PLOS ONE

RESEARCH ARTICLE

Fermentation and its effect on the

physicochemical and sensory attributes of
cocoa beans in the Colombian Amazon
Andrés Felipe Ramı́rez González1¤, Gustavo Adolfo Gutiérrez Garcı́a ID1,2,
Paola Andrea Polanı́a-Hincapié1,3, Luis Javier López ID4, Juan Carlos Suárez ID1,2*
1 Programa de Ingenierı́a Agroecológica, Facultad de Ingenierı́a, Universidad de la Amazonia, Florencia,
Caquetá, Colombia, 2 Centro de Investigaciones Amazónicas CIMAZ Macagual César Augusto Estrada
González, Grupo de Investigaciones Agroecosistemas y Conservación en Bosques Amazónicos-GAIA,
a1111111111 Universidad de la Amazonia, Florencia, Caquetá, Colombia, 3 Programa de Maestrı́a en Sistemas
a1111111111 Sostenibles de Producción, Facultad de Ciencias Agropecuarias, Universidad de la Amazonia, Florencia,
a1111111111 Caquetá, Colombia, 4 Grupo de Investigación en Ciencia y Tecnologı́a de Alimentos-CICTA, Escuela de
a1111111111 Ingenierı́a Quı́mica-Universidad Industrial de Santander, Bucaramanga, Santander, Colombia
a1111111111
¤ Current address: Centro de Investigación La Libertad. Corporación Colombiana de Investigación
Agropecuaria (AGROSAVIA), Villavicencio, Colombia
* [email protected]

OPEN ACCESS

Citation: González AFR, Garcı́a GAG, Polanı́a-

Abstract
Hincapié PA, López LJ, Suárez JC (2024)
Cocoa (Theobroma cacao L.) is the basic raw material to produce chocolate and other
Fermentation and its effect on the physicochemical
and sensory attributes of cocoa beans in the derivatives such as cocoa butter, cocoa powder and cocoa liquor (cocoa paste), which
Colombian Amazon. PLoS ONE 19(10): e0306680. requires a fermentation process that affects its chemical composition and sensory profile.
https://doi.org/10.1371/journal.pone.0306680 The objective of this study was to monitor the biochemical, physical and sensory changes
Editor: Guadalupe Virginia Nevárez-Moorillón, during fermentation of cocoa beans in cocoa bean processing plants in the department of
Universidad Autonoma de Chihuahua, MEXICO Caquetá, Colombia. During fermentation, the temperature of the mass and the pH of the
Received: June 22, 2024 pulp and beans were monitored at the different cocoa bean processing plants (Sites
Accepted: August 26, 2024 ASOACASAN ASA, COMICACAO CMI, COMCAP COC). Also, at two points during fer-
mentation (days 4 and 7), physical properties of the bean were determined, such as vari-
Published: October 3, 2024
ables related to bromatological composition, polyphenolic compounds and antioxidant
Copyright: © 2024 González et al. This is an open
activity as sensory attributes at the different sites. An increase in dough temperature was
access article distributed under the terms of the
Creative Commons Attribution License, which found, however the pH of the cotyledon decreased during the fermentation process and
permits unrestricted use, distribution, and the fat and moisture content varied with fermentation time. At the site level, total polyphe-
reproduction in any medium, provided the original nol content (TPC), total flavonoids (TF), 1,1-diphenyl-2-picrylhydrazil (DPPH) and ferric
author and source are credited.
reducing antioxidant power (FRAP) contents were statistically different, with COC being
Data Availability Statement: All relevant data are different from the other sites. The TPC was higher at the COC site (507 mg gallic acid
within the manuscript and its Supporting
equivalent GAE/g Cocoa) with respect to the other sites (< 360 mg GAE/g Cocoa). The
Information files
TF content followed a similar behavior to TPC, with significant differences between sites
Funding: This study was financially supported by
and differences between fermentation times for ASA. The TF was higher in COC (309.1
the Ministry of Science, Technology and Innovation
project "Fortalecimiento de vocaciones cientı́ficas mg catechin/g cocoa) with respect to CMI (215.6 mg catechin/g cocoa) and ASA (185.7
en jóvenes a través de becas de pasantı́a en la mg catechin/g cocoa). Values in DPPH ranged from 5869.3 to 7781.8 μmol Trolox/g
región centro sur: Caquetá, Amazonas, Putumayo, cocoa and for the FRAP assay ranged from 369.8 to 606.7 mg ascorbic acid AA/g cocoa
Huila, Tolima" financed with resources from the
among the sites. It was found that the time and management of the fermentation process
general royalty’s system and executed by the Vice-
Rectory of Research and Innovation of the has a significant impact on the parameters (biochemical, physical and sensory) of cocoa

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PLOS ONE Fermentation and its effect on the physicochemical and sensory attributes

Universidad de la Amazonia in the form of an beans. Therefore, it is necessary to standardize the fermentation process to achieve a
award received by AFRG. This study was also quality product that meets the needs of the market.
financially supported by the University of Amazon
through the strategy of hiring research assistants
in the research support units at the CIMAZ
Macagual Amazon Research Center in the
framework of the "Pro-development of the
University" stamp of the Amazon" law 1301 of
2009. The funders had no role in study design, data 1. Introduction
collection and analysis, decision to publish, or
preparation of the manuscript.
Cocoa (Theobroma Cacao L.) is the basic raw material to produce chocolate and other deriva-
tives such as cocoa butter, cocoa powder and cocoa liquor (cocoa paste) [1]. This food has sus-
Competing interests: The authors have declared
tained part of the daily diet of people and is currently making inroads in the cosmetics,
that no competing interests exist.
perfumery and pharmaceutical industries [2] with a requirement in the final quality of the
product. Recent studies have reported that grain quality in sensory terms is affected by differ-
ent factors such as the genetics of the material [3], environmental conditions [4], the state of
maturity of the grain and practices in the postharvest stage [5], such as those related to fermen-
tation and drying [6, 7].
The composition and quality of cocoa beans is determined by a set of physical, chemical
and sensory qualities [8]. According to Garcı́a et al. [9] the main biochemical changes that
influence the quality of cocoa beans occur during the fermentation stage, since it is there
where different microbial communities (yeasts, lactic acid and acetic acid bacteria) intervene
in the metabolization of sugars and other compounds [10]. Fermentation is a process in which
fresh cocoa beans are placed in a wooden box for four to seven days. During this process, the
microorganisms metabolize the pulp that surrounds the beans and generate reactions that
result in an increase in temperature, a decrease in pH and death of the embryo [11]. During
fermentation, different biochemical reactions occur on various substances, including antioxi-
dants, polyphenols and methylxanthines [1]. For example, polyphenols are products of the sec-
ondary metabolism of plants, characterized by aromatic rings with hydroxyl groups as
substituents, which are directly related to the sensory properties of chocolate and its positive
effect on human health [12]. Inadequate fermentation of cocoa produces purple and slaty
beans, negatively affecting quality and thus marketing price [13]. However, it has been
reported that beans without fermentation have genotype-specific sensory attributes, mainly
related to bitterness, astringency and acidity, and other attributes depend directly on fermenta-
tion, drying and roasting [14].
Cocoa fermentation is mainly carried out by small producers or collection centers in an
artisanal manner, with little or no technology and without monitoring of processing condi-
tions, which results in low quality beans [15]. These variations during the fermentation process
make the chocolate industry face increasing challenges in maintaining the supply of standard
products with high quality organoleptic properties [16]. This condition in the final product
causes the cocoa bean to be marketed as ordinary cocoa with prices already regulated by an
industry monopolized by large companies, which in the case of Colombia, acquire 90% of the
national production [17], which means that the payment received does not have a sufficient
impact on the welfare of cocoa-growing families [18], which discourages and puts cocoa pro-
duction at risk, increasing the possibility of a change in production activity [19]. Although fer-
mentation is an indispensable stage to ensure optimal bean quality [20], there are deficiencies
in the number, intensity and coverage of studies that determine the effects of the fermentation
process on physicochemical properties related to the content of protein, fat, fiber, functional
and antioxidant capacity of cocoa beans as well as the different sensory attributes.
The study focused on these cocoa bean processing plants since the three associations of
cocoa producers called ASOACASAN, COMICACAO and COMCAP are part of the strategic

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PLOS ONE Fermentation and its effect on the physicochemical and sensory attributes

cooperation agreements with European companies whose objective is the production and mar-
keting of organic chocolate under the principles of fair trade which promotes sustainability
since these associations produce “organic cocoa free of deforestation”. Therefore, the objective
of this study was to monitor the biochemical, physical and sensory changes during fermenta-
tion process of cocoa beans in cocoa bean processing plants in the department of Caquetá,
Colombia. It is expected that the study will provide a scientific basis for decision-making based
on the recognition of the local needs of each plant. With this information, the fermentation
processes carried out by cocoa collection centers in the department of Caquetá will be
improved and standardized, resulting in better bean quality and added value in marketing.

2. Materials and methods

2.1. Study area
The study was carried out in the department of Caquetá, specifically in the cocoa bean process-
ing plants of a) Asociación Orgánica Agrı́cola de San José del Fragua-ASOACASAN (ASA) (1˚
190 43@N 75˚580 22@W), b) Comité de Cultivadores de Cacao en Sistemas Agroforestales del
municipio de San Vicente del Caguán-COMICACAO (CMI) (2˚060 55@N 74˚460 12@W) and c)
Comité de Cacaoteros de los municipios del Paujil y el Doncello-COMCAP (COC) (1˚400 26@
N 75˚160 48@W). The environmental conditions in which these processing plants are found
mainly contrast in the amount of average annual precipitation and the relative humidity (RH),
variables that can affect the drying time. For example, in ASOACAN, which is located further
north in the department of Caquetá, it has an average annual rainfall of 4,200 mm with a RH
of 85% and handles the mass (560 kg of cocoa beans with mucilage) in relation to 7 days of fer-
mentation with 6 subsequent turnings of the anaerobic phase and uses 14 days in the drying
process. COMICACAO, very north of the department of Caquetá, has an average annual rain-
fall of 2,700 mm with a RH of 83%, it handles the mass (270 kg of cocoa beans with mucilage)
very similar to ASOACASAN but with the main difference is that they dry only in 10 days.
Finally, COMCAP, which is located 90 km from COMICACAO to the north and 128 km to
the south with ASOACASAN, presents an average annual rainfall of 3,800 mm with a RH of
82%, carries out mass management (340 kg of cocoa beans with mucilage) with 8 days of fer-
mentation with 7 turning and 12 days of drying. All cocoa processing plants carry out the turn-
ing of the cocoa bean during fermentation every 24 hours and the process of drying the bean
after fermentation in canopies at room temperature.

2.2. Monitoring fermentation and drying of cocoa beans

The cocoa beans were obtained from healthy pods from trees of different universal, national
and regional clones obtained from farms associated with the organizations. Both ASOACA-
SAN and COMICACAO make mixtures of cocoa beans from clones such as CCN51, ICS1,
ICS95, TSH565 and FEAR5, however the difference between these associations is the addi-
tional use in FLE2 and ICS39 for ASOACASAN and COMICACAO, respectively. On the
other hand, COMCAP carries out fermentation processes for cocoa beans obtained from raw
materials. This process was carried out between March and June 2023, when production
peaked. The beans were then fermented and dried according to the protocol of each collection
center. The cocoa beans were placed in wooden crates, which were covered with banana leaves
and natural jute fiber sacks to maintain a homogeneous temperature. These crates were perfo-
rated at the bottom to allow drainage of the cocoa pulp. The fermentation process lasted 168
hours (7 days) and during this time the fermentable mass was turned every 24 hours after 48
hours of fermentation. The turning of the grain was done by passing the dough into the com-
partments of the fermenter box, a process that was carried out using a plastic shovel. This

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PLOS ONE Fermentation and its effect on the physicochemical and sensory attributes

fermentation process was carried out in triplicate, that is, three fermentation bins were used
for each storage center.
During fermentation, the following parameters were monitored daily between 13:00 and
16:00 hours: mass temperature, pH of the grain pulp and pH of the cotyledon. Samples for this
monitoring were taken in the center and at the ends of the bin at a depth of 30 cm. This process
was carried out in triplicate. The temperature of the dough was determined using a digital
thermometer Hl 145 (Hanna Instrument, Woonsocket, RI, USA), the pH of the pulp during
fermentation was measured using a pH tester Hl 98108 (Hanna Instrument, Woonsocket, RI,
USA) and the pH of the cotyledon was performed following the methodology used by Papalex-
andratou [21]. For which, 15 g of randomly selected cocoa beans were taken from the three
points of the bin; then, the pulp and testa were manually removed from the beans with a knife
and the cotyledon was macerated with 30 ml of deionized water in a mortar for one minute
until a homogeneous sample was obtained and the pH was read using a pH tester Hl 98108
(Hanna Instrument, Woonsocket, RI, USA).

2.3. Determination of chemical characteristics of beans during the

fermentation process
2.3.1. Physical analysis. At the end of the fermentation and drying process, a cutting test
was carried out according to the NTC 1252 2021 edition [22] to determine the colorations and
defects present. For this purpose, 100 cocoa beans were randomly selected from each fer-
mented cocoa pod at each collection center, cut longitudinally with the help of a CocoaT Bean
knife (Cocoatown, Alpharetta, Georgia, USA) and the number of beans that were completely
fermented, purple, moldy, damaged by insects, and slaty were quantified.
2.3.2. Bromatological composition. Total fat content was determined by the Randall
method with petroleum ether (AOAC Method No.991.36) [23], using the semi-automatic sol-
vent extractor (SER 148 VELP Scientific, Italy) whose results were expressed as percentage of
fat. Ash content was determined by incinerating the organic matter in a muffle furnace
(1,100˚C, 22.9A Fisher Scientific, Spain) at 550 ± 5˚C until the sample was free of carbon,
cooled in a desiccator and the amount of ash was calculated (AOAC Method No. 930.30) [24],
the results were expressed as percentage of ash. Moisture content was determined using the
methodology proposed by Nuñez et al. [25] with modifications. A crucible was brought to con-
stant weight, 10 g of dry cocoa beans were weighed into the crucible and left in the oven (Her-
atherm OMH400, Fisher Scientific, Spain) at 105 ± 5˚C for 4 hours. The results were expressed
as percent of moisture. Titratable acidity was measured according to AOAC method 939.05
[24]. Extracts of dried cocoa beans (5 g) were homogenized in 50 mL of deionized water, sub-
sequently filtered through filter paper (3hw, 110 mm, 65 g/m2; Boeco, Germany) and titrated
with a standard 0.1 N sodium hydroxide (NaOH) solution to a pH of 8.3. The results were
expressed as percent of acidity.
2.3.3. Polyphenolic compounds. First, the methanolic extract of defatted cocoa beans was
obtained by weighing 3 g and depositing them in falcon tubes, adding 15 mL of methanolic
solution (25% HPLC grade methanol, 24% deionized water and 1% HPLC grade formic acid).
The samples were homogenized for 25 minutes using vortex and ultrasound for 20 minutes.
Subsequently, they were left in darkness for 24 h and centrifuged for 15 min at 4,500 rpm
(4˚C) and filtered with filter paper (3hw, 110 mm, 65 g/m2; Boeco, Germany). With this extract
obtained from each sample, the contents of total polyphenols, total flavonoids, DPPH, FRAP
and methylxanthine contents were analyzed in triplicate. The determination of the total poly-
phenol content was carried out using the Folin-Ciocalteu colorimetric method [26].
Eighteen μL of the extract, 124.5 μL of deionized water, 37.5 μL of Folin-Ciocalteu reagent and

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PLOS ONE Fermentation and its effect on the physicochemical and sensory attributes

120 μL of 7.1% anhydrous sodium carbonate (Na2CO3) were taken. It was left to react for 60
min in the dark at room temperature, after which the absorbance was read at 760 nm. Gallic
acid was used as a standard. Results were expressed as mg gallic acid equivalent (mg GAE)/g
dried cocoa bean. Total flavonoid content was determined by reaction with aluminum chlo-
ride (AlCl3) according to the methodology proposed by Zhishen et al. [27] with slight modifi-
cations. The reaction mixture consisted of 120 μL of deionized water, then 30 μL of the extract
was added, followed by 9 μL of 5% sodium nitrite (NaNO2) (waited 5 minutes), 9 μL of 10%
aluminum chloride (AlCl3) (waited 5 minutes), then 60 μL of 1M sodium hydroxide (NaOH)
(waited 15 minutes) and finally 72 μL of deionized water. It was left to react in the dark at
room temperature for 30 minutes and the absorbance was read at 510 nm. The (+)-catechin
was used as a standard for the quantification of total flavonoids. Results were expressed as mg
catechin equivalent (mgCE)/g dried cocoa bean.
Quantification of methylxanthines and epicatechin were developed on an Ultimate 3000
HPLC, equipped with an auto-injection system and UV-VIS detector, analytical reverse phase
column (Zorbax Eclipse XDB 150mm × 2.1mm) with particle size of 5μm, at 25˚C. All com-
pounds were detected at a wavelength of 273 nm. The mobile phase was water/acetic acid (99.7/
0.3 v/v) (solvent A) and methanol (Solvent B), flow rate 0.5 mL/min. The gradient was as fol-
lows: 0–10 min, 15% linear B; 10.1–18 min, 25% linear B; 18.1–25 min, 30% linear B; 25.1–30
min, 100% linear B; 30.1–35 min, 0% linear B, followed by 5 min of column re-equilibration
before a new injection with an injection volume of 5μL. All analytes were identified and quanti-
fied by the external standard method using calibration curves of the standard substances [28].
2.3.4. Antioxidant activity. The 1,1-diphenyl-2-picrylhydrazil (DPPH) radical scavenging
activity was used according to the method of Brand-Williams et al. [29] with slight modifica-
tions. A stock solution of DPPH (20 mg/L) was prepared in absolute methanol; the absorbance
of the radical was adjusted to 0.3 absorbance units with methanol at 4˚C, then 3 μL of the
extract and 297 μL of the adjusted DPPH solution were taken. It was allowed to react in the
dark for 30 min at room temperature and the absorbance was read at a wavelength of 517 nm.
The results were expressed as Trolox equivalent antioxidant capacity (TEAC) values in μmol
of Trolox (μmol Trolox)/g of dry cocoa beans, constructing a reference curve using Trolox as
an antioxidant. The reducing capacity FRAP (ferric reducing antioxidant power) evaluates the
antioxidant capacity of a sample according to its ability to reduce ferric iron (Fe+3) present in a
complex with 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), to the ferrous form (Fe+3) [30]. The assay
was carried out in a pH 3.6 acetic acid-sodium acetate buffer containing TPTZ and FeCl3.
15 μL of the extract, 15 μL of buffer and 270 μL of FRAP solution were used as samples. It was
allowed to react in the dark for 30 min at room temperature and the absorbance was read at a
wavelength of 590 nm. The FRAP values were expressed as mg ascorbic acid (mg AA/g of dry
cocoa bean), based on a reference curve of ascorbic acid as the primary standard.
2.3.5. Sensory analysis. The cocoa paste samples (cocoa bean roasted and milled) were
analyzed by a sensory panel following the process described by ICONTEC [22]. The roasting
curve was constructed according to bean size and moisture percentage; the roasted beans were
also passed through a grinder and the husk was separated from the nibs. Finally, the nibs were
processed and refined by a melangeur for the manufacture of cocoa paste/liquor. Sensory eval-
uation was carried out with the help of five trained panelists, and the main qualities were deter-
mined: cocoa, acidity, astringency, bitterness, and notes of fresh fruit, brown, floral, wood,
spice and nut, as well as atypical flavors in the samples. The order of perception, intensity,
residual flavor and persistence were established as scores given by the panel. The international
cocoa evaluation scale of excellence [31] was used, which scores from 0 to 10 points, where 0
indicates the complete absence of the evaluated attribute and 10 indicates a very high intensity.

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PLOS ONE Fermentation and its effect on the physicochemical and sensory attributes

2.4. Data analysis and experimental design

A completely randomized plot design with factorial arrangement was used (i. cocoa bean pro-
cessing plants called "Sites" corresponding to ASOACASAN ASA, COMICACAO CMI and
COMCAP COC; and ii. fermentation time, corresponding to samples of cocoa beans obtained
at 4 and 7 days after the start of the fermentation process) using three repetitions with the
objective of analyzing the impact on the physics of the grain, bromatological composition,
polyphenolic compound content and antioxidant activity as sensory attributes. Each repetition
corresponded to a mass of cocoa bean with mucilage carried out independently in each fer-
mentation box at each site from which a sample was obtained for analysis 4 and 7 days after
the start of the fermentation process. The data were analyzed by fitting a linear mixed model
(LMM) where in the fixed factor the sites (ASA, CMI and COC) and the fermentation time
(days 4 and 7) were adjusted, and within the random factor the repetition (mass of cocoa
bean) within the sites. Assumptions of normality and homogeneity of variance were examined
through an exploratory analysis of residuals. In addition, the LSD Fisher test (P< 0.05) was
performed to determine if differences existed between fixed factors. Line graphs were also
made to show the behavior of temperature and pH during fermentation. Subsequently, radar
plots were made to visualize the sensory attributes of the sites by days of fermentation. In addi-
tion, Pearson’s correlation using the “corr” package [32] was employed to establish correla-
tions between antioxidant activity, polyphenolic compounds and methylxanthines. Finally, a
principal component analysis (PCA) was performed to determine the multivariate relation-
ships between the variables evaluated and the study sites. The models were applied using the
statistical software InfoStat [33], PCA and Pearson correlation were performed using the pack-
ages ade4, ggplot2 [34], factoextra [35], FactoMineR [36] in the R language software, using the
RStudio interface [37].

3. Results
3.1. Temperature and pH of fermented cocoa beans
During the fermentation period, the temperature increased until 4 days, reaching an average
of 45˚C, after which it stabilized (Fig 1A). The pH of the pulp did not vary significantly during
the fermentation period, however, there was a small variation at 2 and 3 days between sites
(Fig 1B). The pH in the cotyledon at the beginning of the fermentation process ranged from
5.6 to 6.3 with a steady decrease to 4.5 (Fig 1C).

3.2. Physical properties of dry cocoa beans

The fermentation process between sites varied (Table 1, P<0.05), a situation that resulted in
the number of properly fermented grains; however, no difference was found between fermen-
tation hours. On the contrary, differences between sampling hours were found in the number
of violet and partially fermented grains in ASA and totally fermented grains in COC. Between
sites, the main difference was in the amount of violet and partially fermented grains (Table 1,
P<0.05).

3.3. Component bromatological

In the different variables of the bromatological component, significant differences were found
at the level of fermentation time as well as at the sites (Table 2, P<0.05). For example, fat con-
tent and moisture content varied with fermentation time, being significantly higher at seven
days; the opposite was true for ash (Table 2, P<0.05). As for the titratable acidity level, no dif-
ferences were found between fermentation days.

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PLOS ONE Fermentation and its effect on the physicochemical and sensory attributes

Fig 1. Daily behavior of three variables during cocoa fermentation in three processing plants in the department of
Caquetá. ASA: ASOACASAN, CMI: COMICACAO, COC: COMCAP: a) mass temperature, b) pulp pH, c) cotyledon
pH. Values correspond to means and standard errors (n = 3).
https://doi.org/10.1371/journal.pone.0306680.g001

3.4. Polyphenolic compounds

At the site level, TPC, TF, DPPH and FRAP contents were statistically different, with COC
being different from the other sites (P<0.05, Fig 2). Total polyphenol content was higher at the
COC site (507.05 mg GAE/g Cocoa Fig 2A) with respect to the other sites (< 360 mg GAE/g
Cocoa). The TF content followed a similar behavior to TPC, with significant differences
between sites (P<0.05) and differences between fermentation times for ASA. The TF was
higher in COC (309.1 mg catechin/g cocoa, Fig 2B) with respect to CMI (215.59 mg catechin/g
cocoa) and ASA (185.7 mg catechin/g cocoa). Values in DPPH (Fig 2C) ranged from 5869.3 to

Table 1. Results (%) of the shear test on fermented and dried cocoa beans at two fermentation times.
Sites Fermentation time (days) Fermentation* Completely fermented Partially fermented Violet Mouldy beans Slaty
ASA 4 92 ± 2.0 78 ± 2.31 14 ± 1.15 b 6.67 ± 1.33 b 0 ± 0.0 1.33 ± 0.67
7 92.7 ± 3.33 73.33 ± 9.4 17.3 ± 7.06 a 8.0 ± 4.16 a 1.33 ± 1.3 0 ± 0.0
General Average 92.33 ± 1.74 ns A 75.67 ± 4.45 ns A 15.67 ± 3.28 B 7.33 ± 1.98 B 0.67 ± 0.67 ns A 0.67 ± 0.42 ns A
CMI 4 76.7 ± 8.11 58 ± 8.72 b 18 ± 0.67 b 21.3 ± 6.36 0.7 ± 0.67 1.33 ± 1.33
7 80 ± 2.0 61.3 ± 2.91 a 18 ± 1.33 a 17.3 ± 2.4 1.3 ± 0.67 1.33 ± 0.67
General Average 74.5 ± 3,81 ns B 59.67 ± 4.18 B 18.67 ± 0.67 B 19.33 ± 3.17 ns A 1.0 ± 0.45 ns A 1.33 ± 0.67 ns A
COC 4 68 ± 4.0 37 ± 13.0 b 31 ± 9.0 31 ± 5.0 0 ± 0.0 1 ± 1.0
7 81 ± 9.0 52 ± 2.0 a 29 ± 7.0 19 ± 9.0 0 ± 0.0 0 ± 0.0
General Average 78.33 ± 5.50 ns B 44.5 ± 6.90 C 30.00 ± 4.69 ns A 25.0 ± 5.45 ns A 0.0 ± 0.00 ns A 0.00 ± 0.00 ns A
a, b, c ns
Different letters indicate statistically significant differences by LSD Fisher means test (P< 0.05). Letters means significant and non-significant differences
between the time of fermentation at each site and A, B, C: between the different sites. ASA: ASOACASAN, CMI: COMICACAO, COC: COMCAP. *: Corresponds to the
sum of completely and partially fermented cocoa beans.

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PLOS ONE Fermentation and its effect on the physicochemical and sensory attributes

Table 2. Bromatological composition of fermented and dried cocoa beans at two fermentation times.
Sites Fermentation time (days) Fat (%) Ash (%) Moisture (%) Acidity titratable (mg AA/g cocoa)
ASA 4 40.20 ± 0.28 b 2.77 ± 0.003 4.09 ± 0.01 a 0.40 ± 0.02
7 42.49 ± 0.13 a 2.78 ± 0.003 3.75 ± 0.02 b 0.41 ± 0.003
General Average 41.34 ± 0.53 B 2.77 ± 0.003 ns A 3.98 ± 0.8 A 0.40 ± 0.01 ns C
CMI 4 43.68 ± 0.06 b 2.84 ± 0.05 a 3.16 ± 0.03 0.47 ± 0.01
7 45.47 ± 0.34 a 2.48 ± 0.01 b 3.31 ± 0.14 0.48 ± 0.01
General Average 44.58 ± 0.43 A 2.66 ± 0.8 A 3.23 ± 0.07 ns B 0.48 ± 0.01 ns B
COC 4 42.55 ± 0.74 a 2.75 ± 0.05 a 3.21 ± 0.02 a 0.52 ± 0.003
7 43.74 ± 0.44 b 2.51 ± 0.01 b 3.08 ± 0.01 b 0.55 ± 0.01
General Average 43.15 ± 0.47 A 2.63 ± 0.06 A 3.15 ± 0.03 B 0.53 ± 0.01 ns A

Different letters indicate statistically significant differences by LSD Fisher means test (P< 0.05). Letters a, b, c means significant and non-significant ns differences
between the time of fermentation at each site and A, B, C: between the different sites. ASA: ASOACASAN, CMI: COMICACAO, COC: COMCAP.

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Fig 2. Content of polyphenols, methylxanthines and antioxidant activity in fermented cocoa beans. Different
letters indicate statistically significant differences by LSD Fisher means test (P< 0.05). Letters a, b, c means significant
and non-significant ns differences between the time of fermentation at each site and A, B, C: between the different sites.
ASA: ASOACASAN, CMI: COMICACAO, COC: COMCAP.
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PLOS ONE Fermentation and its effect on the physicochemical and sensory attributes

7781.8 μmol Trolox/g cocoa and for the FRAP assay ranged from 369.8 to 606.7 mg AA/g
cocoa among the sites (Fig 2D). At the methylxanthine level, theobromine was different
between sites and only different at the level of hours of fermentation in COC (P<0.05, Fig 2E).
In the case of caffeine and epicatechin, no differences were found between sites (Fig 2F and
2G); however, only for caffeine there were differences between the fermentation hours for the
three sites, while for epicatechin only COC showed differences in fermentation hours.

3.5. Sensory profile

In general, the presence of complementary attributes such as nutty, spicy, woody, floral and
fresh fruit was found at all sites (Fig 3). It was evident that COC presented higher acidity (5.5)
with respect to ASA (3.67) and CMI (3.33) on the fourth day of fermentation; however, on day
7 acidity decreased in COC (4.5) but increased in CMI (5.0). Astringency and bitterness
increased with days of fermentation in ASA and COC, while astringency and bitterness
decreased with the course of fermentation in CMI.
Fig 4 shows the different positive and negative correlations between the different variables
analyzed. A positive correlation was found between polyphenolic compounds and antioxidants
with theobromine. Likewise, titratable acidity had a positive correlation with total polyphenols,
total flavonoids and variables related to antioxidant capacity (FRAP and DPPH). On the other
hand, caffeine was negatively correlated with DPPH, theobromine and epicatechin (Fig 4).
Principal component analysis explained 43.8% of the variance (PCA, Fig 5), with CP1
explaining 27.2% and opposing the ASA and COC sites by variables such as moisture, titratable
acidity as well as phenolic compound content and antioxidant activity. CP2 explained 16.6%,

Fig 3. Sensory profile of cocoa liquor in three processing plants: a. ASA: ASOACASAN, b. CMI: COMICACAO, c. COC:
COMCAP.
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PLOS ONE Fermentation and its effect on the physicochemical and sensory attributes

Fig 4. Correlation analysis between the different variables analyzed. The numbers shown presented significant correlations (P<0.05)
in the color gradient from red to blue showing negative and positive correlation, respectively.
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opposing different sensory attributes such as cocoa and bitterness. According to the Monte-
Carlo test, the sites explained 24.9% of the variance (Fig 5).

4. Discussion
4.1. Temperature and pH of fermented cocoa beans
An increase in temperature was found in the fermentation mass due to the activity carried out
by the yeasts involved in the metabolization of sugars in the cocoa pulp (exothermic process),
to produce ethanol and other metabolities (other alcohols, esters, aldehydes and ketones), a

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PLOS ONE Fermentation and its effect on the physicochemical and sensory attributes

Fig 5. PCA projection of variables related to bromatological composition, polyphenol content, methylxanthines and antioxidant activity in
cocoa beans fermented in different cocoa bean processing plants in the department of Caquetá, Colombia. ASA: ASOACASAN, CMI:
COMICACAO, COC: COMCAP. Contribution of each of the variables evaluated in the PC1/PC2 principal components of PCA, the gradient from
green to red means from greater to lesser contribution.
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process that releases heat and increases the fermentation temperature [38]. It was observed
that from the fifth day of fermentation the temperature did not vary, reaching a temperature
between 40–50˚C, a desirable range for good fermentation [39]. Although the environmental
temperature was not a condition that varied, a significant increase in the temperature of the
cocoa fermentation mass was found in the first three days in ASA, this is possibly due to a
greater amount of cocoa bean in mucilage that was used to ferment which influenced main-
taining the temperature resulting from the exothermic reaction during the anaerobic phase in
the fermentation of the cocoa mass [38]. Situation that has been evidenced in studies where
the behavior of temperature is analyzed in different places simultaneously in the departments
of Huila, Santander and Antioquia, Colombia which were different between sites [16]. On the
other hand, from the third day of fermentation the mass recorded an increase in pH, a situa-
tion attributed to the decrease by leaching of citric acid contained in the pulp [40] and the
decrease of volatile organic acids leading to an increase in pH in the fermentation mass [41] as
to the metabolization of citric acid by the yeasts [42]. This decrease in pH within the first few
days at all sites is attributed mainly to the diffusion of organic acids in the bean produced by

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PLOS ONE Fermentation and its effect on the physicochemical and sensory attributes

lactic acid bacteria [43] and together with the high temperatures, which lead to embryo death,
triggering a series of biochemical changes that greatly impact the development of bean flavor
and color. Also, this behavior was considered [44] to be because of turning the cocoa mass
after 48 hours, because it favors aeration and the growth of acid-acetic bacteria [45]. Finally,
the pH of the cotyledon at the end of fermentation in the three sites was very similar ASA
(4.33), CMI (4.59) and COC (4.63), these results are very important since some studies men-
tion that pH above 5.5 at the end of fermentation indicates lower quality fermented beans [46],
however, Calvo et al. (2021) [16] mentions that pH ranges in cotyledon between 4.8 and 5.2
indicates a good fermentation process. In this study, fermentation at CMI and COC were the
closest to the conditions. Recent studies [47, 48], in addition to showing the decrease in grain
pH during the fermentation process, showed changes in this variable between locations. For
example, the pH of the cotyledon decreased consistently throughout the fermentation, reach-
ing final values of 5.1 ± 0.4 in location A and 5.6 ± 0.4 in location F, with no significant differ-
ence in the variations after 72 h of fermentation.

4.2. Physical properties of dry cocoa beans

Variation was found in the number of fermented grains according to the category. For exam-
ple, the increase in the percentage of fermented and completely fermented beans (brown) are
mainly associated with anthocyanin degradation [49] because of gradually increasing tempera-
ture in the fermentation box [50]. Purple cocoa beans are characteristic of an inadequately fer-
mented bean and brown beans are typical of optimal fermentation [51], a situation that was
present in the CMI and COC sites. The percentage of partially fermented beans presented sig-
nificant differences between sites, with higher values in COC. The presence of partially fer-
mented beans is produced by some factors such as: reduction in the amount of turning in the
cocoa mass during fermentation or due to the location of the beans in the fermentation
box [52]. For this reason, several studies mention the importance and impact of turning on the
physical characteristics of cocoa beans [53]. The effect of the site on the quality of fermentation
has been documented by different studies [47, 48, 54, 55] which mention an impact on the
number of beans with complete fermentation, for example Murcia-Artunduaga et al. [55] they
mention that the municipality of Tarqui (Huila, Colombia) presented the best results of physi-
cal characteristics of the cocoa beans, while Oporapa (Huila, Colombia) presented a high ten-
dency to have “pasilla” beans. In the different locations (Manabı́ and Los Rı́os provinces in
Ecuador [48]) in which the incidence of fermentation on the physical quality of cocoa beans
was evaluated, a significant variation was found in relation to a greater proportion of well-fer-
mented beans (24.7 ± 13.4%) and slightly fermented grains (58.3 ± 6.7%) between the sites, the
above product of temperature variations.

4.3. Component bromatological

An increase in fat content was evidenced with increasing days of fermentation at all sites
(P<0.05), the data obtained agree with the study of Millena et al. [56] who found an increase
in fat content from the fifth day of fermentation. The increase in fat contents during fermenta-
tion days can be attributed to the interaction between acidity and temperature during fermen-
tation, leading to the alteration of lipid bonds [57]. On the other hand, the values in ash
percentage were below 3%, values that coincide with those reported by Calvo et al. [16], like-
wise, this decrease in ash content during fermentation was reported by Afoakwa et al. [58].
This behavior is explained by the loss of water-soluble minerals drained during the fermenta-
tion process [59]. Moisture contents varied significantly in all sites, being ASA with the highest
values (3.98%) with respect to CMI and COC, it is believed that this behavior is due to the

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PLOS ONE Fermentation and its effect on the physicochemical and sensory attributes

geographical location and precipitation of the site, because ASA is located in the Andean-
Amazonian transition with higher precipitation than the other sites included in the study;
according to Bomdzele and Molua [60] high precipitation can slow grain drying and increase
moisture content. The decrease in grain moisture content during fermentation at the COC site
is explained by the increase in temperature allowing moisture to diffuse outward and by the
effect of turning the fermentable mass [61]. The increase in acidity percentage is associated
with the increase of lactic acid and acetic acid during the fermentation days [16]. These acids
enter the cotyledon generating biochemical reactions and causing embryo death [62].

4.4. Polyphenolic compounds

Total polyphenol content was higher at the COC site (507.05 mg GAE/g cocoa) compared to
the other sites (< 360 mg GAE/g cocoa), the highest COC contents are mainly attributed to
beans from hybrid cocoa plantations found in the study area. According to Jonfia-Essien et al.
[63] hybrid cocoa beans have higher polyphenol levels and antioxidant activity than clones,
due to their genetic variability. Regarding total polyphenol values, he results of the present
study were much higher than those reported by Ramón et al. (2022) [4] in fermented and
roasted beans (210.2 mg GAE/g cocoa), with the difference that in our study, the cocoa beans
evaluated were not roasted, only fermented and dried. According to Urbańska et al. [64] the
polyphenol content may decrease due to the oxidation of these compounds by the effect of
high temperatures in the roasting process and interaction with Maillard reaction products [65,
66]. In addition to the above, a decrease in total polyphenol content was observed at all sites
with increasing fermentation time, which is attributed to the effect of increasing temperature
in the fermentation box [16], although this decrease has also been reported due to the influence
of polyphenol oxidase enzyme activity during fermentation and drying [67]. In general terms,
we posit that grains that have undergone adequate fermentation have lower polyphenol con-
tents. This is desirable to achieve pleasant sensory profiles, because higher polyphenols content
is associated with astringency [68], while anthocyanins are linked to the violet color of unfer-
mented grains [69]. Both aspects are considered undesirable in chocolate production [70, 71].
However, their decrease has a proportional effect on antioxidant activity, a desirable feature
from the functional point of view of chocolate to decrease the risk of oxidative stress-mediated
diseases [72].
Total flavonoids content (TF) followed a similar behavior that TPC, with significant differ-
ences between sites (P<0.05) and differences between fermentation times for ASA. The TF
was higher in COC (309.1 mg catechin/g cocoa) with respect to CMI (215.6 mg catechin/g
cocoa) and ASA (185.7 mg catechin/g cocoa). Our results showed changes in TF contents
because of fermentation days, this explains that flavonoids also decreased during fermentation
as did polyphenols, corroborating what was found by Hu et al. [73] in their research on chemi-
cal properties in roasted and unroasted cocoa beans, in which they mention that flavonoids are
more sensitive to high temperatures than polyphenols. The values found in TF in our study
were higher than those reported by da Silva Oliveira et al. [74] and like those found by Cuéllar-
Álvarez et al. [75] in Copoazú (Theobroma grandiflorum) beans with different fermentation
days. The literature primarily reports the total flavonoid content extracted from fermented
and roasted grains; hence, our values are higher than those reported by many authors, because
in our study the grains used for physical and chemical properties analysis did not go through
the roasting process. Zaman et al. [76] mentions that roasting causes the contents of flavo-
noids, flavanols, flavones and anthocyanins to decrease with increasing time and temperature
during roasting. While flavonoids are important bioactive compounds that have been associ-
ated with their anti-inflammatory properties, they have a negative effect on the sensory

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PLOS ONE Fermentation and its effect on the physicochemical and sensory attributes

attributes of the bean; however, chocolate manufacturers today pay great attention to declaring
their products as functional foods [77]. This opens the door to discussions regarding the man-
ufacture of chocolates with fermented/roasted beans, fermented/unroasted beans or fer-
mented/roasted beans enriched with encapsulated polyphenols.

4.5. Sensory profile

In general, complementary attributes such as nutty, spicy, woody, floral and fresh fruit were
present in all sites, which are categorized as fine cocoa aroma and flavor [78]. The sensory eval-
uation revealed that, in all sites and days of fermentation, cocoa notes obtained intensity scores
higher than 6, however, our results contrast with those reported by Calvo et al. [16] who found
values below 5 in cocoa samples from three different regions in Colombia. Also, Romero and
Pabón [79] mentions that beans with high cocoa flavor intensity indicate a successful process-
ing (fermentation and drying), although the main attributes are mostly influenced by genotype
[80]. In fermentation and drying, pyrazine contents increase because they are compounds of
thermal origin and increase with increasing temperature [81], some studies mention that pyra-
zines are related to cocoa flavor [8].
It was evidenced that COC presented higher acidity (5.5) with respect to ASA (3.7) and
CMI (3.3) on the fourth day of fermentation, however, on day 7 acidity decreased in COC
(4.5), but increased in CMI (5.0), this decrease in acidity is mainly due to acetic acid volatiliza-
tion during fermentation and drying because of temperature [82]. The increase in acidity dur-
ing fermentation at CMI was mainly attributed to the presence of volatile acids, generated as a
product of over fermentation and poor drying [58, 83]. Acidity is an important attribute, as
high levels of acidity in cocoa samples can be detrimental to bean quality [84]. Astringency
and bitterness increased with fermentation days in ASA and COC, this behavior was associated
with the effect of a possible over fermentation, we also believe that the increase in astringency
is due to a deficiency in the turnovers of the fermenting mass [85, 86] behavior that contrasts
with a good fermentation [87] while astringency and bitterness decreased with the course of
fermentation at CMI, this decrease is characteristic of a good fermentation process [88].
This flavor is acquired mainly in the roasting process, because it has a direct effect on the
development of flavors in the cocoa beans through various reactions, including Maillard reac-
tions, which contribute to the presence of nutty flavor and aroma [89]. Rodriguez-Campos
et al. [84] concluded that 6 days of fermentation are sufficient to produce volatile compounds
with desirable flavor notes in cocoa, conclusions that contrast with the results of this study,
given that the profiles obtained here had flavor notes characteristic of over fermentation in
ASA, although favorable notes in COC and CMI with 7 days of fermentation. In addition, it is
known that cocoa flavor and complementary attributes are influenced by the genetic potential
of cocoa, but also by postharvest practices (fermentation and drying) [90].

5. Conclusions
The management of the fermentation process has a significant impact on the different charac-
teristics (biochemical, physical and sensory) of the cocoa beans. A similar trend of fermenta-
tion mass variables was found at all sites where cotyledon pH decreased during the
fermentation process and fat and moisture content varied with fermentation time. At the site
level, COC was different from the other sites specifically for total polyphenol content (TPC),
total flavonoids (TF), DPPH and FRAP. The TPC was higher in the COC site (507 mg GAE/g
Cocoa) with respect to the other sites (< 360 mg GAE/g Cocoa). The TF content followed a
similar behavior to TPC, with significant differences between sites and differences between fer-
mentation times for ASA. The TF was higher in COC (309.1 mg catechin/g) with respect to

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PLOS ONE Fermentation and its effect on the physicochemical and sensory attributes

CMI (215.6 mg catechin/g) and ASA (185.7 mg catechin/g). Values in DPPH ranged from
5869.3 to 7781.8 μmol Trolox/g cocoa and for the FRAP assay ranged from 369.8 to 606.7 mg
AA/g cocoa among sites. Complementary attributes such as nutty, spicy, woody, woody, floral
and fresh fruit were observed at all sites. Astringency and bitterness increased with days of fer-
mentation at ASA and COC, while astringency and bitterness decreased with the course of fer-
mentation at CMI.

Supporting information
S1 File. Variables physicochemical and sensory attributes of cocoa beans.
(XLSX)

Acknowledgments
We also thank the following collaborators: Asociación Orgánica Agrı́cola de San José del Fra-
gua (ASOACASAN), Comité de Cultivadores de Cacao en Sistemas Agroforestales del munici-
pio de San Vicente del Caguán (COMICACAO) and Comité de Cacaoteros de los municipios
del Paujil y el Doncello (COMCAP) for providing the benefit facilities to carry out this study.
Finally, we would like to thank Corporacion Econexus Colombia (INSITU) for their support
in the tasting of the samples.

Author Contributions
Conceptualization: Andrés Felipe Ramı́rez González, Gustavo Adolfo Gutiérrez Garcı́a,
Juan Carlos Suárez.
Data curation: Andrés Felipe Ramı́rez González, Paola Andrea Polanı́a-Hincapié,
Luis Javier López, Juan Carlos Suárez.
Formal analysis: Andrés Felipe Ramı́rez González, Juan Carlos Suárez.
Funding acquisition: Gustavo Adolfo Gutiérrez Garcı́a, Juan Carlos Suárez.
Investigation: Andrés Felipe Ramı́rez González, Juan Carlos Suárez.
Methodology: Andrés Felipe Ramı́rez González, Gustavo Adolfo Gutiérrez Garcı́a,
Paola Andrea Polanı́a-Hincapié, Juan Carlos Suárez.
Project administration: Juan Carlos Suárez.
Resources: Juan Carlos Suárez.
Software: Juan Carlos Suárez.
Supervision: Gustavo Adolfo Gutiérrez Garcı́a, Paola Andrea Polanı́a-Hincapié,
Luis Javier López, Juan Carlos Suárez.
Validation: Andrés Felipe Ramı́rez González, Gustavo Adolfo Gutiérrez Garcı́a,
Paola Andrea Polanı́a-Hincapié, Luis Javier López, Juan Carlos Suárez.
Visualization: Andrés Felipe Ramı́rez González, Gustavo Adolfo Gutiérrez Garcı́a,
Paola Andrea Polanı́a-Hincapié, Luis Javier López, Juan Carlos Suárez.
Writing – original draft: Andrés Felipe Ramı́rez González, Gustavo Adolfo Gutiérrez Garcı́a,
Paola Andrea Polanı́a-Hincapié, Luis Javier López, Juan Carlos Suárez.
Writing – review & editing: Andrés Felipe Ramı́rez González, Gustavo Adolfo
Gutiérrez Garcı́a, Paola Andrea Polanı́a-Hincapié, Luis Javier López, Juan Carlos Suárez.

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PLOS ONE Fermentation and its effect on the physicochemical and sensory attributes

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