Valorization of Cocoa Bean Shell into High-value Bioactive Extracts as Functional Food Ingredients Using Green Extraction Techniques: Enzyme-assisted and Ultrasound-assisted Methods

This study investigated the extraction of bioactive compounds from cocoa bean shell (CBS) using a combined enzymatic treatment of pectinase, xylanase, and cellulase (PXC), followed by ultrasonication-assisted extraction. The optimized aqueous extracts were subsequently converted into powders by freeze-drying and spray-drying, and the resulting products were evaluated for storage stability, biological activities, and cytotoxicity. The results indicated that an enzyme concentration of 1.0% (v/v) combined with an ultrasonication amplitude of 80% produced the highest yields of bioactive compounds, including total phenolic content (TPC; 286.97 ± 1.01 mg GAE/g), total flavonoid content (TFC; 42.20 ± 0.28 mg CAE/g), and procyanidin content (7.76 ± 0.30 mg PC/g). Antioxidant activities, assessed by DPPH and FRAP assays, were also maximized under these conditions, reaching 132.31 ± 0.72 and 125.88 ± 0.24 µM Trolox equivalents/g, respectively. Freeze-dried extracts exhibited significantly higher retention of bioactive compounds and antioxidant activities compared with spray-dried extracts. High-performance liquid chromatography analysis revealed glycitin as the predominant isoflavone in freeze-dried CBS extract powder (0.18 ± 0.00 mg/g). During 12 weeks of storage at 25°C, the freeze-dried powder maintained relatively stable CIE color parameters, while moisture content, water activity, bioactive compound levels, and antioxidant activities gradually but significantly decreased ( p < 0.05). The CBS extract also demonstrated tyrosinase and collagenase inhibitory activities, with IC₅₀ values of 1.32 ± 0.07 and 1.26 ± 0.02 mg/mL, respectively. Moreover, all tested concentrations showed no cytotoxic effects, with cell viability exceeding 100%. Overall, CBS exhibits strong potential as a sustainable source of antioxidant and anti-aging agents for functional food and nutraceutical applications.