## 2 Materials and Methods
### Thermal Stability of the Cell-Free Supernatants  
The heat resistance of the CFSs was measured to know whether the antimicrobial activity of CFSs would be maintained if they were added to foods that would subsequently receive a pasteurization or sterilization treatment (commonly used by the food industry). These treatments were used as a reference to select the intensity (temperature and time) of the different heat treatments (from 10 to 120 min at 70, 90, 110, and 120 \({}^{\circ}\)C).  
For low temperatures (70 \({}^{\circ}\)C and 90 \({}^{\circ}\)C), heat treatments were carried out in a thermoblock (Eppendorf, Hamburg, Germany), and, for high temperatures (110 \({}^{\circ}\)C and 121 \({}^{\circ}\)C), an oil bath (Leyro Instruments, San Sebastian de los Reyes, Spain) was used. A small portion of CFS was added in preheated liquid to ensure the initial temperature, which was measured by the temperature sensor Checktemp 1 HI98509 (HANNA instruments, Limena, Italia). After the preset intervals, the eppendorfs or vials were removed and introduced in cold water. Afterward, the indicator micro-organisms _L. monocytogenes_ and _L. parabuchneri_ were exposed to the treated CFSs following the agar well diffusion methodology.  
Finally, taking as a reference the halo obtained by agar well diffusion assay (Section 2.1.1) of the untreated CFSs against _L. monocytogenes_ and _L. parabuchneri_, a formula (Equation (2)) was applied to determine the percentage of antimicrobial activity maintained by each CFS following exposure to various combinations of temperatures and durations.  
\[\%\ loss\ antimicrobial\ activity=\frac{\textit{halo diameter }\left(mm\right)_{\textit{before heat treatment}}-\textit{halo diameter }\left(mm\right)_{\textit{after heat treatment}}}{\textit{halo diameter }\left(mm\right)_{\textit{before treatment}}}\times 100 \tag{2}\]  
Following the representation of the percentage of the retained antimicrobial activity of the CFS throughout time at each temperature, a linear regression was obtained to estimate the loss of antimicrobial activity of the CFS under commercial heat treatments applied by the food industry.  
Whole-Genome Sequencing of the Strains, Identification of AMR Genes, and Identification of Biosynthetic Gene Clusters  
The sequenced genome of the _Bacillus subtilis_ CNTA 517 strain was obtained through whole-genome sequencing (WGS) using an Illumina iSeq platform (NASERTIC, Pamplona, Spain). Genomic DNA was extracted from 1 mL of fresh culture using the GenElute(tm) bacterial Genomic DNA kit (Merck KGaA), and tagmented with the Nextera XT DNA library prep kit. The 2 \(\times\) 150-bp paired-end sequencing reads generated were pre-processed by removing adapters, short reads (<140 bp), and poor quality reads (Phred score < 28) using Trimmomatic v.0.39 [37]. After the quality of the filtered clean reads was checked with FastQC v.0.12.1 [38], de novo genome assembly was performed with Shovill v.1.1.0 [39] using the SPAdes assembler with default parameters. Quast v.5.2.0. [40] was used to evaluate the quality of the assembly. Then, 24 long contigs (>500 base pairs) were generated, with 99.24% of reads successfully mapped to the assembly.  
The assembled contigs in FASTA format were screened for antimicrobial resistance genes (ARGs) using the ABRicate v. 1.0.1 tool [41] and the CARD [42,43] and ResFinder [44,45] with a minimum identify and coverage values of 80%. The assembled contigs were additionally screened for mobile genetic elements using the MobileElementFinder v.1.0.3 tool [46,47]. Finally, antiSMASH v.6.1.1. [48] was used to predict the type and location of secondary metabolite biosynthesis gene clusters within the assembled genomes.