Tepache is a spontaneously fermented, pineapple-based beverage whose microbial consortium remains poorly characterised at the level of individual isolates. Spontaneous fermentation remains the norm for comparable beverages across sub-Saharan Africa, and the resulting batch-to-batch variability obstructs standardisation. This study identified bacterial and yeast isolates from traditional and modified tepache formulations, the latter containing Nigerian botanicals, and screened them for the genes carrying out core fermentative reactions. Genomic DNA was extracted from four isolates, and the 16S rRNA gene and internal transcribed spacer (ITS) region were amplified and sequenced. Polymerase chain reaction screening targeted the yeast genes ADH1, PDC and SUC2 and the bacterial lactate dehydrogenase (D-LDH and L-LDH) and scr sucrose-utilisation loci. Sequence analysis identified both yeast isolates as Saccharomyces cerevisiae, matching strains ZP 541 (98.40%) and NCIM3107 (97.60%). The bacterial isolate from the modified beverage matched Bacillus subtilis CICC10148 at 98.70%, whereas that from the traditional beverage matched Lacticaseibacillus sp. at 95.10%, an identity supporting assignment at the genus level only. Both yeast isolates carried ADH1 (179 bp), PDC (195 bp) and SUC2 (approximately 1320 bp), which define a complete genotypic route from sucrose hydrolysis to ethanol. Both bacterial isolates yielded D-LDH and scr genes as multiple fragments, whereas L-LDH amplified only in the traditional-beverage isolate. The isolates therefore carry a gene complement matched to the sucrose-rich substrate, providing a genotypic rationale for their evaluation as defined starter cultures.
