Prior to optimization, the experimental fermentation time was defined as 36h. The experiments were carried out at the center point at 12, 24, 36, and 48 h. The results indicated that yeast biomass increases with increased fermentation time. However, the activity of phytase when the yeast was cultured in both substrates increased only up to 36 h (0.55 ± 0.01 U/mL and 0.051 ± 0.004 in sodium phytate and 4-NPP substrates, respectively) and decreased significantly after 48 h.