Simulated Gastrointestinal Digestion of Cocoa Powder (INFOGEST): Methylxanthine Bioaccessibility, and Antioxidant Properties

Abstract

Cocoa (Theobroma cacao L.) is a rich source of bioactive compounds, including methylxanthines (theobromine and caffeine) and polyphenols, which are known for their stimulants, antioxidants, and cardioprotective properties. In this study, an ultrasound-assisted extraction (UAE) method was developed and optimized for methylxanthine recovery from commercial cocoa samples using a Box-Behnken design and response surface methodology (BBD-RSM). The optimal conditions for this experiment were as follows: 67.81% EtOH, 50.10 °C, 36.02% amplitude, 0.3 s⁻¹ cycle, and 10 minutes of extraction. Three samples (C-1, C-2, and C-4) were selected for in vitro digestion (INFOGEST 2.0) to assess the bioavailability and stability of bioactive compounds during gastrointestinal transit. Across the oral, gastric and intestinal phases, accumulative methylxanthine bioavailability reached more than 85%, indicating matrix-dependent release and possible co-extracted modulators. Antioxidant capacity, measured by DPPH and ABTS assays, was high in the pre-digestive phase but decreased significantly after digestion, highlighting the impact of gastrointestinal conditions on the functionality and stability of cocoa bioactives. The present study highlights the differential bioaccessibility of metylxantines and the pivotal role of formulation factors in determining their gastrointestinal fate. The findings of this study provide a robust foundation for substantiating the formulations of cocoa-based products, underscoring the significance of conducting comprehensive studies that encompass not only the content of bioactive compounds but also their bioaccessibility.