| Summary: | The aim of this present study was to optimize the fermentation conditions (time and temperature) of <i>amasi</i> (a Southern African fermented dairy product) using response surface methodology (RSM), and to determine the physicochemical properties, as well as the microbial composition, using next generation sequencing. Fermentation time and temperature were optimized to produce different <i>amasi</i> samples and different parameters, including pH, total soluble solids (TSS), total titratable acids (TTA), and consistency. All the variables studied were found to show significant (<i>p</i> ≤ 0.05) changes with increasing fermentation time and temperature. Numerical optimization was used to obtain the optimal fermentation conditions for <i>amasi</i>; based on RSM, it was 32 °C for 140 h, while with k-means clustering, it was 25 °C for 120 h. Under both conditions for the optimal samples, the pH reduced from 6.64 to 3.99, TTA increased from 0.02 to 0.11 (% lactic acid), TSS decreased from 9.47 to 6.67 °Brix, and the consistency decreased from 23 to 15.23 cm/min. Most of the identified bacteria were linked to lactic acid bacteria, with the family <i>Lactobacillaceae</i> being the most predominant in <i>amasi, </i>while in raw milk, <i>Prevotellaceae </i>was the most abundant. The fermentation conditions (time and temperature) had a significant influence on the parameters investigated in this study. Results of this study could provide information for the commercialization of quality <i>amasi</i>.
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