Biodiversity of NSLAB in Traditional Serbian Cheeses and Stepwise Functional Characterization of Selected Autochthonous Lactobacillus Strains Through Technological, Probiotic and Intestinal Epithelial Interaction Assessment

Traditional cheeses represent complex microbial ecosystems and valuable reservoirs of autochthonous lactic acid bacteria (LAB) with functional potential. This study characterized the biodiversity of non-starter LAB (NSLAB) isolated from traditional Serbian raw-milk household cheeses and selected promising autochthonous lactobacilli through a stepwise approach combining a safety assessment, technological characterization, probiotic evaluation and an in vitro interaction with Caco-2 intestinal epithelial cells. From 56 presumptive LAB isolates, RAPD-PCR differentiated 37 distinct genotypes, all of which were taxonomically identified by 16S rRNA gene sequencing. In line with the specific focus of this study on autochthonous lactobacilli, all seven isolates taxonomically assigned to this genus were selected for further characterization. Safety assessment showed that two strains (28.6%) formed biofilms and were excluded, whereas the remaining five isolates complied with EFSA antibiotic susceptibility criteria. These candidates exhibited strain-dependent degradation of αs1- and β-casein fractions and moderate milk acidification. Four strains survived simulated gastrointestinal transit, showed antagonistic activity against foodborne pathogens and adhered to Caco-2 cells without inducing cytotoxicity. Lactiplantibacillus plantarum ZI237 showed the broadest autophagy-related transcriptional response and NQO1 induction. Limosilactibacillus fermentum KT upregulated MUC2, MUC5, NQO1 and HBD-1 but reduced Occludin protein abundance, indicating a mixed epithelial response. Both strains reduced p62 protein levels, supporting modulation of autophagy-related pathways. Overall, Lpb. plantarum ZI237 emerged as the most promising candidate, whereas Lm. fermentum KT requires further evaluation before application in functional dairy products.