Lactobacillus helveticus 34.9 was isolated from a sample of Romanian home-made fermented milk, producing both surface layer proteins and a class III bacteriocin. The present study aimed to investigate the biological and functional role of the S-layer in correlation with its probiotic properties. The presence of S-layer proteins resulted in various degrees of co-aggregation of L. helveticus 34.9 with pathogens and with other lactic acid bacteria, but the removal of these proteins reduced the co-aggregation with all the tested strains. Moreover, the S-layer proved to be involved in cell wall hydrophobicity and cellular protection during freeze-drying. In the simulated passage through the gastrointestinal tract, S-layer depleted cells exhibited increased vulnerability, with greater viability loss in low pH and pepsin treatment compared to control cells. Subsequently, in the small intestine simulation, these cells lost all viability, underscoring the vital role of extracellular proteins for cell protection. The morphological effects of these treatments were observed by scanning electron microscopy. Severe structural damage was noticed when the S-layer was absent, including loss of cell shape and integrity as well as many ghost cells emptied of their content. Finally, the elimination of surface proteins reduced the interaction between L. helveticus 34.9 and mammalian cells.

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 Lactiplantibacillus, Lactococcus, and Leuconostoc 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 ribA, gad, and α-amy, while genes associated with bacteriocin (nisA, pln) and folate (folK) 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 Lactiplantibacillus plantarum and Lactococcus lactis strains, exhibiting strong inhibitory effects. Bacteriocin production was confirmed for three Lact. lactis 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.

Romanian borş, a traditional fermented wheat bran beverage, is produced through spontaneous fermentation and represents a complex microbial ecosystem. Despite its cultural importance and presumed health benefits, its microbial ecology and functional potential remain poorly characterized. The present study aimed to elucidate the microbial community structure of borş and link it to functional traits relevant to fermentation performance and food functionality by integrating culture-independent sequencing with culture-dependent isolation and functional characterization. A total of 32 borş samples (12 commercial and 20 homemade) were analyzed. Amplicon-based sequencing revealed a microbiome dominated by lactic acid bacteria (LAB), with lactobacilli accounting for the majority of the bacterial communities and Lactobacillus amylolyticus being identified as the most prevalent and abundant species. The yeast communities were mainly composed of fermentative taxa, including Pichia kudriavzevii and Kluyveromyces marxianus . Lactobacillus amylolyticus and P. kudriavzevii were also the most frequently isolated species among bacteria and yeasts, respectively. These results highlighted a strong adaptation of the microbial isolates to starch-rich cereal substrates and underscored the central role of these microorganisms in wheat bran fermentation for borş production. Whereas the sequencing-based analyses showed no significant differences in overall diversity between the commercial and homemade borş samples, the cultivation-based results indicated a higher bacterial richness in the commercial products. Notably, the culture-dependent method captured substantially fewer taxa, highlighting the complementary nature of the two approaches. Of a total of 101 bacterial strains (88 LAB and 13 acetic acid bacteria) isolated, many exhibited rapid growth and strong acidification capacity, reaching pH values below 4.5 within 12 h. A functional screening revealed that 21 % of these strains displayed α-amylase activity, 65 % phytase activity, and 50 % β-glucosidase activity, highlighting their capacity to metabolize cereal substrates and enhance the nutrient availability of borş. All strains showed antibacterial activity against at least one indicator bacterium tested, with a universal inhibition of Listeria monocytogenes . Overall, Romanian borş harbored a lactic acid bacteria-dominated core microbiome with a significant functional diversity. These findings underscored its potential as a rich source of functional and technologically important strains for application in starter and protective culture development.