Identification and Characterisation of Lactic Acid Bacteria from “Torta del Casar”, a Semi-Soft Artisanal Cheese

“Torta del Casar” is a traditional Spanish cheese made from raw ewe’s milk under Protected Designation of Origin (PDO) regulations. Its unique characteristics result from the use of vegetable rennet and an abundant indigenous microbiota, primarily lactic acid bacteria (LAB). Since PDO rules permit only autochthonous microorganisms, identifying specific LAB strains is a fundamental first step for developing starter and protective cultures. This study aimed to characterise the LAB population of 22 cheeses from different industries and assess batch-to-batch variability. Physicochemical analysis revealed that some batches exceeded PDO pH limits, highlighting the potential need for standardised acidification. Species-level identification was performed via 16S rRNA sequencing, while RAPD-PCR was used for strain-level differentiation. Lactiplantibacillus plantarum was the predominant species, followed by Leuconostoc mesenteroides. RAPD-PCR differentiated a total of 20 strains across all species, revealing high intra-specific diversity. Leu. mesenteroides exhibited higher variability compared to the more homogenous clusters found within Lpb. plantarum. Although batches from the same industry showed low strain overlap, specific strains of Lpb. plantarum (G5-3, G4-1) and Leu. mesenteroides (S2-2, G5-2, L4-1) were shared across different batches or industries. These autochthonous strains are promising candidates for further evaluation as starter cultures, since they could potentially contribute to product standardisation and safety while preserving the traditional sensory profile of “Torta del Casar”.