| Summary: | The undefined mixed starter culture (UMSC) is used in the manufacture of cheeses. Deciphering UMSC microbial diversity is important to optimize industrial processes. The UMSC was studied using culture-dependent and culture-independent based methods. MALDI-TOF MS enabled identification of species primarily from the <i>Lactococcus</i> genus. Comparisons of carbohydrate metabolism profiles allowed to discriminate five phenotypes of <i>Lactococcus</i> (<i>n</i> = 26/1616). The 16S sequences analysis (V1–V3, V3–V4 regions) clustered the UMSC microbial diversity into two <i>Lactococcus</i> operational taxonomic units (OTUs). These clustering results were improved with the DADA2 algorithm on the housekeeping <i>purR</i> sequences. Five <i>L. lactis</i> variants were detected among the UMSC. The whole-genome sequencing of six isolates allowed for the identification of the <i>lactis</i> subspecies using Illumina<sup>®</sup> (<i>n</i> = 5) and Pacbio<sup>®</sup> (<i>n</i> = 1) technologies. Kegg analysis confirmed the <i>L. lactis</i> species-specific niche adaptations and highlighted a progressive gene pseudogenization. Then, agar spot tests and agar well diffusion assays were used to assess UMSC antimicrobial activities. Of note, isolate supernatants (<i>n</i> = 34/1616) were shown to inhibit the growth of <i>Salmonella</i> ser. Typhimurium CIP 104115, <i>Lactobacillus sakei</i> CIP 104494, <i>Staphylococcus aureus</i> DSMZ 13661, <i>Enterococcus faecalis</i> CIP103015 and <i>Listeria innocua</i> CIP 80.11. Collectively, these results provide insightful information about UMSC <i>L. lactis</i> diversity and revealed a potential application as a bio-protective starter culture.
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