Screening of Functional Properties of Lactic Acid Bacteria Isolated from Animal Rennets and Their Associated Cheeses and Whey

This study investigated the diversity and functional potential of lactic acid bacteria isolated from traditional lamb rennet, cheese, and whey collected from seven artisanal sheep farms in southern Romania. A total of 31 samples were analyzed, yielding 118 Gram-positive, catalase-negative isolates. Following dereplication by rep-PCR and 16S rRNA gene sequencing, 63 unique strains were identified across nine genera, with <i>Lactiplantibacillus</i>, <i>Lactococcus</i>, and <i>Leuconostoc</i> being the most prevalent. Strain distribution varied by sample type and manufacturer, with rennet and whey showing greater species diversity than cheese. Technological characterization showed strain-dependent differences in acidification and growth in cow's and goat's milk. Genetic screening revealed a high prevalence of functional genes such as <i>rib</i>A, <i>gad</i>, and <i>α-amy</i>, while genes associated with bacteriocin (<i>nis</i>A, <i>pln</i>) and folate (<i>fol</i>K) production were less common. Most strains carried multiple functional genes, indicating a genetic potential for diverse functional traits. Antibacterial activity assays demonstrated that nearly all strains inhibited at least three indicator pathogens, with ten strains, particularly <i>Lactiplantibacillus plantarum</i> and <i>Lactococcus lactis</i> strains, exhibiting strong inhibitory effects. Bacteriocin production was confirmed for three <i>Lact. lactis</i> strains. Exopolysaccharide (EPS) production was confirmed in two strains, with yields varying by growth medium and sucrose supplementation. Overall, the results underscore the rich microbial diversity and promising biotechnological potential of LAB from traditional Romanian dairy ecosystems, supporting their use in food fermentation and functional product development.