Optimization of pH and Temperature in a Simplified Peptone-Based Medium for Enhanced Recombinant Brazzein Expression in Pichia pastoris

Brazzein is a sweet-tasting protein with high stability across a wide range of pH and temperature conditions. This study aimed to develop a simplified peptone-based medium (PSM) for the recombinant expression of brazzein in Pichia pastoris X-33 and to evaluate the effect of two inoculum concentrations (5%, 10%, and 15%) on cell growth and protein production in flask fermentations. Subsequently, fermentation was scaled up to a 2 L bioreactor using PSM and a 10% inoculum, achieving a yield of 0.196 g·L−1 after 216 h of induction. These results demonstrate that the PSM medium promotes robust biomass growth and efficient brazzein expression, representing a cost-effective alternative to conventional complex media. Additionally, the effect of pH (5.0, 5.5, and 6.0) and temperature (20, 25, and 28 °C) on brazzein production was evaluated, revealing that fermentation at pH 5.0 and 28 °C resulted in the highest protein concentration (0.422 g·L−1, unpurified). Finally, kinetic models based on the Monod and Luedeking–Piret equations were developed to describe the relationship between biomass formation, substrate consumption, and recombinant protein production.