Phenolic Profile and Antioxidant Activity of Chocolates Supplemented with Bioactive Ingredients

The growing demand for functional foods has stimulated the development of chocolate matrices enriched with bioactive ingredients. This study aimed to optimize the extraction of phenolic compounds using hydro-organic solvents of different polarities, characterize the phenolic and alkaloid profile of distinct chocolate formulations by HPLC-DAD, and evaluate whether fortification with microencapsulated vitamins E and D, zinc, and omega-3 fatty acids (EPA and DHA), either alone or combined with partial replacement of cocoa butter by a structuring oil, enhances the extractable bioactive compounds and in vitro antioxidant activity. Five formulations were evaluated: control chocolate (C), choc-olate containing vitamin microcapsules (T1), chocolate with DHA/EPA microcapsules (T2), lipid-modified chocolate with structuring oil (T3), and chocolate combining microcapsules with lipid modification (T4). Phenolic compounds were extracted using 50% ethanol, 70% methanol, and 70% acetone. Among the tested solvents, 70% methanol showed the highest extraction efficiency, enabling broader detection of bioactive compounds. HPLC-DAD analysis revealed compounds characteristic of cocoa-based matrices, including epicatechin, gallic acid, vanillin, and traces of procyanidins, as well as the methylxanthine alkaloids theobromine and caffeine. The T4 formulation showed a greater abundance of extractable compounds compared to the other formulations. Antioxidant activity was evaluated using DPPH radical scavenging and β-carotene/linoleic acid bleaching assays. T4 exhibited the highest antioxidant performance, with 21.8% DPPH inhibition (EC₅₀ = 2.5 mg/mL) and the highest optical density in the β-carotene assay, indicating enhanced protection against lipid peroxidation. These findings demonstrate that the combination of microencapsula-tion and lipid phase modification improves antioxidant functionality and supports the development of value-added functional chocolates.