Study on the Thermal Stability of the Sweet-Tasting Protein Brazzein Based on Its Structure–Sweetness Relationship

Brazzein (Brz) is a sweet-tasting protein composed of 54 amino acids and is considered as a potential sugar substitute. The current methods for obtaining brazzein are complicated, and limited information is available regarding its thermal stability. In this study, we successfully expressed recombinant brazzein, achieving a sweetness threshold of 15.2 μg/mL. Subsequently, we conducted heat treatments at temperatures of 80, 90, 95, and 100 °C for a duration of 2 h to investigate the structural changes in the protein. Furthermore, we employed hydrogen–deuterium exchange coupled to mass spectrometry (HDX-MS) to analyze the effect of heating on the protein structure–sweetness relationships. Our results indicated that the thermal inactivation process primarily affects residues 6–14 and 36–45 of brazzein, especially key residues Tyr8, Tyr11, Ser14, Glu36, and Arg43, which are closely associated with its sweetness. These findings have significant implications for improving the thermal stability of brazzein.