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 stimulant, antioxidant, and cardioprotective properties. In this study, an ultrasound-assisted extraction (UAE) method was developed and optimized for methylxanthine recovery from commercial cocoa powders and cocoa-based drinking chocolate mixes (powdered products) 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. In parallel, three samples (C-1, C-2, and C-4) were selected for in vitro digestion (INFOGEST 2.0) to assess the bioaccessibility and stability of bioactive compounds during gastrointestinal transit. Across the oral, gastric and intestinal phases, accumulative methylxanthine bioaccessibility reached more than 85%, indicating matrix-dependent release and possible co-extracted modulators. Antioxidant capacity, measured using DPPH assay, was high prior to digestion and showed phase-dependent changes during INFOGEST digestion, with lower values in the oral phase followed by higher measured antioxidant capacity in the gastric and/or intestinal phases, consistent with digestion-driven release of antioxidant compounds. The present study highlights the differential bioaccessibility of methylxanthines and the pivotal role of formulation factors in determining their gastrointestinal fate. Overall, the study establishes a sustainable, food-grade UAE workflow as a scalable starting point for obtaining methylxanthine-enriched cocoa extracts, while providing mechanistic insight into methylxanthine bioaccessibility in cocoa powder-based products during INFOGEST digestion; further work is required to translate these conditions into nutraceutical/functional ingredients (e.g., stability during concentration/drying, sensory and regulatory aspects).