EFFECTS OF POST-HARVEST PROCESSING AND EXTRACTION TECHNIQUES ON COCOA (Theobroma cocoa L.) PHENOLIC COMPOUNDS AND ALKALOIDS

Cocoa and its derivatives are known worldwide for their health benefits due to the bioactive compounds in their composition, mainly polyphenols and alkaloids. These compounds are affected by post-harvest processing, i.e., fermentation and drying, and there are several methodologies for their extraction and quantification, with no consensus on the most suitable. This study aimed to develop a protocol for the extraction and quantification of 16 cocoa bioactive compounds ((+)-catechin, (-)-epicatechin, procyanidins A2, B1, B2, and C2, theobromine, caffeine, theophylline, quercetin, quercetin-3-O-glucopyranoside, quercetin-3-O-α-L-arabinoside, luteolin, protocatechuic acid, cyanidin-3-arabinoside, and cyanidin-3-galactoside) in unfermented and fermented beans, using methanol and acetone as solvents. The extracts were analyzed by UHPLC-MS/MS. The major compounds identified in cocoa beans before and after fermentation were (in mg/g), respectively, theobromine (29.14±0.08; 14.47±0.50), (-)-epicatechin (20.37±0.44; 14.22±0.49), procyanidins C1 (11.55±0.09; 7.19±0.04), B2 (8.83±0.04; 4.96±0.09), and caffeine (3.86±0.01; 1.29±0.04). For unfermented beans, an exhaustive extraction in six steps was more efficient for the quantification of all compounds, especially theobromine (61%) and (-)-epicatechin (66%). Before processing, they are present in larger quantities and stored in pigmented cells of the seed cotyledons. Interestingly, for fermented beans, the six-step extraction allowed for higher quantifications only for procyanidins B2, C1, and cyanidin-3-arabinoside (18.5%, 35%, 37%, respectively), and the influence of the extraction type was less significant (p<0.05), possibly due to the fermentation inducing the rupture of subcellular structures, the oxidation of polyphenols, and the diffusion of alkaloids to the bean shell, reducing concentrations and favoring the accessibility of these compounds. Protocatechuic acid was identified as a biomarker of fermentation since its concentration increases during the processing. We demonstrate that cocoa bioactive compounds can be underestimated depending on the extraction techniques. We also highlight that an exhaustive six-step extraction using solvents with different polarities is a good strategy for recovering phenolic compounds and alkaloids in cocoa samples.