Journal of Food Measurement and Characterization

https://doi.org/10.1007/s11694-022-01620-x

ORIGINAL PAPER

Effect of a semi‑automated fermentation system on the physical

and chemical characteristics of Theobroma cacao L. grown
in the northern Ecuadorian Amazon
Remigio Armando Burbano‑Cachiguango1 · Reinier Abreu‑Naranjo2 · Carlos Estuardo Caicedo‑Vargas1 ·
César Andrés Ramírez‑Romero1 · Andrés Santiago Calero‑Cárdenas1 · Erika Magaly Llumiquinga‑Marcillo3 ·
Melany Ruiz‑Urigüen3,4

Received: 14 March 2022 / Accepted: 6 September 2022

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022

Abstract
Ecuador is one of the main cocoa exporters in Latin America and the top producer of high-quality fine cocoa (~ 65%) in the
world. However, there is a lack of knowledge regarding the physicochemical properties of cacao grown locally in the Ecuado-
rian Amazon region, since these properties have seldom been studied. In addition, it is necessary to improve the postharvest
process adapted to the environmental conditions of the Amazon region. This study evaluated the effect of a semi-automatic
fermentation system on the main physical and chemical characteristics [weight of 100 beans, cotyledon pH, fermentation
percentage, total fat content, and total polyphenol content (TPC)] of Theobroma cacao L. (Nacional and Super Árbol). A
full factorial design ­22 was used to determine the influence of the factors studied: genotype and fermentation method (semi-
automatic and Rohan boxes). The influence of the fermentation method had a highly significant (p < 0.05) positive effect,
while the variety of cocoa did not significantly influence the fermentation percentage (p > 0.05). The cocoa beans fermented
by the semi-automatic method met the quality parameters for fine aroma cocoa according to the Ecuadorian standard. This
method achieved a 10% reduction in fermentation time compared to the traditional system (Rohan boxes). The values obtained
for the cotyledon pH (5.85) and TPC (52.8 mg GAE/g) stand out as being directly related to the organoleptic characteristics
of chocolate and its derivatives. This indicates that the semi-automatic method can be applied to both cocoa genotypes.

Keywords Theobroma cacao L. · Cocoa postharvest · Fermentation quality · Fermented cocoa · Semi-automatic method

Introduction
Ecuador is one of the main cocoa exporters worldwide and is
the world’s top producer of high-quality fine cocoa. Accord-
ing to estimations by the International Cocoa Organization
* Reinier Abreu‑Naranjo
rabreu@uea.edu.ec
1
Central Experimental Station of the Amazon, National
Institute of Agricultural Research (INIAP), Vía Sacha ‑ San
Carlos a 3 km, Joya de los Sachas, Orellana, Ecuador
2 most widely cultivated, with a predominance of 95% of
Departamento de Ciencias de la Vida, Universidad Estatal
Amazónica (UEA), Vía Tena km 2 ½, Puyo, Pastaza,
Ecuador
3
Core Lab de Ciencias Ambientales, Universidad San
Francisco de Quito USFQ, Quito, Ecuador
4
Ingeniería Ambiental, Politécnico, Universidad San
Francisco de Quito USFQ, Quito, Ecuador
(ICCO) for the 2020/2021 season, Ecuador was set to reach
a production of 350 kt, contributing almost 40% of the total
production from the Americas (885 kt) [1]. Cocoa culti-
vation represents one of the most important produces for
Ecuador’s economy. Production is diversified throughout
the country’s three mainland regions: the coastal region is
the main producer; followed by the Highland region; and
then the Amazon [2]. In the local cocoa industry, four geno-
types are notable: Forastero, Criollo, Trinitario (a hybrid
between Criollo and Forastero), and Nacional cacao (NC),
also known as “Arriba” [3]. The Forastero variety is the
worldwide production, but its organoleptic properties are
poor. NC beans have an excellent flavor and aroma, normally
classified as “fine cocoa flavor”, providing unique and spe-
cific characteristics to the organoleptic profiles of chocolate
products. [4].

13
Vol.:(0123456789)

Moreover, there are other lesser-known varieties grown
locally, such as the Super Árbol variety (ESS), which is a
clone-type Trinitario cocoa originating from the northern
Ecuadorian Amazon region. This variety is characterized by
its high production (between 700 and 940 kg/ha per year),
good quality, and resistance to pests such as Crenipellis per-
niciosa and Moniliophthora roreri [5]. Nevertheless, this
information is mostly based on empirical knowledge due to
the scarcity of studies, especially concerning the produc-
tion and postharvest processes, and physicochemical quality
aspects of ESS beans.
Cocoa bean fermentation is a crucial stage in the produc-
tion of high-quality chocolate. The fermented bean micro-
biology implies a succession of microbial activities and
enzymatic conversions inside the cocoa beans, which play a
determining role in the flavor, aroma, and color [6]. One of
the changes that occurs is the release of anthocyanin-type
polyphenols (water-soluble pigments), responsible for the
bean changing color from purple to brown [7]. Epicatechin
and catechin are oxidized to quinones and the condensation
of proteins and polyphenols occurs, decreasing astringency
[8]. Often, one of the main problems encountered in the
cocoa production process is an insufficient fermentation of
the beans, which causes the final product to be poor in qual-
ity due to inadequate control of the fermentation and drying
process [6, 9].
Among the fermentation methods used by producers on
the Ecuadorian coast, the most common are: jute sacks,
bearing or heaps, and wooden boxes [9, 10]. However, the
Ecuadorian Amazon region has different environmental con-
ditions from the coast. Therefore, changes are necessary in
the post-harvest process in order to guarantee the quality
of the cocoa beans in accordance with market standards.
In this sense, the objective of this work was to evaluate the
effects introducing semi-automated control changes in the
fermentation system on the physical and chemical properties
of two cocoa genotypes (NC and ESS) grown in the northern
Ecuadorian Amazon.
Materials and methods
Samples
The cocoa beans used in this study were collected from the
Joya de Los Sachas zone, Orellana, Ecuadorian Amazon
region. This zone has an average altitude of 273 m.a.s.l., an
average rainfall of 3125 mm/year, an average annual tem-
perature of 27 °C, and a mean relative humidity of 79.5%.
The ESS and NC samples were supplied by the CacaoExport
Company and Central Experimental Station of the Amazon,
National Institute of Agricultural Research (INIAP, by its
acronym in Spanish), respectively.
13
R. A. Burbano‑Cachiguango et al.
The sample preparation for laboratory analysis was
performed using INIAP’s official method for the analysis
of chemical parameters associated with cocoa quality, as
proposed by Espín and Samaniego [11]. Briefly, the sam-
ples were pre-dried at 65 °C for 3 days until a value of ~ 7%
humidity in a forced-air oven (Thermo Scientific, OV700F,
USA) was reached. Then, the shell was manually removed
and discarded. The remaining part of the seed was frozen
at − 18 °C for 24 h, then ground and sieved using a No. 20
mesh to achieve a particle size of 0.85 mm. They were then
placed in plastic bags and stored at − 18 °C until analysis.
Fermentation processes involved
For the fermentation of cocoa beans (NC and ESS), two
methods were studied: Rohan boxes and semi-automatic, to
which humidity and temperature measurement and control
elements were incorporated. Figure 1 shows the flow dia-
gram of the semi-automatic process used.
The semi-automatic fermentation system consisted of
measuring and controlling the temperature and relative
humidity during pre-drying and aerobic fermentation to
avoid the growth of molds and fungi that would negatively
influence the quality of the fermented cocoa beans. The
anaerobic phase was as follows: the fresh cocoa samples
were placed in plastic drawers where they remained for
36 h at an ambient temperature of 25–35 °C and a relative
humidity of 70–80%. After the first 24 h, stirring was car-
ried out to achieve a more homogeneous fermentation and
a uniform temperature distribution. Subsequently, the sam-
ples were briefly washed to extract part of the mucilage and
stop the anaerobic phase. Then the samples were subjected
to pre-drying for 24 h at temperatures of 35–40 °C and a
relative humidity of 60%. Later, in the aerobic fermentation
phase, the cocoa beans were transferred to a chamber with
thermal insulation for 48 h, where the temperature increased
to ~ 60 °C due to the effect of fermentation; the drawers
were changed every 6 h to achieve a uniform fermentation.
Finally, the cocoa beans were dried.
Rohan box fermentation was carried out according to
the methodology suggested by Jiménez Barragán et al.
[12]. Briefly, this method involves laurel-wood boxes of
100 × 100 × 15 cm, divided into 25 compartments that each
have the capacity to ferment up to 2.5 kg of cocoa beans.
The fermentation period was 120 h, with 2 mass removals
at 24 and 48 h. Subsequently, the material was sun-dried as
per the local producers’ typical procedure.
Analytical determinations
The physicochemical characterization was carried out
according to the official method for the analysis of chemi-
cal parameters associated with cocoa quality [11] in the
Effect of a semi‑automated fermentation system on the physical and chemical characteristics…
Pre-drying
Water (24 h, 40 oC,
Removal at 24h
humidity ~ 60%)
Cocoa beans
Anaerobic fermentation Washed
(36 h)
Heat source
Fig. 1  Flowchart of a semi-automatic fermentation system for cocoa beans
INIAP’s Food Quality Laboratory. The main methods are
briefly described below.
Weight of 100 beans 100 fermented and dried beans
were randomly collected from Joya de las Sachas, then
weighed on a precision balance (Model CG 4102C, Citi-
zen Scale (I) Pvt. Ltd., Maharashtra, India). Their weights
were recorded in grams.
Cotyledon pH was determined using a pH meter (Model
PT-380, Boeco, GmbH + Co., Hamburg, Germany) accord-
ing to AOAC Method 970.21 [13].
The fermentation percentage or cutting test was estab-
lished by using the methodology described by Beckett [14]
to evaluate the color inside the kernel. The change in the
internal coloration of the bean (from purple to brown) is
indicative of a well-fermented bean that has acquired the
precursors of flavor and aroma.
Total fat content was determined according to AOAC
Method 963.15 [13]. Cocoa powder (5 g) was wrapped in
a cellulose paper cartridge and placed in the Soxhlet appa-
ratus (Glassco Laboratory Equipments Pvt. Ltd., Hary-
ana, India) using petroleum ether (180 mL) for 8 h were
concentrated. Subsequently, the solvent was eliminated in
a rotary vacuum evaporator (Model: RE200A, Yamato,
Tokyo, Japan). The total fat was determined using Eq. 1:
W1 − W2
Total fat content(%) = ∗ 100, (1)
W of 0.94 mL/min. The oven temperature was programmed
where ­W1 = Weight of the ball with fat (g), ­W2 = Weight of
empty balloon tared (g), W = Sample weight (g).
The total polyphenol content (TPC) was determined
using Folin–Ciocalteu’s Method [15] with slight modifi-
cations. Briefly, 1 g of defatted cocoa sample was mixed
Aerobic Drying
fermentation (12 h, ~50 oC,
(48 h, ~50 oC) humidity ~ 60%)
Fermented and dried
cocoa beans
with methanol (80 mL, 70% v/v) for 45 min with mag-
netic stirring. Subsequently, it was filtered using What-
man No. 4 filter paper in a volumetric flask (100 mL)
and the volume was adjusted with methanol (70% v/v) to
obtain the extract. A sample extract (1 mL), distilled water
(6 mL), and Folin Ciocalteu reagent (1 mL) were mixed
and allowed to stand for 3 min. Then, a sodium carbonate
solution (2 mL, 20% w/v) was added and heated to 40 °C
in a water bath for 2 min. The absorbance was measured
using a spectrometer (Perkin Elmer Lambda 25 UV/Vis-
ible Spectrophotometer, PerkinElmer Inc., Waltham, MA,
USA) at 760 nm. Results were expressed in Gallic acid
equivalents per gram of extract (mg GAE/g).
Analysis in gas chromatography coupled to mass spec-
trometry (GCMS) was carried out to obtain a qualitative
chemical characterization of hydroalcoholic extracts of
cocoa (70:30, MeOH:H 2O). Approximately 10 mL of
hydroalcoholic extract of cocoa was concentrated in a
rotary evaporator (Buchi, R-100) until methanol was elimi-
nated. The sample was resuspended with 1 mL of chloro-
form for subsequent injection into the GCMS.
Analyses were performed in a Shimadzu GCMS-
QP2010 Ultra gas chromatograph, equipped with a TR-
Wax capillary column (30 m, 0.25 mm, 0.25um) based
on a 100% polyethylene glycol (PEG) stationary phase.
Helium was employed as a carrier gas with a constant flow
as follows: 180 °C for 1 min, from 180 to 270 °C at 5 °C/
min, held at 270 °C for 5 min. The injector temperature
was held at 250 °C. The ion source was set at 200 °C and
interface at 250 °C. Full-scan spectra were collected from
35 to 700 m/z.
13
 R. A. Burbano‑Cachiguango et al.

Experimental design cocoa beans and fermentation time using the semi-automatic
method of fermentation.
It is possible to investigate a series of variables and their
effects using a factorial design. The simplest factorial design
involves two factors, each at two levels (­ 22). The advan- Results and discussion
tage of the two-factor study is a reduction in the number
of experimental runs while still being as precise as other Model fit and analysis of the effect of the factors
designs in terms of estimating the effect. The factorial design
provides mean contrasts, thus assigning statistical power to Table 2 shows the main parameters of the ANOVA for the
the effect estimates [16]. A two-level factorial experiment model applied to the data regarding the fermentation of
design (TLFD) (type 2­ 2) for main effects with 3 replicas cocoa beans.
was used with a total of 12 experimental runs. This allowed The p-value (p < 0.05) obtained showed that the model
the estimation of the effects of the two factors considered is significant and able to describe the behavior of the fac-
independent variables (fermentation method and genotype tors considered, with determination coefficients of ­R2 (0.90),
type). As a response variable, the fermentation percentage ­R2-adjusted (0.86), and R ­ 2-predicted (0.78). These values
was selected. The experimental planning was performed are within the range suggested by Anderson and Whitcomb
using the Design-Expert software, Version 12 (Stat-Ease, [16] for these types of studies. With respect to the independ-
USA), as well as ANOVA for the analysis of the results. The ent variables studied, the fermentation method was highly
experimental planning for the factorial design is presented in significant with a positive effect on the selected response
Table 1, which also includes the response variable. variable (p < 0.002). Meanwhile, the cocoa variety did not
significantly influence (p-value = 0.8145) the fermentation
Statistical analysis percentage. This indicates that the semi-automatic method
can be applied to both cocoa genotypes. In the Pareto chart
All analytical determinations were made in triplicate in Fig. 2, the significance of the factors and their interaction
and expressed as mean values with standard deviation can be graphically corroborated using the Bonferroni Limit
(Mean ± SD). Significant statistical differences between criterion.
data sets were evaluated by ANOVA with Tukey’s Test Figure 2 shows that the fermentation method and the
(p < 0.05) using Origin 2020b software (OriginLab Corpora- interaction of the two independent variables are highly sig-
tion, Northampton, Massachusetts, USA). In addition, Pear- nificant, exceeding the determined Bonferroni Limit. The
son’s test was used to determine the degree of correlation positive calculated effects are green, whereas the negative
between the main quality parameters of NC (A) and ESS (B) ones are red. In this way, the significance of the changes
introduced as part of the semi-automatic fermentation sys-
tem was statistically validated as having a positive influence.
Table 1  Factorial design (­22) setting for the variables: fermenta-
tion method (A), genotype (B), and the experimental results of the Main physicochemical characteristics of CN and ESS
response variable (fermentation percentage) cocoa bean fermentation using a semi‑automatic
system or Rohan Method
Run* Fermentation method (A) Genotype (B) Fermenta-
tion (%)
(C) In Table 3, the values of the main parameters related
to the quality of cocoa fermentation are shown for the
1 Rohan NC 87
2 Semi-automatic ESS 97
3 Semi-automatic NC 88 Table 2  Main parameters of the ANOVA of the factorial design for
the factors: fermentation method and cocoa genotype
4 Rohan NC 89
5 Rohan NC 88 Parameter Sum of df Mean squares F value p value
6 Semi-automatic ESS 98 squares Prob > F
7 Semi-automatic ESS 95 Model 419.67 3 139.89 24.69 0.0002
8 Rohan ESS 80 A-méthod 243.00 1 243.00 42.88 0.0002
9 Semi-automatic NC 87 B-genotype 0.33 1 0.33 0.059 0.8145
10 Rohan ESS 77 AB 176.33 1 176.33 31.12 0.0005
11 Semi-automatic NC 93 Pure error 45.33 8 5.67
12 Rohan ESS 83 Total cor- 465.00 11
rected
*Not randomized

13
Effect of a semi‑automated fermentation system on the physical and chemical characteristics…
Fig. 2  Pareto chart that visualizes the significance of the effects
regarding the factors evaluated in the fermentation process (Color fig-
ure online)
semi-automatic method and Rohan Method applied to the
two varieties of cocoa studied. In general, between the
determined values, no significant differences were found
(p < 0.05), regardless of the fermentation method used or
variety of cocoa.
The weight of 100 beans (Table 3) was higher for the NC
variety in both fermentation methods. However, the maxi-
mum value was obtained for the NC variety when the Rohan
Box Method was applied (over 130 g), a value established in
Ecuador’s INEN 176 standard [17] for the classification of
high-quality “Arriba Superior Summer Selecto (A.S.S.S.)”
fine cocoa. Meanwhile, for the semi-automated fermentation
system, lower values were obtained, hence a medium and
low-quality classification for NC and ESS, respectively, for
fine cocoa. These results are consistent with those reported
by Álvarez, Tovar [18], who in their study, when apply-
ing different types of fermenters, also found differences
Table 3  Main physicochemical Parameter
parameters of fermented cocoa
beans (NC and ESS) using
semi-automatic and Rohan box
methods Fermentation time (h)
Weight of 100 beans (g)
Cotyledon pH
Total fat (%)
TPC (mg GAE/g)
Means with the same letters in the same row do not present significant differences according to Tukey’s
Test (p < 0.05)
between the weight values of 100 beans. In the same study,
the authors suggested that in addition to the fermentation
method, cocoa variety is directly related to bean size and
weight.
Only the fat content showed significant differences under
the effect of genotype change. Regarding the variability of
the fat percentage results, a wide range of values can be
found in the literature, such as: 20.31% for cocoa type
CCN-51 and 41.44% for clone DIRCYT-C255, as reported
by Chang, Torres [19]; 50.7% for traditional criollo cocoa
grown in Chuao in Venezuela [20]. Various studies attribute
this variation in values to the cocoa variety and environmen-
tal factors [21, 22].
As for TPCs (Table 3), it was found that they did not dif-
fer significantly (p > 0.05) for the changes of fermentation
method and genotype investigated. These results regarding
fermentation method are consistent with a previous study
carried out by Samaniego et al. [4], in which the authors sug-
gest that the fermentation method does not influence TPC.
The variation in TPC is more conditioned to other factors,
such as fermentation time, geographic location, genotype,
ripeness of beans, and harvest time [23, 24]. It is impor-
tant to highlight that when the semi-automatic fermentation
method was applied, the quality parameters obtained for the
fermented beans were comparable to when the traditional
method (Rohan box) was used, with the advantage of reduc-
ing the fermentation time by 12 h.
The linear relationship of the chemical parameters
(weight of 100 beans, cotyledon pH, TPC, total fat, and fer-
mentation percentage) vs. fermentation time was determined
using an analysis of the Pearson correlation. Figure 3 shows
the results obtained from the Pearson correlation analysis.
In general, the results of the connections had comparable
values. The fermentation time and fermentation percentage
had a perfect positive confirmation (r = 1); similarly, in the
ESS variety, the value was 0.99. A high negative correlation
(0.8 < r < 0.99) between the following variables—weight of
100 beans, cotyledon pH, and TPC vs. fermentation time—
and fermentation percentage was found for both varieties
studied. Total fat content was the only variable for which
the results showed a moderate correlation (0.4 < r < 0.59)
Semi-automatic Rohan
NC ESS NC ESS
108 120
125.3 ± 15.2b 100.3 ± 14.3b 146.2 ± 16.5a 128.4 ± 12.2b
5.92 ± 0.4a 5.85 ± 0.2a 5.10 ± 0.3a 5.90 ± 0.6a
42.0 ± 2.1b 48.1 ± 0.7a 44.1 ± 1.4b,c 46.6 ± 0.5ª,c
50.0 ± 4.8a 52.8 ± 4.2a 49.4 ± 3.3a 53.1 ± 4.1a
13
 R. A. Burbano‑Cachiguango et al.

Fig. 3  The Pearson correlation coefficients of the main quality parameters of NC (A) and ESS (B) cocoa beans and fermentation time using a
semi-automatic fermentation method

or a weak correlation (0.2 < r < 0.39) with the other chemi-
cal parameters, for both the NC genotype and the ESS. The
results agreed with those reported by Akoa et al. [25], who
acknowledged a strong negative correlation between pH and
fermentation percentage. Similarly, a strong negative corre-
lation was observed between polyphenols and fermentation
percentage, comparable with results obtained by Caporaso
et al. [26]. As with those authors’ results, the current results
showed a strong decrease of polyphenols occurring during
cocoa bean fermentation, which started to decrease signif-
icantly at 72 h. The degree of fermentation is commonly
measured by assessing color changes inside the cocoa bean,
or by using TPC as an analytical parameter [26, 27].
The GCMS chromatograms (for more details see sup-
plementary material S1) showed the same compounds for
ESS cocoa beans samples under Rohan box or semiauto-
matic fermentation, and for NC coupled to semiautomatic
fermentation, but not for NC cocoa beans with Rohan box
fermentation (Table 4). Caffeine and theobromine, which
are methylxanthine compounds, were the main compounds
measured in all samples, with theobromine being the most
common and abundant, a characteristic in fermented cocoa
beans [28]. In addition to caffeine and theobromine, some
methyl esters were found in the NC fermented in Rohan
boxes. According to Allmaier and Pittenauer [29], cocoa
beans have an approximate 55% lipid content composed
of a predominant fraction of triglyceride molecules spe-
cies (oleic, stearic, palmitic, and linoleic acid), some of
which were detected by GCMS in the samples analyzed
(Table 4).

Table 4  Compounds identified by GC–MS in cocoa beans (NC and ESS) using semi-automatic and Rohan box fermentation
Compound Rohan box Semi-automatic
NC ESS NC ESS
Area (%) RT (min) Area (%) RT (min) Area (%) RT (min) Area (%) RT (min)

Hexadecanoic acid, methyl ester 1.88 4.46

Heptadecanoic acid, 16-methyl-, methyl ester 0.7 3.79
9-Octadecenoic acid, methyl ester, (E)- 1.69 3.56
1,2-Benzenedicarboxylic acid, butyl 2-methylpropyl 0.19 2.97
ester
n-Hexadecanoic acid 0.83 3.72 1.72 4.33
Caffeine 33.74 5.03 35.59 4.69 37.13 4.79 42.76 4.66
Theobromine 60.97 7.68 62.69 7.26 62.87 6.89 57.24 7.37

13
Effect of a semi‑automated fermentation system on the physical and chemical characteristics…

Main quality parameters of cocoa beans

110.4 ± 2.7a,b
105.9 ± 7.5a,b
98.8 ± 6.8b,c
95.2 ± 8.3b,c
94.3 ± 7.2b,c

61.6 ± 6.1d
48.5 ± 5.0d
47.8 ± 7.6d
120.9 ± 5.1a

83.0 ± 7.5c
during fermentation using the semi‑automatic
method

ESS
Fermentation is an essential step in the attainment of opti-

TPC (mg GAE/g)

mum-quality cocoa beans for the production of chocolate

104.5 ± 4.1a,b
100.1 ± 1.8a,b
with the flavor and aroma that the markets demand. The

97.4 ± 1.9b
94.4 ± 3.8b

55.7 ± 6.5d
51.4 ± 2.6d
109.2 ± 3.2a
108.6 ± 2.9a
107.6 ± 2.2a

78.1 ± 3.4c
values of the main quality parameters of cocoa beans during
the fermentation of NC and ESS genotypes using the semi-

NC
automatic method are shown in Table 5.
The fermentation percentage results were statistically sig-

6.36 ± 0.1a,b
6.25 ± 0.3a,b
6.12 ± 0.2a,b
6.07 ± 0.3a,b
6.02 ± 0.3a,b
6.01 ± 0.1a,b
5.94 ± 0.1a,b
5.92 ± 0.4a,b
nificant (p < 0.05), principally for the samples taken every

5.85 ± 0.1b
6.53 ± 0.1a
24 h, while the values determined every 12 h did not vary
significantly (p > 0.05). This behavior was maintained up to

ESS
the 84th h of fermentation; no significant differences were
found between the values obtained for the samples at 84 and

Cotyledon pH

6.27 ± 0.1a
6.37 ± 0.1a
6.39 ± 0.2a
6.32 ± 0.1a
6.29 ± 0.1a
6.21 ± 0.1a
6.12 ± 0.2a
5.98 ± 0.5a
5.96 ± 0.3a
5.90 ± 0.4a
108 h for both cacao varieties, although the value continued
to increase until ~ 89% at 108 h. Values above 90% would

NC
indicate over-fermentation and therefore a loss of quality
[18]. The Ecuadorian standard NTE INEN 176 (2021-02)

Means with the same letters in the same column do not present significant differences according to Tukey’s Test (p < 0.05)
“Cocoa beans. Requirements” establishes 75% as a mini-

42.7 ± 1.4a
42.0 ± 1.7a
42.4 ± 2.1a
42.2 ± 1.4a
42.1 ± 0.9a
42.9 ± 1.1a
41.6 ± 1.2a
41.7 ± 1.9a
41.4 ± 1.3a
42.0 ± 2.1a
mum value of fermented beans in order to classify them as
high-quality fine cocoa (A.S.S.S). This value was reached

ESS
using the semi-automatic method at 84 h. The fermentation
percentages obtained in this study are comparable to those
Total fat (%)
Table 5  Main quality parameters of cocoa beans during fermentation using the semi-automatic method

47.5 ± 1.6a
46.5 ± 2.5a
48.1 ± 2.6a
48.2 ± 3.1a
48.6 ± 2.5a
49.2 ± 0.8a
47.4 ± 2.2a
47.0 ± 1.2a
48.7 ± 0.9a
48.1 ± 0.7a
reported by Álvarez et al. [18] for Trinitario-type cocoa
(89%) using wooden boxes but over 5 days of fermenta-
tion. Nonetheless, they were higher than the values (64.0,
NC

63.3, 73.3, and 64.3%) reported by Fernández et al. [30]

for the jute sack, bearing or heap, wooden box, and plastic
108.6 ± 11.1a
105.8 ± 10.4a
105.1 ± 11.3a
104.3 ± 14.9a

102.2 ± 11.1a
101.3 ± 10.5a
100.7 ± 10.5a
100.3 ± 14.3a
120.7 ± 9.2a

103.0 ± 9.0a
bucket methods, respectively. These results suggest that the
changes introduced in the semi-automatic method provide an
Weight of 100 beans (g)
ESS

adequate fermentation of the cocoa beans with a reduction

in fermentation time.
In general, the variables weight of 100 beans, total fat,
144.8 ± 12.9a
136.1 ± 10.4a

132.2 ± 11.6a
132.0 ± 10.2a

125.3 ± 15.2a
131.9 + 11.8a
and cotyledon pH did not vary significantly (p < 0.05) during
149.2 ± 9.6a

135.9 ± 9.5a
133.8 ± 4.4a
132.4 ± 9.4a

the fermentation period.

An excess of acidity can be detrimental to the final quality
NC

of the cocoa beans, while lower total acidity values (higher

cotyledon pH) are related to good-quality cocoa. The coty-
ledon pH results in this study showed a tendency to decrease
16.3 ± 1.5b,c
26.3 ± 1.5c,d

78.3 ± 5.6f,g
8.3 ± 0.6 a,b

33.0 ± 2.0d

88.1 ± 2.5g
51.0 ± 4.6e
73.3 ± 3.5f

85. ± 4.7g

with fermentation time, a behavior that has been previously

reported by Puerto et al. [31]. This can be attributed to the
ESS

0.0a

increased presence of non-volatile (Citric, lactic, and malic)

Fermentation (%)

and volatile (Acetic, isovaleric, and isobutyric) acids pro-

11.0 ± 1.7a,b
21.7 ± 2.9b,c
32.7 ± 5.5c,d

46.7 ± 8.6d,e

duced mainly by mucilage degradation and microbial activ-

76.3 ± 8.3f,g
62.3 ± 6.5e,f
39.3 ± 8.4d

84.0 ± 5.0g
89.3 ± 3.2g

ity, which are later absorbed into the cotyledon. They bring
about an increase in acidity and consequently a decrease in
0.0a
NC

pH [32]. Jinap and Dimick [32] evaluated 39 cocoa sam-

ples from 13 countries and proposed a classification where
Fermentation

Ecuador was included among the countries with a high pH

time (h)

range (5.50–5.80). However, a review recently published by

108

Loureiro et al. [33] considered not only the geographical

12
24
36
48
60
72
84
96
0

13

region but also soil properties on the influence of acidity
and cotyledon pH. They suggested that values higher than
5 (cotyledon pH) indicate optimum quality values. The
cotyledon pH values for cocoa beans cultivated in yellow
argisol dystrophic latosolic (pH 6.3) and red-yellow argisol
dystrophic typic soil (pH 5.8) stand out. These soil types
are typical of humid zones like the cocoa region of Bahia
in Brazil with cotyledon pH of 6.01. This characteristic is
shared with the northern Ecuadorian Amazon, where the
samples for this study were taken. The cotyledon pH results
obtained for NC and ESS beans using the semi-automatic
method are comparable.
Regarding TPC (Table 5), ESS initially showed a higher
TPC than NC beans; however, at the end of the fermentation
process, the TPC decreased by 60.5 and 52.9%, respectively.
This suggests that polyphenol oxidase (PPO) oxidation and
anthocyanin degradation reactions were more pronounced
for ESS beans. Both genotypes showed the greatest variation
in TPC value from 72 to 96 h of fermentation. A decrease
in TPC is directly related to organoleptic quality, since by
decreasing the content of these compounds, the astringency
of the beans decreases and the pleasant flavor of the cocoa
bean improves [34]. Therefore, adequate levels of these
compounds must be established such that they do not result
in poor sensory quality nor lose their antioxidant potential.
According to Fernández et al. [30], a value of around 50 mg
GAE/g could be the reference for achieving this desired
balance.
The TPC results in the semi-automated system for the
two evaluated cocoa genotypes are consistent with those
observed by Fernández et al. [30] and comparable to those
previously obtained by Samaniego et al. [4] for NC samples
from the following three areas in the northern Ecuadorian
Amazon region: Coca (49.4 mg GAE/g), Loreto (43.6 mg
GAE/g), and Joya de los Sachas (50.4 mg GAE/g).
Conclusions
The changes introduced in the proposed fermentation sys-
tem (semi-automatic) positively influenced the fermentation
percentage, whereas the genotype factor did not present sta-
tistically significant differences (p < 0.05). This suggests that
the system can be applied to other varieties of cocoa and
reduce the fermentation time compared to the traditional
system (Rohan boxes). The physicochemical quality param-
eters determined in the cocoa beans fermented by the semi-
automatic method are within the range established in the
Ecuadorian standard in order to classify the cocoa as higher
quality fine cocoa. Defined trends can be observed in the
results for the variables analyzed, which are consistent with
the values reported in the literature. The values obtained for
cotyledon pH and TPC stand out, because they are directly
13
R. A. Burbano‑Cachiguango et al.
related to the organoleptic characteristics of chocolate and
its derivatives. Nonetheless, further studies should be car-
ried out into cocoa’s organoleptic properties, as well as, a
more in-depth chemical characterization concerning specific
components produced in cocoa fermentation.
Supplementary Information The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 11694-0​ 22-0​ 1620-x.
Acknowledgements The authors would like to thank the National
Institute of Agricultural Research (INIAP) and Universidad Estatal
Amazónica (UEA) for providing all necessary facilities and purchasing
the software Design Expert Version 12 (Number of series 9847-9696-
7992-6750) and Origin 2020b. The authors are indebted to Ms. Helen
Pugh for her extensive proofreading of the manuscript. We thank the
Core Lab for Environmental Sciences of Universidad San Francisco
de Quito for the GC-MS chemical characterization of hydroalcoholic
extracts of cocoa.
Author contributions RAB-C: conceptualization, performed the exper-
iments, analyzed and interpreted the data, and writing—original draft.
RA-N: conceptualization, methodology, validation and analysis tools
or data writing—review & editing, and supervision. CEC-V: performed
the experiments, and analyzed and interpreted the data. CÁR-R: per-
formed the experiments, and analyzed and interpreted the data. ASC-
C: performed the experiments, and analyzed and interpreted the data.
EML-M: GC–MS chemical characterization of hydroalcoholic extracts
of cocoa. MR-U: GC–MS chemical characterization of hydroalcoholic
extracts of cocoa funding and data analysis, and manuscript editing.
All authors reviewed and approved the final version of the manuscript.
Declarations
Conflict of interest The authors declare no conflict of interest.
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