact with human epithelial cells. Instead of using the protective cultures, this study proposes the use of the CFS, showing their potential as biopreservatives in the food industry. The antimicrobial spectrum exhibited by the cell-free supernatants of the selected strains was primarily directed against Gram-positive bacteria, encompassing both pathogenic (_L. monocytogenes_) and spoilage (_L. parabuchneri_) micro-organisms. The observed antimicrobial activity in all cases can be attributed to the production of bacteriocin-like inhibition substances (BLISs) (and/or lipopeptides in the specific case of _B. subtilis_ CNTA 517). These compounds demonstrate stability across various pH levels and temperature fluctuations, making them amenable to be used in products subjected \begin{table}
\begin{tabular}{c c c c} \hline \hline  & \begin{tabular}{c} **\% of Antimicrobial Activity** \\ **Cost after the In-Container** \\ **Milk Sterilization Treatment** \\ **(110 \({}^{\circ}\)C/20 min)** \\ \end{tabular} & \
\begin{tabular}{c} **\% of Antimicrobial Activity** \\ **Lost after Botulinum Cooking** \\ **(121 \({}^{\circ}\)C/3 min)** \\ \end{tabular} &
\begin{tabular}{c} **\% of Antimicrobial Activity** \\ **Lost after Autoclaved** \\ **Treatment (121 \({}^{\circ}\)C/15 min)** \\ \end{tabular} \\ \hline _Policoccus acidilactici_ CNTA 1059 & 8.18 & 3.73 & 11.06 \\ _Lacilplantobacillus plantarum_ CNTA 600 & 13.92 & 13.12 & 22.17 \\ _Levilacobacillus brevis_ CNTA 1374 & 8.36 & 4.73 & 9.02 \\ _Bacillus subtilis_ CNTA 517 & 21.67 & 9.44 & 28.70 \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Estimation of the lost antimicrobial activity of the cell-free supernatants (CFSs) after two examples of commercial sterilization treatments. to thermal preservation treatment, even at sterilization temperatures. Nevertheless, it is essential that we note that these results solely depict the _in vitro_ capacity, with the subsequent step involving the testing of these CFSs in diverse food matrices to extend shelf life and potentially reduce or eliminate the need for additives or intense treatments. Therefore, further research is imperative to position CFSs as a viable clean-label alternative for food companies. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app14104016/s1](https://www.mdpi.com/article/10.3390/app14104016/s1)]([https://www.mdpi.com/article/10.3390/app14104016/s1](https://www.mdpi.com/article/10.3390/app14104016/s1)), Figure S1: Depiction of the inhibition zone achieved by each tested culture-free supernatant; Figure S2: Growth over time of the selected indicator microorganisms in presence of the CFS; Figure S3: The cytotoxic effect of _Bacillus subtilis_ CNTA 517 cell-free supernatant (CFS); Table S1: Potential protective culture species from CNTA collection employed for the preliminary screening; Table S2: Potential protective culture micro-organisms' growth conditions; Table S3: Pathogenic and spoilage micro-organisms chosen as indicator strains and growth conditions; Table S4: Antimicrobial activity of the cell-free supernatants (CFSs) against pathogenic and spoilage strains with a well diameter of 7.20 mm; Table S5. Antibiotic resistance genes identified in _Bacillus subtilis_ CNTA 517; Table S6: Antimicrobial activity of the cell-free supernatants (CFSs) after a heat treatment of 110 \({}^{\circ}\)C and 121 \({}^{\circ}\)C at different times; Table S7. Lost antimicrobial activity of the cell-free supernatants (CFSs) after heat treatments at 110 and 121 \({}^{\circ}\)C.  
Conceptualization, N.G.-M., L.E., R.V.