Comparative Extraction of Polyphenolic Co-Pigments and Proteins from Annatto (Bixa orellana L.) Seed By-Products

Annatto (Bixa orellana L.) seed by-products (ASBs), generated after industrial pigment extraction, remain a largely underexplored source of bioactive compounds, including residual carotenoids, phenolics, and proteins. This study compares three extraction strategies-maceration (ME), ultrasound-assisted extraction (UAE), and subcritical water extraction (SWE)-for the recovery of these compounds from ASB within a green processing framework. Extraction efficiency was assessed based on the yield, protein recovery, total carotenoid content (TCC), total phenolic content (TPC), antioxidant capacity (DPPH, ABTS, FRAP), and phenolic profile (HPLC-DAD). SWE at 140 °C (40 bar, 1:30 g/mL) achieved the highest extraction yields and compound recovery (proteins: 80.0 ± 3.22 mg/g extract; TCC expressed as bixin equivalents: 1.60 ± 0.07 mg/g extract; TPC: 17.96 ± 0.90 mg GAE/g extract). Phenolic profiling identified gallic and protocatechuic acids as major constituents, with significantly higher concentrations in SWE extracts. However, discrepancies between spectrophotometric and chromatographic data suggest the presence of unidentified phenolics. Cytotoxicity assays using Caco-2 cells indicated no significant effects at concentrations up to 10 µg/mL. While SWE demonstrates superior extraction performance, further studies on protein functionality, pigment stability, and bioavailability are required to validate food and nutraceutical applications. This work highlights ASB as a promising resource for circular bioeconomy strategies.