Determination of Fermentable Hidden Sugars in Energy Drinks via Crabtree-Based Yeast Bioassay

This study aimed to evaluate the actual fermentable sugar content of a commercially available energy drink by employing the Crabtree effect in Saccharomyces cerevisiae as a biological indicator system. To eliminate confounding effects, caffeine was removed through liquid–liquid extraction prior to experimentation. Yeast cultures were exposed to five different conditions: low glucose (5 g 100 mL⁻¹), high glucose (40 g 100 mL⁻¹), labeled glucose equivalent to the beverage (11 g 100 mL⁻¹), caffeine-removed energy drink, and a glucose-free control. Real-time monitoring of oxygen consumption, carbon dioxide release, pH dynamics, and ethanol formation was performed using a tri-sensor fermentation monitoring system. Intracellular redox balance was assessed through NADH/NAD⁺ quantification at 0.5 min intervals. The low-glucose group exhibited limited oxygen depletion and moderate CO₂ production, consistent with predominantly respiratory metabolism. In contrast, the high-glucose group showed rapid oxygen depletion and pronounced CO₂ accumulation, confirming a switch to fermentative metabolism. The labeled-glucose condition displayed an intermediate response, characterized by partial oxygen utilization followed by moderate fermentation. Strikingly, the caffeine-removed energy drink induced the strongest fermentative profile, with CO₂ production reaching 91 %, pH dropping to 5.4, and ethanol formation occurring at 2.5 min despite oxygen levels above 50 %. Furthermore, the NADH/NAD⁺ ratio in this group increased earlier and more sharply than in all other conditions, indicating glycolytic flux exceeding mitochondrial capacity. These findings demonstrate that the tested energy drink contained considerably higher fermentable sugar levels than what is declared on its label. The integration of gas exchange data, pH measurements, ethanol detection, and redox balance strongly support the applicability of the Crabtree effect as a functional bioassay for hidden sugar detection in beverages. This approach not only complements chemical analyses but also offers a biologically relevant method for assessing metabolic impact. From a regulatory perspective, the results highlight the necessity for stricter labeling practices and underscore potential public health risks associated with undeclared sugars in energy drinks.