Spontaneous fermentation of motoho, a Southern African non-alcoholic sorghum beverage, results in products with inconsistent microbiological and sensory quality. We aimed to identify the microorganisms involved in the fermentation of motoho by using culture-dependent techniques as well as culture-independent
polymerase chain reaction (PCR) screening and matrix-assisted laser esorption/ionisation time-of-flight analysis (MALDI-TOF). Lactobacillus, Candida, Rhodotorula and Geotrichum species were identified. Sodium dodecyl sulfate olyacrylamide gel electrophoresis (SDS-PAGE) was used to evaluate the protein profiles of the isolated Lactobacillus species which produced protein bands of 14 Da to 160 kDa, similar to those of other lactic acid bacteria isolated from various foods. A sensory panel evaluated and found significant differences (p<0.05) between the mouth feel, aroma and flavour of the traditional and modified motoho, with the latter being preferred. The microorganisms identified in this study could be used as starter cultures to optimise upscaled production of motoho. SIGNIFICANCE : Traditionally fermented products have variable quality and the microorganisms isolated in this study could be used to decrease the variability in this fermented sorghum beverage.