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Effect of fermentation and drying on cocoa polyphenols Barbara Albertini, Aurélie Schoubben, Davide Guarnaccia, Filippo Pinelli, Mirco Della Vecchia, Maurizio Ricci, Gian Carlo Di Renzo, and Paolo Blasi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01062 • Publication Date (Web): 18 Jun 2015 Downloaded from http://pubs.acs.org on July 2, 2015

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EFFECT OF FERMENTATION AND DRYING ON COCOA POLYPHENOLS

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Barbara Albertini1, Aurélie Schoubben1, Davide Guarnaccia2, Filippo Pinelli3, Mirco Della

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Vecchia4, Maurizio Ricci1, Gian Carlo Di Renzo5, Paolo Blasi6*

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Department of Pharmaceutical Sciences, University of Perugia (Italy);

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Laerbium Pharma S.r.l., Via Togliatti n. 73/A - 06073 Corciano, Perugia (Italy);

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European Network “The chocolate way” [http://www.thechocolateway.eu/];

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Accademia Maestri Cioccolatieri Italiani, Via Sass Muss, 6 32037 Sospirolo, Belluno (Italy);

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Department of Surgical and Biomedical Sciences, S.M. della Misericordia Hospital, S. Andrea

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delle Fratte, 06156 Perugia (Italy).

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School of Pharmacy, University of Camerino, Via Sant’Agostino 1, 62032, Camerino (Italy).

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*Corresponding Author: Paolo Blasi, e-mail: [email protected]; phone +39 0737402289, fax:

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+39 0737637345.

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Abstract

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Cocoa seed polyphenols have demonstrated interesting beneficial effects in humans. Most of

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polyphenols contained in fresh seeds are chemically modified during fermentation, drying, and

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cocoa powder or chocolate production. The improvement of these procedures to obtain a high-

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polyphenol-content cocoa is highly desirable. To this aim, a field investigation on the effect of

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fermentation and natural drying on fine flavor National cocoa (cacao Nacional) was performed.

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Cocoa seeds were fermented for 6 days and, every day, samples were sun dried and analyzed for

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polyphenol content and antioxidant power. During the first 2 days of fermentation, Folin-Ciocalteau

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and FRAP test evidenced a significant reduction of polyphenol content and antioxidant capacity,

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respectively. Changes during the following days of fermentation were less significant. Epicatechin,

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the most studied member of the catechin family, followed a similar pathway of degradation. Data

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confirmed the high impact of fermentation and drying on cocoa seed polyphenols. Fermentation and

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drying are, in the one hand, necessary to obtain cocoa flavour and palatability but, in the other hand,

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are strongly compromising polyphenol content. To obtain high-polyphenol-content cocoa, the

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existing fermentation, drying, and manufacturing protocols should go through a systemic review to

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understand and modify the critical steps.

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Keywords: Theobroma cacao L., cocoa, chocolate, polyphenols, catechin, epicatechins, cocoa

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fermentation and drying.



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Introduction

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Cocoa seeds, the seeds contained in the fruits of the plant Theobroma cacao L., and the products

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derived from its transformation have been known for millennia. At the moment, the oldest traces of

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the human use of cocoa have been found in Mesoamerica and date back 2000 B.C.1-3 while the

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beginning of domestication is still controversial and contended between Central and South

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America1,4-7. Well known is the use of cocoa by Mayans and Aztecs that consumed it in form of

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unsweetened dark beverage8. The use of cocoa and cocoa derived products has continued through

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the centuries and many beneficial and/or curative effects on humans have been described. Only

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recently, thanks to the advancement of the analytical technologies and the availability of in vitro, in

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vivo, and clinical studies, the mechanism underlying these effects has been attributed to the

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presence of a plethora of antioxidant compounds9. Among them, the most representatives are

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catechins, anthocyanins, and proanthocyanins, with (-)-epicatechin being the most abundant

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polyphenol. The peculiarity of cocoa is the high content of polyphenols per dry weight that is

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superior to other beverages and foods including tea and wine10,11. Naturally, cocoa polyphenol

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content varies with the plant variety12, climate and soil characteristics13,14, harvesting15,

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fermentation16, drying time method17, as well as storage and transportation. In chocolate,

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polyphenol content is negatively influenced by different manufacturing steps, such as alkalinization,

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roasting, and conching18-20. On the other hand, fermentation, alkalinization, roasting, and conching

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are the major steps contributing to the development of the characteristic color and flavors of cocoa

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and chocolate14.

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Among the preventive and/or therapeutic effects reported in literature, cocoa and/or high-cocoa-

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content chocolate contributed to reduce blood pressure, glycemic and liver pattern during

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pregnancy21, improved cognitive function, reduced blood pressure and insulin resistance in elderly

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subjects with mild cognitive impairment22, and showed multiple disease-modifying properties in

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Alzheimer's disease23,24. Several short-term intervention studies have also reported beneficial health 3 ACS Paragon Plus Environment

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effects against oxidative stress and chronic inflammation, risk factors for cancer and other chronic

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diseases25.

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More clear evidences are probably limited by the small amount of studies focused on the

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bioavailability of the different cocoa polyphenols in humans. Investigations on the effect of product

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processing, interactions with other components of the diet, and host factors on polyphenol

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bioavailability have not been well defined in the scientific literature26,27.

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For a reliable use of cocoa and cocoa derived products as food supplements or medicines, a deep

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understanding of the negative effect of the manufacturing steps mentioned above on polyphenols

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amount is needed. To obtain standardized semi-finished or finished products, the whole procedure

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(from the agronomical techniques to the manufacturing processes) should be strictly controlled and

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certified. This is mandatory to obtain a consistent and qualified product. At the moment we are far

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from having a complete understanding and control of these factors: for instance time and mode of

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pulp preconditioning may differ according to traditions, fermentation may be performed for

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different periods of time in heaps, wood boxes, fermentation baskets, or juta sacs in diverse climatic

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conditions14. In addition, cocoa pods harvesting from small farmers is difficult to standardize since

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traditional empirical “standards” on pod collection, preconditioning, and fermentation are extremely

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well established and difficult to eradicate.

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With the aim of identifying the procedures that impair the production of a high-polyphenol-content

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cocoa (intending a cocoa with a natural polyphenol content higher than the currently available), a

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study on the impact of fermentation on total phenolic content (TPC), catechins and epicatechins

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content, and total antioxidant capacity (TAC) has been performed. This is the beginning of a

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systematic study aimed at the standardization and certification of the fine flavor National cacao,

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form Ecuador variety. cacao Nacional as well as at the production of a high quality cocoa naturally

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enriched in polyphenols with its characteristic taste and flavor. 4 ACS Paragon Plus Environment


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Materials and methods

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Materials

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Folin-Ciocalteu reagent, potassium persulfate, gallic acid, 6-hydroxy-2,5,7,8-tetramethyl-2-

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carboxylic acid (Trolox), 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonate) diammonium salts

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(ABTS) and 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), were purchased from Sigma Aldrich (Milan,

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Italy). Methanol (MeOH), hexane, acetic acid (AcOH), hydrochloridric acid (HCl) and sodium

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carbonate were obtained from J.T. Baker (Milan, Italy). Ferric chloride was bought from Carlo Erba

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(Milan, Italy). Ultrapure water was obtained from Human Power 1 system (Human Corporation,

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Caserta, Italy). All solvents used were of the highest purity grade commercially available.

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Cocoa harvesting, fermentation and drying

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Mature pods (the fruit of Theobroma cacao L.) from the variety Arriba Nacional were obtained

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from farmers located in the Canton Babahoyo (Los Ríos, Ecuador). No reliable information on the

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days of pod storage after harvesting was available. Fermentation and drying were performed at the

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collection centre of Pueblo Nuevo in the Municipality of Babahoyo (geographical coordinates of

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Babahoyo; Latitude: -1 49' 00'', Longitude: -79 31' 00'') (Canton Babahoyo, Los Ríos, Ecuador)

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(Figure 1). T. cacao L. fruits used in this study were from a variety known as Arriba Nacional or

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simply cacao Nacional.

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Pods were opened and the seeds (120 Kg) covered of their mucilaginous pulp were deposited for

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fermentation in a wood box made of Cordia alliodora (R. & P.) Oken28, also known as mountain

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laurel29. Fermentation mass was covered with banana leafs and wood to maintain fermentation

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temperature and avoid arthropod contamination. Temperature of the fermenting seeds was recorded



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every day measuring it in three different zones of the mass. Temperature near the fermentation box

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was recorded as well.

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The fermenting mass was manually mixed once a day for at least 10 minutes while every 2 days the

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mass was moved from an upper box to a lower one. The fermentation had a duration of 6 days and

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every day (including day zero: non-fermented seeds) three samples of 300 g were randomly

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withdrawn from the mass and dried.

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Since the experimentation was performed during the dry season (May 31, 2014 - June 11, 2014),

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sun drying was chosen. Natural seed drying was performed in the collection centre by laying the

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seeds on a suspended net situated under a plastic canopy. Seeds were mixed three times a day and

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left to dry for 5 days. The temperature under the drying structure was recorded every day in

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triplicate. After the 5 days of fermentation, cocoa seeds were collected in plastic bags and frozen

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until use.

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Extraction of polyphenols

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Cocoa beans were ground to obtain a fine powder that was defatted using n-hexane. 150 mL of n-

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hexane were added to 30 g of cocoa powder and the mixture was kept under magnetic stirring for 2h

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to remove the fat components30. The suspension was filtered through a paper filter and the solid

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material (defatted cocoa powder) was washed with 50 mL of n-hexane and then dried at room

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temperature. Polyphenol extraction was performed using a mixture of MeOH:H2O:AcOH

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(79.2:20:0.8 v/v/v) as solvent31. Defatted cocoa powder (0.5 g) was added to 200 mL of extraction

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mixture and maintained under stirring for 3 hours at room temperature. Clear polyphenol solution,

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obtained by filtration, was used for polyphenol content and antioxidant activity analysis.


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Total Polyphenol content

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Folin-Ciocalteu assay

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Folin-Ciocalteu reagent was diluted 10-fold and 750 µL of diluted reagent was added to 100 µL of

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the solution containing cocoa extract. The mixture was incubated at room temperature for 10

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minutes then 750 µL of sodium carbonate aqueous solution (2% w/v) was added. The mixture was

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incubated at room temperature, in the dark for 3h before to read the absorbance at 765 nm using an

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ultraviolet/visible (UV/VIS) Agilent 8453 spectrophotometer (Agilent, Waldbronn, Germany).

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Different times of sample incubation have been reported in literature before spectrophotometric

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analysis32. Gallic acid was used as a standard. Calibration curve was prepared in concentration

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range of 2.382-19.062 µg/mL and the results obtained were expressed as mg of gallic acid

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equivalents/g of defatted cocoa (mgGAE/gDC).

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Ferric reducing antioxidant power test

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This test was performed to determine the TAC and it is based on the reducing capacity of

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antioxidant compounds to reduce Fe3+ to Fe2+ forming a complex [TPTZ-Fe(II)] that absorbs at a

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wavelength of 593 nm. Ferric reducing antioxidant power (FRAP) reagent was prepared by mixing

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2.5 mL of a HCl (0.04 M) solution of TPTZ (0.3 % w/v) with 2.5 mL of a ferric chloride aqueous

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solution (0.3 % w/v) and 25 mL of acetate buffer (0.3 M, pH= 3.6). 100 µL of ultrapure water and

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100 µL of cocoa extract solution were added to 1.5 mL of the FRAP reagent. This mixture was

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incubated for 4 minutes at room temperature before reading the absorbance at 593 nm. Trolox was

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used as a standard. Calibration curve was elaborated in a concentration range of 0.794-6.353 µg/mL

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and the results obtained were expressed as mg of Trolox equivalents/g of defatted cocoa

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(mgTE/gDC)32.



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Catechins and epicatechins quantification

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HPLC separation was performed as previously reported33 using an amide C18 column, 5 µm, 250 x

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4.6 mm i.d. (MetaChemTechnologics Inc., CA, USA). For LC/ESI-MS experiments an Agilent

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1100 SeriesLC/MSD system (Agilent Technologies, Waldbronn, Germany), equipped with a

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quaternary pump, diode-array detector, column compartment temperature control, degasser, MSD

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ion trap with an electrospray ionization source.

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ABTS assay

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This analytical methodology, based on the capacity of the antioxidant compounds to inhibit the

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oxidation of ABTS to radical cation ABTS+•, allows determining the TAC. An aqueous solution of

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ABTS (20 mL, 3.6 % w/v) was added to 10 mL of an aqueous solution of potassium persulfate

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(0.2% w/v) to obtain the ABTS+•. The solution was incubated in the dark at room temperature

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overnight and then diluted with methanol to an absorbance of 0.7±0.05 at 734 nm. 10 µL of cocoa

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extract solution were added to 1 mL of diluted ABTS+• solution and incubated for 2 minutes before

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reading the absorbance at 734 nm. Trolox was used as a standard in a concentration range from 0 to

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3.6 µg/mL. In this case increasing the amount of Trolox (antioxidant compound) decreases the

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absorbance. Results obtained with this assay were expressed as inhibition percentage of ABTS

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oxidation due to antioxidant compounds34.

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Results

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Cocoa harvesting, fermentation and drying

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The genetic origins of the different T. cocoa L. varieties domesticated and nowadays cultivated is

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not completely elucidated and is still under discussion1,3-6,35. Criollo seems the first to be cultivated

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but, due to its extreme sensitivity, hybrids with foreign genotypes (i.e., Forastero) have been created

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and named Trinitario35. Criollo and Forastero groups correspond to the 2 subspecies T. cacao ssp.

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cacao and T. cacao ssp. spharocarpum, respectively. The Forastero group is composed of

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extremely different populations from different origins and trees are generally discriminated by pod

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morphology. The pods collected for the field experiment were from trees of Arriba Nacional, a

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hybrid between Criollo and Forastero belonging to the Trinitario group4,5.

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Pods were delivered to the collection centre for 2 consecutive days and the following day the fruits

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were opened and the seeds collected to perform the fermentation.

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The first day of fermentation the pulp covering the seeds became liquid and draws off from holes

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drilled at the bottom of the box. The temperature of the mass did not increase in the first 24 hours.

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Between 24 and 48 hours, the temperature of the mass raised up to about 43°C, remaining between

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43 and 45°C for the following 3 days and then starting to decrease after day 5, indicating the end of

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the fermentation (Figure 2).

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Five days of natural drying allowed to obtain a residual humidity below 7 % (w/w) necessary for a

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good conservation and to avoid mould development. Only the unfermented sample (day 0) seemed

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susceptible to mildew. Natural drying has been seen to be adequate to achieve the desired cocoa

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seed residual humidity if performed during the dry season.

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Total Polyphenol content

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Folin-Ciocalteau analytical protocol has been modified with respect to data reported in the scientific

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literature. In the reports describing this methodology there is no consensus on different variables,

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such as: reaction time, incubation temperature and wavelength of analysis. Different Authors

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conditioned the sample at room temperature for 4532, 6036 or 12037 minutes before reading the

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absorbance at 765 nm, while others incubated the mixture for 15 min at 50°C before analysis38.

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Preliminary kinetics experiments allowed setting the preconditioning to 3 hours at room

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temperature in the dark as the optimal incubation time length. The reaction was monitored by

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analysing the absorption spectra in the range 300-800 nm every 60 seconds for 3 hours. The

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reaction was faster during the first hour; it began to decrease, slowing down without stopping or

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reaching a plateau, after two hours. The absorption peak during the first hour was not symmetrical,

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maximum of absorption slightly deviated from 765 nm and lower standard concentrations absorbed

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below 0.1 (A.U.). Calibration curves were built by plotting the absorbance at 765 nm of different

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standards after 40, 60, 120, and 180 minutes. An incubation of 3 hours permitted to have higher

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sensitivity, lower standard errors and higher r2 (0.99833)of the extrapolated curves.

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Folin-Ciocalteau and FRAP test methods gave the same trend but different results in terms of the

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relative amount of total polyphenols (Figure 3). Even though it is not easy (and maybe not possible)

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to compare the two methods and the results obtained (Folin-Ciocalteau uses gallic acid equivalents

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while FRAP test trolox equivalents) unfermented seeds showed similar amount of total polyphenol

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with a value around 60 g/Kg. The results of both methods indicate that the first 2 days are the most

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critical for the loss of polyphenols. Folin-Ciocalteau recorded about 20 gGAE/gDC after 6 days of

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fermentation and 5 of drying while, according to the results of FRAP test, less than 10 gTE/gDC

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were detected at the end of the fermentation/drying process (Figure 3).


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Catechins quantification

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Eight different antioxidant compounds of the catechins family, namely catechin, epicatechin,

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catechin gallate, epicatechin gallate, epigallocatechin, epigallocatechin gallate, gallocatechin,

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gallocatechin gallate39, as well as gallic acid, were quantified by HPLC. Catechins and epicatechins

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were present in large amount in the cocoa samples (Figure 4) while the other compounds above

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mentioned were present in concentrations below 5 mg/Kg and were not of interest in our study.

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Epicatechins have been found in the range of g/KgDC while catechins were present in the range

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between 100 and 20 mg/KgDC (Figure 4). The concentration trend during fermentation is

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superimposable to that of the TPC analysed using Folin-Ciocalteau methodology with a surprising

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high concentration recorded the fourth day of fermentation (Figure 3). The experimental setup used

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in this work does not allow any further speculation on the origin of this data. At the moment, even

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though unlikely, it can be explained just accounting for non-uniformity in the fermentation mass

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and/or in the sample of day 4.

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Discussion

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Fermentation is a crucial process in the production of cocoa and chocolate because it is responsible

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for the development of flavors and flavor precursors that confer to cocoa its characteristic

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bouquet40. At the same time, fermentation contributes to the elimination of the astringency and the

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bitter taste characteristics of fresh unfermented cocoa seeds. Fermentation inhibits germination and

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contributes to the mixing of enzymes with substrates by eliminating the compartmentalization of the

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seeds, a step necessary to allow enzymatic reactions.

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It has been reported that the optimal fermentation length is generally 5 or 6 days for Forastero14.

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The first day, the sweet mucilaginous pulp adhering the seed surface liquefies and drains out while

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the temperature of the fermenting mass remains low. Temperature rises 24-48 hours after the

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beginning of the fermentation process. The sugar of the seed pulp contributes to initiate what is

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known as “external fermentation” that, under anaerobic conditions, produces ethyl alcohol and

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acetic acid. “Internal fermentation” comprises all the biochemical changes that happen in the

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cotyledons. Polyphenols undergo enzymatic oxidation by polyphenol oxidase and condensate in

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high molecular weight tannins.

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Independently of the employed analytical method (Folin-Ciocalteau or FRAP test), data showed an

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initial content of total polyphenols of ~ 60 mg/gDC. Previous studies have reported a high variation

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in polyphenol content in seeds from different T. cocoa L. varieties and/or cultured in different

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regions12,41. Contents as high as ~80 mgGAE/gDC have been recorded for the variety Forastero

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(Ivory Coast), Amazon (Columbia), and Amazon hybrid (Ecuador)42 while other Authors have

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found amounts of polyphenols in the range 34-60 and 45-60 mg/gDC15,43.

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Most of polyphenols (80-90%) were lost during the first 48 hours but the remaining amount can be

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considered high12,44. Polyphenol loss is generally ascribed to multiple factors: diffusion of soluble

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polyphenols into fermentation sweating, enzymatic oxidation, and non-enzymatic oxidation, the 13 ACS Paragon Plus Environment

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latter happening especially during sun drying. The additional fermentation days do not seem to

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affect in a significant way the content of polyphenols.

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The present experimental setup contemplated 5 days of natural drying (sun drying) for each sample.

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The loss of polyphenols in the samples 1-6 is due to the combined negative effects of fermentation

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and drying while the polyphenols in unfermented sample (sample 0) have been affected by the sole

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drying. However, it cannot be neglected that polyphenol loss could be due to a certain degree of

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fermentation occurring during drying . The process of sun drying might reduce the content of

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polyphenols because of non-enzymatic oxidation45. Polyphenols and especially epicatechin, the

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most abundant compound of the catechin family, was surely present in larger amount in fresh

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seeds41,46.

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The loss of epicatechins followed the trend of total polyphenols with the maximum lost during the

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first 2 days. At the end of fermentation and drying, epicatechin concentration was reduced by

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approximately 75%.

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The concentration of epicatechin in fermented cocoa beans seems to be more variable than the

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concentration of total polyphenols41,43,47. Kim and Keeney41 reported values ranging from about 2.5

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to 16.5 mg/gDC while Wilson et al. have found concentrations of 3.0-3.3 mg/gDC43.

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Considering the lack of knowledge on eventual pod storage and the data available in literature, it

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seems that the pods were not freshly harvested even though a strong reduction in pulp volume was

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not evidenced during fruit opening. The low content of epicatechins could be due to the time lag

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between pod collection and the beginning of the fermentation process48,49.

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Preliminary results confirmed the high impact of fermentation and drying on the content of

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polyphenols in cocoa seeds. Being the first and mandatory step necessary to obtain cocoa and

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chocolate bouquet and palatability, attention should be paid to these 2 steps to obtain high-

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polyphenol-content cocoa. 14 ACS Paragon Plus Environment


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Due to the discordance of the data reported in literature, systemic investigation aimed at a full

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characterization of the whole cocoa transformation process is mandatory. To have high significance,

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investigations should consider and analyse all the possible variables influencing the initial

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polyphenol content and should avoid artefacts generated from unreal working conditions, such as

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microfermentation. Knowledge of agronomic techniques, plants location, soil and climate

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conditions, T. cocoa L. variety, maturation stage and pod conditioning are of fundamental

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importance to know polyphenol variability in the starting material.

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To produce high-polyphenol-content cocoa and to build a successful certification protocol, all the

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factors mentioned have to be carefully controlled and standardized.

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Acknowledgment

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The Authors are indebted to the Istituto Italo-Latino Americano for financial and logistic support

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during the experimental part performed in the municipality of Babahoyo (Ecuador). The authors

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would like to acknowledge the Mayer of Babahoyo, Mr. Johnny Terán Salcedo and the Ing.

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Jonathan Romero Jacome for the support during the scientific activities.

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FIGURE CAPTIONS

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Figure 1. Map of Ecuador. The municipality of Babahoyo, in the Province of los Ríos, is

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indicated by the red spot. Galapagos Islands are not reported in the map.

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Figure 2. Mean temperatures recoded in the fermentation mass (grey columns) and outside the

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fermentation box (white columns). Bars indicate the standard deviations.

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Figure 3. Results of Folin-Ciocalteau and FRAP tests. Results are expressed in mg of gallic

461

acid equivalents per g of defatted cocoa (mg GAE/g DC) in Folin-Ciocalteau assay and in mg

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of trolox equivalents per g of defatted cocoa (mg TE/g DC) in FRAP test. Bars indicate the

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standard deviations.

464 465

Figure 4. Plots showing the amount of catechins and epicatechins during fermentation. Bars

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indicate the standard deviations.

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Figure 5. Results of the ABTS assy. Data are reported as percentage of inhibition of the

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oxidation reaction. Bars indicate the standard deviations.

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.

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Effect of Fermentation and Drying on Cocoa Polyphenols - Journal of

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