The aim of this study was to use different concentrations of lemon juice and honey to improve the formulation of a green tea water kefir (GTWK) beverage by applying a central composite design (CCD). Honey’s concentration was 10–50% and lemon juice concentration was 1–5%, these were used as the independent factors, whereas pH, bacteria and yeasts’ count, total phenolic content, % DPPH^. scavenging activity, and overall acceptability were used as the dependent factors. The optimal concentration of honey and lemon juice for highest microbial count, antioxidant activities and overall acceptability was 42.85% and 1.771%, respectively. The analysis of variance revealed that the model was well-fitting, with R² ranging from 87.27% to 96.95%, adj-R² ranging from 78.17% to 94.26% and a non-significant lack of fits. The optimized fermented beverage showed antibacterial potential against Echerichia coli ATCC11229, Staphylococcus aureus ATCC6538 and Salmonella typhimirium ATCC14028 strains. The anti-inflammatory activity was evaluated on CaCo-2 and RAW 264.7 cells. According to ELISA assay, a significant decrease (p < 0.05) in TNF-α concentration was found after inflammatory stimulation, from 1205.41 ± 55.87 pg/mL to 478.17 ± 69.12 pg/mL.

Based on nucleotide sequences, we re-identified representative samples of Phlebotomus perfiliewi originating from two different biogeographical areas of Tunisia, whose populations had previously been identified based on morphological criteria. A partial region of the mitochondrial DNA cytochrome b gene was targeted, and sandfly species was determined by analogy with DNA sequences available in the GenBank database via a BLAST analysis, taking into account the query coverage and percentage identity. The recognized species presents the most substantial homology with the analyzed sequence. The results of the molecular identification showed complete agreement with the morphological identifications, and Phlebotomus perfiliewi is genetically variable.

This study aimed to isolate and characterize microorganisms from water kefir beverage for their functional properties. Five lactic acid bacteria (LAB) strains were isolated: three Leuconostoc citreum strains (LB4, LB6, LB13) and two Lactococcus lactis strains (LB5, LB25), identified via 16S rRNA sequencing, along with three Saccharomyces cerevisiae yeast strains (Y7, Y9, Y10), confirmed by 18S rDNA sequencing. Due to the high genetic and phenotypic similarity within each species, one representative strain from each (LB4, LB5, Y9) was selected for further analysis. These strains showed potential probiotic properties, including tolerance to acid and bile, high auto-aggregation, and hydrophobicity. The LAB strains were sensitive to gentamicin, and their supernatants inhibited the growth of tested pathogenic bacteria. The cumulative probiotic potential (CPP) scores were 93.33% for Lc. citreum LB4 and L. lactis LB5, and 100% for S. cerevisiae Y9. Furthermore, the fermentation potential of these strains was evaluated in a green tea beverage using three co-culture formulations. Among the formulations tested, the BF1 beverage, fermented by F1 (40% LB4, 40% LB5, and 20% Y9), demonstrated optimal physicochemical, microbiological, and sensory properties. Notably, while the individual strains did not show anti-inflammatory activity, the BF1 beverage formulation exhibited this effect, suggesting a synergistic interaction during fermentation.