## 3 Results and Discussion
### Thermal Stability of the Cell-Free Supernatants  
To assess the suitability of these CFSs for application in food products, different pasteurization and sterilization treatments were explored. Examples of pasteurization treatments are 72 \({}^{\circ}\)C/15 s [82] for milk pasteurization or 90-95 \({}^{\circ}\)C for 15-30 s (High Temperature Short Time--HTST), a common treatment applied to the stabilization of citric juices [83]. Pasteurization treatments assayed at 70 and 90 \({}^{\circ}\)C for even 90 min did not induce a significant (\(p>0.05\)) reduction in the antimicrobial activity of the tested CFSs. Therefore, all the CFSs of the four protective cultures could be considered suitable to be added to products subjected to any kind of pasteurization treatment during their production. In the case of industrial sterilization treatments, three different practices were selected as examples to apply the equations obtained from the laboratory treatments: an in-container sterilization of milk at 110 \({}^{\circ}\)C for 20 min, 3 min botulinum cooking at 121 \({}^{\circ}\)C (commonly applied to canned food), and an autoclaving processing of 15 min at 121 \({}^{\circ}\)C (commonly \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Region** & **Type** & **Most Similar Known Cluster** & **Similarity** \\
**(contig:from-to)** & & & \\ \hline
01:93826-115524 & epilepeptide & thailandstatin A & 10\% \\
01:335810-377228 & other & bacilysin & 100\% \\
01:380214-401825 & sactipeptide & subtilisin A & 100\% \\
01:618860-639606 & CDPS & pulcherriminic acid & 100\% \\
01:922093-973870 & NRP-metallophore, NRPS & bacillibactin & 100\% \\
02:317794-358891 & T3 PKS & 1-carbapen-2-em-3-carboxylic acid & 16\% \\
02:407348-429246 & terpene & & \\
02:502119-579867 & NRPS, beatlactone & fengycin & 100\% \\
02:644080-758852 & transAT-PKS, NRPS, T3 PKS, PKS-like & bacillaene & 100\% \\
03:1-21708 & NRPS & - & - \\
03:430506-451309 & terpene & - & - \\
05:199657-265045 & NRPS & surfactin & 78\% \\
05:395661-418614 & sactipeptide, ranhipeptide & sporulation killing factor & 100\% \\
10:1-6820 & NRPS & peligpiperin & 37\% \\
14:1-1179 & NRPS & & \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Identified secondary metabolite regions in the genome (assembled contigs) of _Bacillus subtilis_ CNTA 517 using AntiSMASH with strictness ‘relaxed’. applied to some materials in contact with food). Treatments at 110 and 121 \({}^{\circ}\)C for 90 min did result in some loss of activity, depending on both the specific treatment and the tested CFS (Table S6). For the in-container sterilization, the estimated loss of antimicrobial activity would range between 8 and 21%, maintaining 79-92% of the activity. For the botulinum cooking, it would result in a loss of activity ranging from 3% to 13%, so 87-97% of the antimicrobial activity would be retained. When the CFSs were autoclaved, the loss of activity ranged between 9 and 28% (Table 5). These results confirmed the findings from various authors regarding the thermal stability of the CFSs (or BLIS) of the bacteria when treated at 100 \({}^{\circ}\)C. However, they diverged when subjected to treatments at 121 \({}^{\circ}\)C. Both [PERSON] et al. [84] and [PERSON] et al. [85] reported no antimicrobial activity after the treatment at 121 \({}^{\circ}\)C. This contrasts with what was obtained with the CFS of _P. acidilactici_ strain CNTA 1059. The results of the thermal stability of CFS _L. plantarum_ CNTA 600 aligned with [PERSON] et al. [86], who described as thermostable the bacteriocin produced by a strain isolated from traditional sourdough even after treatment at 121 \({}^{\circ}\)C for 15 min. However, [PERSON] et al. [87] reported the loss of antimicrobial activity of two _L. plantarum_ strains after treatment at 121 \({}^{\circ}\)C for 15 min. The thermostability of the strain _L. brevis_ CNTA 1374 at 121 \({}^{\circ}\)C was not supported by [PERSON] et al. [87], who did not describe any antimicrobial activity after a treatment of 121 \({}^{\circ}\)C for 15 min of another strain of L. brevis.