Development and Characterization of a Pilot-Scale Model Cocoa Fermentation System Suitable for Studying the Impact of Fermentation on Putative Bioactive Compounds and Bioactivity of Cocoa

Abstract

Cocoa is a concentrated source of dietary flavanols—putative bioactive compounds associated with health benefits. It is known that fermentation and roasting reduce levels of native flavonoids in cocoa, and it is generally thought that this loss translates to reduced bioactivity. However, the mechanisms of these losses are poorly understood, and little data exist to support this paradigm that flavonoid loss results in reduced health benefits. To further facilitate large-scale studies of the impact of fermentation on cocoa flavanols, a controlled laboratory fermentation model system was increased in scale to a large (pilot) scale system. Raw cocoa beans (15 kg) were fermented in 16 L of a simulated pulp media in duplicate for 168 h. The temperature of the fermentation was increased from 25–55 °C at a rate of 5 °C/24 h. As expected, total polyphenols and flavanol levels decreased as fermentation progressed (a loss of 18.3% total polyphenols and 14.4% loss of total flavanols during fermentation) but some increases were observed in the final timepoints (120–168 h). Fermentation substrates, metabolites and putative cocoa bioactive compounds were monitored and found to follow typical trends for on-farm cocoa heap fermentations. For example, sucrose levels in pulp declined from >40 mg/mL to undetectable at 96 h. This model system provides a controlled environment for further investigation into the potential for optimizing fermentation parameters to enhance the flavanol composition and the potential health benefits of the resultant cocoa beans.

Functional role of yeasts, lactic acid bacteria and acetic acid bacteria in cocoa fermentation processes

“…Acetic acid migrates into the cotyledon tissue with the water stream upon initiation of seed germination during the initial phase of fermentation of the cocoa pulp-bean mass through a pressure-induced opening of the shell (testa or seed coat) at the micropyle and hilum of the swelling seeds. The colour and flavour of the cured beans are determined to a large extent by the leakage of purple anthocyanins, causing bleaching of the cocoa beans, and by the oxidation of polyphenols and their polymerization into highmolecular-mass compounds as well as condensation and complex formation with proteins, oligopeptides, free amino acids and monosaccharides (forming complex tannins), depending on the fermentation time (and pod storage/pulp-preconditioning) (Wollgast and Anklam 2000;Nazaruddin et al 2006;Camu et al 2008b;Suazo, Davidov-Pardo and Arozarena 2014;Albertini et al 2015;De Taeye et al 2016;Evina et al 2016;Hue et al 2016;Mayorga-Gross et al 2016;Racine et al 2019). The bitterness and astringency of the raw cocoa beans are reduced due to the leakage of flavonoid polyphenols [flavanols (flavan-3-ols or catechins) and proanthocyanidins (oligomers of flavan-3,4-diols)] and methylxanthine alkaloids (1,3,7-trimethylxanthine or caffeine and 3,7-dimethylxanthine or theobromine), besides effects originating from the formation of insoluble tannins.…”

“…Strategies for their recovery can nevertheless be envisaged, either from the pulp drainage or the cocoa beans. To this end, laboratoryscale (2 L) and pilot-scale (15 L) model fermentation processes in polypropylene boxes, placed in an incubator with controlled temperature and aeration, have been proposed, using a cocoa pulp simulation medium (CPSM; Lefeber et al 2010) and rehydrated unfermented dry cocoa beans, to assess the impact of controlled fermentation conditions on bioactive compounds present in cocoa beans (Lee et al 2019;Racine et al 2019). Finally, it is unclear if the fat content of the cocoa beans varies during fermentation, but its content should be as high as possible, as the cocoa bean fat (cocoa butter) is a basic ingredient of the final chocolate (Lima et al 2011;Ndife et al 2013;Trognitz et al 2013;Krähmer et al 2015;Servent et al 2018;Sirbu et al 2018).…”

Flavanol Polymerization Is a Superior Predictor of α-Glucosidase Inhibitory Activity Compared to Flavanol or Total Polyphenol Concentrations in Cocoas Prepared by Variations in Controlled Fermentation and Roasting of the Same Raw Cocoa Beans

“…The raw unfermented cocoa beans were rehydrated, fermented, and dried based on previously established pilot-scale cocoa fermentation protocols described by Racine et al [30] and Lee et al [31] with modifications. The unfermented cocoa beans (32 kg) were rehydrated in plastic fermentation boxes (polypropylene, Sterilite, Townsend, MA, USA) by submersion in distilled, deionized (DI) water (45 L) for 24 h at room temperature.…”

“…The bean pH was determined every 24 h based on the method that was described by Racine et al [30]. A representative cut test was performed on a sample of whole beans from each of the four fermentation runs (2 cool batches, 2 hot batches).…”

“…Polyphenol-rich extracts were made from raw beans, the fermented cocoa beans, and powders for all seven treatments, as described previously [13,30]. The Folin-Ciocalteu colorimetric assay was used to approximate the total phenolic content of the freeze-dried cocoa extracts, and total flavanols were measured by the 4-dimethylaminocinnamaldehyde (DMAC) colorimetric assay, as previously described in Dorenkott et al [12].…”