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The aim of this present study was to optimize the fermentation conditions (time and temperature) of amasi (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 amasi 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 (p ≤ 0.05) changes with increasing fermentation time and temperature. Numerical optimization was used to obtain the optimal fermentation conditions for amasi; 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 Lactobacillaceae being the most predominant in amasi, while in raw milk, Prevotellaceae 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 amasi.

Influence of Fermentation Conditions (Temperature and Time) on the Physicochemical Properties and Bacteria Microbiota of Amasi