Engineering acid- and heat-stable sweet proteins for beverage applications

Overview

This research program develops modified sweet proteins that retain their high-intensity sweetness when exposed to the acidic pH and elevated temperatures typical of beverage manufacturing and shelf storage. Sweet proteins such as brazzein, curculin, monellin, and thaumatin offer natural, calorie-free sweetness but degrade under low pH and heat, limiting their use in mainstream beverage products. By applying computational design and targeted protein modification, the program aims to produce robust sweetener candidates that withstand harsh processing while preserving taste profile and functional properties. The work targets the beverage industry’s growing need for natural, high-potency sweeteners that survive pasteurization, carbonation, and acidic formulations.

Technical specifications

Approach and methodology:

• Saturation mutagenesis performed on four sweet proteins: brazzein (988 mutations), curculin (2,109 mutations), monellin (1,824 mutations), and thaumatin (3,933 mutations)
• Structural calculations used to predict changes in thermal stability, with computed ΔΔG values validated as reliable predictors of mutation-driven stability changes
• Identification that substituting negatively charged residues with non-negatively charged amino acids is an efficient strategy to improve thermal stability
• Promising mutation sites catalogued for future rational protein modification

Future development pipeline:

• Computational identification of structural elements responsible for instability at low pH and heat
• Rational design targeting degradation-susceptible regions with minimal impact on sweetness
• Gene cloning and expression in bacterial or yeast host systems
• Biophysical characterization of structural changes and stability gains
• Sensory evaluation of sweetness intensity, solubility, and taste profile
• Simulated beverage-condition testing, including low pH and heat treatments analogous to industrial beverage processes
• Time-course monitoring of structural integrity, sweetness retention, and stability using analytical techniques

Technology readiness level

The program is at an early-to-mid stage of development. Saturation mutagenesis screening and computational stability predictions have been completed across four sweet proteins, yielding validated mutation strategies and candidate sites. The next phase involves cloning, expression, biophysical validation, sensory testing, and simulated beverage-condition trials over an estimated three-year timeline. Current readiness corresponds to early proof-of-concept for the design rules, with engineered variants yet to be expressed, characterized, and validated under real-world beverage processing conditions.