**1. Introduction**

Flavanols are the most abundant substances of polyphenols found in cocoa, with a degree of polymerization ranging from monomers to polymeric proanthocyanidins [1]. Therefore, unfermented cocoa bean is composed of 1.3–3.3% methylxanthines [2] and of about 6% condensed flavan-3-ols [3]; as a result, it is has been listed as the 4th richest dietary source of polyphenols [4]. Cocoa is one of the top crops in Colombia in terms of economic impact, with a national production record of 59,665 tons in 2019 according to National Cacao Producers Federation. There is also a government initiative aiming at a switch from cocaine to cocoa and to join forces with the private sector to enhance Colombia's competitiveness at an international level, which has been established in the comprehensive national program for the replacement of illegal crops [5].

Polyphenols have gained increasing attention as supplements and additives in functional foods due to their nutraceutical properties and beneficial health properties. The recovery of polyphenols from cocoa and its by-products through several technologies such as maceration [6], microwave [7], and pressurized liquid extraction [8] has been previously reported. Most of them use powder cocoa beans obtained from processing operations consisting mainly of (a) drying and particle size reduction pretreatment, (b) degreasing, and (c) recovery of secondary metabolites. During the pretreatment stage, low drying temperatures and short times are preferred to avoid oxidation of flavonoids while small particle size is suggested to increase mass transfer.

The total fat content of whole cocoa beans is over 50% (on a dry basis) [9], which constitutes a barrier for the release of polyphenols from the cells; therefore, a degreasing process is commonly employed that can include pressing of the beans, or using a solvent extraction (i.e., hexane, chloroform, petroleum ether or other non-polar solvents) [10,11] to achieve a final fat content lower than<12 wt.% [12]. When defatting using solvents, an additional step for residual solvent removal is thus needed. Supercritical fluid CO2 can be used, which leaves no residue in the final product, but it is more expensive [13].

During the last stage, the extraction yield can vary not only due to the pre-treatment step, but also due to the type of solvent, contact time, temperature, solid to solvent ratio, the structure of the solid matrix and pH [14]. Although several solvents can be used to extract cocoa polyphenols, polar solvents approved by food regulations agencies (i.e., FDA and EFSA) for human consumption (that is, water, ethanol or a mixture of both) are preferred.

Some patented processes can be found in the literature for producing cocoa polyphenol concentrate; these include a series of sequential steps consisting on inhibition of enzymatic browning (blanching with hot water or steam), pressing and supercritical CO2 extraction for fat removal or degreasing with hexane over 6 h [13], reduction of particle size (<500 um) using cryogenic milling (under −5 ◦C), and extraction of polyphenols using hot water [13], 60% 2-propanol [15], acetone/water/acetic acid [16] and aqueous methanol [17].

These are good examples showing that no single universal extraction processes can be employed for the extraction of polyphenols from different plant sources [18]. Therefore, the goal of the present work was to establish the optimal experimental conditions to enhance higher recovery of polyphenols from cocoa beans without degreasing. Thus, a suitable method that was reproducible for large-scale production (0.05–10 L), cheap, used food grade solvents and reliable for food applications was developed based on ultrasound-assisted solid-liquid extraction using aqueous ethanol. The best operational conditions for the pre-treatment process (drying and particle size reduction to avoid the degreasing) and for the recovery of polyphenols were established. In addition, chemical characterization and antioxidant activity of the cocoa polyphenol extract were also studied.

#### **2. Material and Methods**

#### *2.1. Reagents*

All the chemicals used were analytical or reagent grade and were not purified further. Folin-Ciocalteu reagent, gallic acid, sodium carbonate, ascorbic acid, l-cysteine, sodium phosphate monobasic monohydrate (NaH2PO4·H2O), disodium hydrogen phosphate (Na2HPO4), caffeine, and theobromine were obtained from Sigma Aldrich (St. Louis, MO, USA). (+)-Catechin hydrate (≥99%; ASB-000003310), (−)-epicatechin (≥99%; ASB-00005127), procyanidin B2 (≥90%; ASB-00016231) were purchased from ChromaDex Inc. (Irvine, CA, USA). Acetonitrile, methanol, (HPLC-grade), ethanol, *n*-hexane, citric acid and formic acid were acquired from Merck (Merck, Germany). Deionized water (18 MΩ/cm) from an Aqua Solution system (Aqua solution, Inc. Jasper, Georgia, USA) was used for the preparation of all solutions.
