Purpose: Scientific information regarding the microbial content and functional aspects of fermented beverages traditionally produced in certain parts of Europe are scarce. However, such products are believed to have some health benefits and might contain functional bacterial strains, such as probiotics. The aim of the study was to identify such lactic acid bacteria strains isolated from water kefir and, for the first time, from braga, a Romanian fermented beverage made of cereals. Methods: Lactic acid bacteria (LAB) were identified to species level based on (GTG)₅-PCR fingerprinting and 16S rRNA gene sequencing. Selected strains were screened for their antibacterial activity and probiotic potential. Results: Eight isolates belonging to seven Lactobacillus species were recovered from the two drinks. The identification of LAB involved in the fermentation of braga (Lactobacillus plantarum, Lactobacillus fermentum, and Lactobacillus delbrueckii) is firstly reported here. Five of the Lactobacillus isolates showed antibacterial activity against pathogenic bacteria, including Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, and Salmonella enterica. Moreover, most of them showed a good resistance to pH 2.5 and some survived at high concentrations of bile salts (up to 2%). Two L. plantarum isolates were able to inhibit all the indicator strains, and showed the best viability (about 70%) after a sequential treatment simulating the passage through the gastrointestinal tract. Conclusion: Based on the results, the most promising candidates for designing new probiotic products are: L. plantarum BR9 from braga and L. plantarum CR1 from water kefir.

At present, there is an increasing interest in beverages of non-dairy origin, as alternatives to those based on milk, but having similar health-promoting properties. Fermentation with specific bacteria or consortia may enhance the functionality of these products. In our study, selected lactic acid bacteria, that have been previously shown to possess functional properties (antimicrobial activity, probiotic potential), were used for the fermentation of wheat bran combined with root vegetables. Strains were investigated for their safety, while the obtained beverages were characterized in terms of microbial content, physical, chemical, nutritional, and functional properties. None of the strains harbors virulence genes, but all of them possess genes for survival at low pH, starch metabolism, and vitamin biosynthesis. Three strains (Lactiplantibacillus plantarum BR9, L. plantarum P35, and Lactobacillus acidophilus IBB801) and two substrates (5% wheat bran with 10% red beetroot/carrots) were selected based on a preliminary assessment of the beverage’s sensory acceptability. These strains showed good growth and stability over time in the stored beverages. No enterobacteria were detected at the end of fermentations, while the final pH was, in most cases, below 3.5. Free phenolics, flavonoids, and DPPH scavenging effect increased during fermentation in all drinks, reaching 24h values that were much higher than in the unfermented substrates. Most of the obtained drinks were able to prevent the growth of certain pathogens, including Listeria monocytogenes ATCC 19111, Salmonella enterica ATCC 14028, Staphylococcus aureus ATCC 25923, and Escherichia coli ATCC 25922. The obtained beverages would combine the nutritiveness of the raw ingredients with the beneficial effect of fermentation (increasing shelf life, health-promoting effect, pleasant flavor, etc.). They would also fill a gap in the non-dairy probiotics sector, which is constantly increasing due to the increasing number of vegan people or people that cannot consume dairy products.

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.

To characterize S-layer proteins produced by four lactobacilli isolated from Romanian artisan fermented products. Four lactobacilli strains have been shown to produce S-layer proteins, both under optimal and stressfull conditions. The presence of S-layer proteins was confirmed by transmission electron microscopy. Removal of S-layer proteins caused a loss of the bacterial resistance to stress conditions and of the autoaggregation ability. Liquid chromatography–mass spectrometry analysis identified peptides corresponding to Slp M sequence in case of Levilactobacillus brevis 403, and peptides corresponding to Slp A sequence in case of Lactobacillus helveticus 34.9. The analysis confirmed molecular masses of ∼51 and 48 kDa, respectively, for the two proteins, and gave information about their pI, of about 9.4–9.6. A specific PCR amplification was obtained for the genome of Lact. helveticus 34.9 with slpA primers, and the amplicon sequence was 95.31% identical to slpA gene. Our findings indicate that certain environmental stress conditions can induce the S-layer production, which helps the producing cells to survive under unfavorable conditions.