Terroir Rediscovered: A Polyphasic Approach to Autochthonous Lactic Acid Bacteria as Functional and Microbial Heritage Resources in Portuguese PDO Cheeses

Portuguese Protected Designation of Origin (PDO) cheeses from Azeitão and Nisa harbor unique autochthonous microbial communities integral to their terroir, sensory identity, and authenticity. Rediscovering and preserving this microbial heritage is essential for safeguarding traditional cheesemaking practices and unlocking the biotechnological potential of indigenous microorganisms. This study used a polyphasic approach to isolate, preserve, and functionally characterize lactic acid bacteria (LAB) from these cheeses, aiming both to conserve autochthonous microbial resources and evaluate their suitability for food applications and preliminary probiotic-related traits. From 2304 bacterial isolates, 119 representative LAB were selected and screened for safety based on hemolytic and gelatinolytic activities and antimicrobial susceptibility. Technological potential was assessed through acidification, proteolytic and lipolytic activities, and antagonism against foodborne pathogens, while probiotic traits were evaluated via tolerance to acidic pH and bile salts, survival under simulated gastrointestinal conditions, and auto- and co-aggregation. Thirty-nine isolates exhibited desirable technological traits, including proteolytic and lipolytic activities and broad-spectrum pathogen antagonism. Among these, three isolates (two Leuconostoc mesenteroides and one Enterococcus hirae) combined desirable technological traits with survival under simulated gastrointestinal conditions, representing preliminary multifunctional candidates. These findings reveal Azeitão and Nisa PDO cheeses as living reservoirs of terroir, where indigenous LAB rediscovered through this approach represent both a key component of Portugal’s microbial heritage and a source of functional cultures for sustainable dairy innovation.