Current Research in Food Science 6 (2023) 100494

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Current Research in Food Science

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Changes in bioactive compounds during fermentation of cocoa (Theobroma

cacao) harvested in Amazonas-Peru
Denny Cortez a, c, Luz Quispe-Sanchez a, Marilu Mestanza a, Manuel Oliva-Cruz a, Ives Yoplac b,
Cesar Torres c, Segundo G. Chavez a, *
a
Instituto de Investigación del Desarrollo Sustentable-Ceja de Selva (INDES-CES), Universidad Nacional Toribio Rodríguez de Mendoza (UNTRM), Amazonas, 01001,
Peru
b
Facultad de Ingeniería Zootecnista, Agronegocios y Biotecnología, Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas, Chachapoyas, 01001, Peru
c
Facultad de Ciencias, Universidad Nacional de Piura, Piura, 20002, Peru

A R T I C L E I N F O A B S T R A C T

Keywords: Cocoa (Theobroma cacao) is the main raw material for the production of chocolate; it is considered the food of the
UHPLC gods, as it possesses a diversity of bioactive compounds beneficial to human health. The abundance of bioactive
Bioactive compounds, among others, is conditioned by the post-harvest processing of cocoa beans, and fermentation is a
Cocoa
major step in this regard. Consequently, this research evaluated the changes in phenolic compounds and
Antioxidant
Fermentation
methylxanthines occurred in the fermentation of Criollo and CCN-51 cocoa beans, varieties of great commercial
interest for the cocoa-growing areas of Peru. For this purpose, samples were taken every 12 h of cocoa beans
under fermentation for 204 h in which phenols (gallic acid, caffeic acid, catechin, and epicatechin) and meth­
ylxanthines (theobromine, caffeine and theophylline) were quantified by ultra-high performance liquid chro­
matography (UHPLC); total polyphenols by Folin Ciocalteu; antioxidant capacity by DPPH free radical capture
method; total anthocyanins; pH; titratable acidity; and fermentation rate of beans. We found that during
fermentation, phenolic content, antioxidant activity, and methylxanthines of cocoa beans decreased; on the other
hand, the anthocyanin content increased slightly. Indeed, at distinctly degree, fermentation influences bioactive
compounds in cocoa beans, depending on the variety cultivated.

1. Introduction
In recent decade, there has been increasing interest in studying cocoa
and its derivatives as superfoods, not only for their unique sensory
characteristics, but also for their nutritional compounds beneficial to
human health. Cocoa is rich in bioactive compounds, predominantly
methylxanthine compounds such as theobromine and caffeine, as well as
polyphenols such as flavan-3-ol monomers with anthocyanins and
proanthocyanidins as the most abundant (Carrillo et al., 2014; Dugo
et al., 2018). Nonetheless, these compounds in high amounts can in­
crease astringency and bitterness in foods, there by the interest of
reducing the content without significantly affecting potential bioactivity
in the food (Carrillo et al., 2014).
Phenolic compounds account for 12–18% of the total compounds,
and their main precursors are amino acids and phenolic acids
(Cádiz-Gurrea et al., 2020). Cocoa represents one of the highest natural
sources of phenolic compounds compared to tea and red wine, which
requires further studies for optimal utilisation. Many studies have re­
ported on the potential beneficial effects of these phytochemicals.
Martin and Ramos (2021); Strat et al. (2016); Weikart et al. (2022) have
demonstrated important biological effects in in vitro studies related to
cardiovascular diseases, mitigation of chronic inflammation and obesity.
Álvarez-Cilleros et al. (2020) have shown that phenolic compounds
benefit intestinal integrity and inflammation in diabetic rats. In bio­
assays Żyżelewicz et al. (2016) and Żyżelewicz et al. (2020) conclude
that polyphenols in cocoa are potential modulators of insulin signalling
with possible anti-obesity properties; high antioxidant capacity in the
blood, ability to inhibit lipid peroxidation in the heart and increase
hepatic reduced glutathione concentrations. In addition, daily con­
sumption of dark chocolate leads to lower human blood pressure

* Corresponding author.
E-mail addresses: dennycortezcordova@gmail.com (D. Cortez), luz.quispe.epg@untrm.edu.pe (L. Quispe-Sanchez), marilu.mestanza@untrm.edu.pe
(M. Mestanza), manuel.oliva@untrm.edu.pe (M. Oliva-Cruz), ives.yoplac@untrm.edu.pe (I. Yoplac), ctorresd@unp.edu.pe (C. Torres), segundo.quintana@untrm.
edu.pe (S.G. Chavez).

https://doi.org/10.1016/j.crfs.2023.100494
Received 29 January 2023; Received in revised form 17 March 2023; Accepted 22 March 2023
Available online 4 April 2023
2665-9271/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
D. Cortez et al. Current Research in Food Science 6 (2023) 100494

(Maskarinec, 2009).
The agents responsible for the synthesis of bioactive compounds
during cocoa post-harvest processing are reducing sugars, peptides, and
amino acids (Toker et al., 2020). They are also influenced by factors of
genotype, physiological maturity of the bean, geographical origin, and
processing (fermentation and drying) (Hernández-Hernández et al.,
2018). Consequently, changes in bioactive compounds during cocoa
fermentation may also depend on the cocoa variety.
Fermentation of cocoa beans is one of the most important post-
harvest processes, as it not only influences on bean physical changes,
but also contributes to the development of the aroma and flavour pre­
cursors of cocoa (Marseglia et al., 2019; Schwan and Wheals, 2004).
During this process, cocoa beans are exposed to the action of microor­
ganisms (yeasts and bacterias) and enzymes, responsible for degrading
and metabolising substrates (Marseglia et al., 2014), the effectiveness of
which will determine the quality of the derived products (Schwan and
Wheals, 2004). In addition, it has been determined that microbial re­
actions occurring in the mucilaginous pulp trigger biochemical reactions
inside the grain, altering its phenolic composition and other bioactive
compounds (Cruz et al., 2015).
Some changes occurring in fermentation are well reported in the
literature. Yeasts and lactic acid bacteria initiate the fermentation pro­
cess by degrading the sugars in the pulp and converting them mainly
into ethanol. This exothermic process increases the temperature of the
cocoa mass and the oxygen tension, producing acetic acid from ethanol
(Afoakwa et al., 2012; Delgado-Ospina et al., 2020). During this process,
pH decreases and titratable acidity increases, besides, it generates em­
bryo death and cell wall rupture by the effect of ethanol and acetic acid.
There is also a decrease in the bitterness and astringency of the cocoa
beans caused by a decrease in phenolic content due to the activity of
phenol oxidase (Barišić et al., 2019; Taeye et al., 2017).
Despite the importance of fermentation for the quality of cocoa in the
food industry, this operation is still an artisanal process on cocoa farms,
using wooden boxes covered with leaves, plastic, jute, etc. Normally, the
fermentation process lasts 6 days under environmental conditions in the
Peruvian Amazon (Gutierrez et al., 2022), and the changes that occur in
many respects are advantageous (sensory profile, some bioactive com­
pounds). However, poorly executed processes can be disadvantageous,
as phenolic compounds and antioxidant activity of the cocoa bean
decrease (Melo et al., 2021; Oracz et al., 2015).
There is limited information on the behaviour of bioactive com­
pounds during cocoa fermentation addressing the changes that bioactive
compounds undergo on cocoa farms. In this context, the objective of this
study was to evaluate changes in phenolic compounds, methylxanthines,
and antioxidant activity during Criollo y CCN-51 cocoa fermentation
harvested in Amazonas-Peru.
2. Materials and methods
2.1. COCOA beans
were collected every 12 h from three equidistant points of the box during
the fermentation process and placed in low density polyethylene bags to
be transported with gelpacks to the Plant Biotechnology Laboratory
where they were stored at − 24 ◦ C until further evaluation.
2.3. pH and temperature of fermentation
The pH was measured according to the method of AOAC. Official
(1990) and temperature of fermentation was measured every 12 h by
inserting the thermometer into the fermentation heap.
2.4. Titratable acidity
From the solution prepared for pH measurement, titratable acidity
was measured according to AOAC 981.12/90 (AOAC. Official, 1990).
NaOH was used as neutralisation solution and phenolphthalein as in­
dicator. The acidity was expressed in mL of 0.1 M NaOH needed to
neutralise the acids present in 100 g of fresh sample.
2.5. Preparation of extracts for analysis
Samples were defatted following the methodology of Ioannone et al.
(2015) with some modifications. For this purpose, they were thawed at
room temperature for 1 h, then 10 g each were crushed with the addition
of liquid nitrogen and sieved at 850 μm (sieve No. 40). We weighed 0.5 g
of the samples in 15 mL centrifuge tubes. After that, 3 mL of hexane was
added in the fume hood; next step, the tubes were shaken horizontally at
300 revolutions per minute (RPM) for 10 min and centrifuged at 2078 G
(G-force) for 15 min. The supernatants were discarded and the contents
of the tubes were defatted 3 more times. Finally, after the last defatting
the precipitates were left to dry for 24 h in dark.
From the defatted samples, methanolic extracts were prepared ac­
cording to the methodology described by Melo et al. (2021) with some
modifications. For this, 100 mg of the defatted samples were added to
centrifuge tubes containing 10 mL of methanol-water (80:20, v/v), then
shaken horizontally at 300 RPM for 15 min on an orbital shaker and
centrifuged at 2078 G (G-force) for 30 min. The supernatants (liquid
phase) were filtered with filter paper (Watman No. 40) and stored at
− 24 ◦ C in absence of light. All samples were run in triplicate.
2.6. Determination of phenolic compounds and methylxanthines
Solutions of individual standards were prepared: gallic acid,
Table 1
Range of phenolic and methylxanthine standards concentrations, retention time
and linear equations.
Compound Concentration range Standard curve
(μg/mL)
Retention Linear equation (R2)
time

The pods of Criollo cocoa and clone CCN-51 (Castro Naranjal Phenolics compounds
Collection) were collected from plots located in the village of “Naranjos Gallic acid 5–300 06.9 y = 8336.3x - 6110.1
(0.999)
Alto”, Utcubamba, Amazonas (S 5◦ 44‫יי‬16.52 ‫י‬, W 78◦ 20‫יי‬29.363 ‫ )י‬and
Catechin 1–20 12.8 y = 36.366x - 9.2388
(S 5◦ 43‫יי‬26.64 ‫י‬, W 78◦ 20‫יי‬33.65 ‫)י‬, respectively. The fruits were opened (0.999)
with knives in situ to remove the kernels from the cobs, then only the Caffeic acid 1–20 15.3 y = 70.517x - 20.703
kernels in optimum state of maturity and free of diseases were selected, (0.999)
then they were transferred in bags to the district of Cajaruro where Epicatechin 1–20 15.9 y = 42.693x - 10.304
(0.999)
fermentation was carried out.

Methylxantines
2.2. Fermentation of cocoa beans
Theobromine 1–20 09.1 y = 202.37x - 43.541
(0.999)
Criollo and CCN-51 cocoa beans were placed in 12 × 12 × 12 cm Theophylline 1–20 10.9 y = 175.24x - 32.894
wooden boxes with holes at the bottom for mucilage drainage, and 10 kg (0.999)
of each variety were fermented for 9 days covered with jute bags. After Caffeine 1–20 14.1 y = 160.26x - 30.87
(0.999)
48 h, the heap was daily stirred until the end of fermentation. Samples

2
D. Cortez et al.
theophylline, theophylline, catechin, epicatechin, caffeic acid, caffeine,
and theobromine in concentrations (Table 1).
The methanolic extracts and standards were filtered through 0.45 μm
filters and placed in amber chromatography vials. The determination of
phenolic compounds and methylxanthines was carried out based on the
methodology described by Cruz et al. (2015) with some adaptations.
Ultra-high-performance liquid chromatography (UHPLC) equipment
(Chromatograph, Agilent Technologies, Santa Clara, California, USA)
equipped with a Zorbax Eclipse Plus C18 column (Agilent Technologies,
Santa Clara, California, USA) of 4.6 × 250 mm and 5 μm particle size,
coupled to a diode array detector (Agilent Technologies, Santa Clara,
California, USA), an autoinjector and software was used. The injection
volume was 20 μL, the run time was 22 min, the C18 Column temper­
ature was 26 ◦ C and using in the mobile phase the solvents (A): 2% acetic
acid in water and (B): a mixture of acetonitrile, water and acetic acid
(400:90:10 v/v/v) filtered under vacuum using 0.22 μm filters. The
mobile phase gradient used was: 0–2 min, 0.4 mL/min, 90% A, 10% B;
2–5 min, 0.3 mL/min, 88% A, 12% B; 5–8 min, 0.4 mL/min, 86% A, 14%
B; 8–10 min, 0.4 mL/min, 84% A, 16% B; 10–12 min, 0.5 mL/min, 82%
A, 18% B; 12–22 min, 0.5 mL/min, 90% A, 10% B. The compounds were
monitored by UV detection at 280 nm wavelength. Identification of the
bioactive compounds was performed using the retention times of the
above mentioned standards and quantification by interpolation of the
linear equations of the standards (Table 1). The results were expressed in
mg standard equivalent/g cocoa on dry base (d.b.).
2.7. Fermentation index
We followed the methodology proposed by Granato and Savío
(2016). The absorbance at 460 nm and 535 nm of the sample extracts
was measured and the fermentation index was calculated using equation
(2.7).
Aλ460
Fermentation index = Eq. (2.7)
Aλ535
2.8. Total polyphenols
Total phenolic compounds were determined by the Folin-Ciocalteu
colorimetric assay following the methodology described by Cadena
and Herrera (2008) with some modifications. 50 μL of each methanolic
extract was placed in test tubes, then 1.5 mL of aqueous Folin-Ciocalteu
solution (1:10 v/v) was added followed by 1.5 mL of Na2 CO3 at 7.5%
(diluted in deionised water); the solutions were vortexed for approxi­
mately 30 s and left in an oven at 50 ◦ C for 5 min to generate the re­
action. Finally, the absorbances of the samples were read in the
UV–Visible spectrophotometer (UV-VIS PEAK Instruments Inc, 16223
Park Row, Houston, USA) at a wavelength of 765 nm. The quantification
of total phenols was obtained from the standard curve obtained for gallic
acid (y = 2.4253 x + 0.0436) at concentrations of 0.01–0.1 mg/mL, and
R2 = 0.9949. Results were expressed as mg gallic acid equivalent/g
cocoa (d.b).
2.9. Antioxidant capacity
The determination of antioxidant capacity was performed using the
2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical uptake technique
following the methodology described by Batista et al. (2016) with some
modifications. DPPH solution was prepared (20 mg/L methanol) until
reaching the absorbance of 0.5–516 nm. We added 77 μL of the meth­
anolic extracts were added into test tubes containing 3 mL of DPPH
solution, then vortexed for 30 s and allowed to stand for 15 min in the
dark. After this time, the absorbance of the reactions was measured at a
wavelength of 516 nm in a UV-VIS spectrophotometer. The antioxidant
capacity was expressed as percentage inhibition, calculated by equation
(2.9).
3
Current Research in Food Science 6 (2023) 100494
Where, A0 : absorbance of DPPH solution, Am : absorbance of meth­
anol and As : absorbance of sample extract. The results were expressed in
mg Trolox equivalent/g cocoa (d.b.), for which a linear Trolox calibra­
tion curve (y = 231.88x – 2.7837) was used at concentrations of
0.025–0.25 mg/mL and a linearity of R2 = 0.9946.
(A0 − Am ) − (As − Am )
% of inhibition = x 100 Eq. (2.9)
(A0 − Am )
2.10. Total anthocyanins
Total anthocyanins were assessed following the methodology
described by Granato and Savío (2016). The absorbance of the meth­
anolic extract was measured at 530 nm. The concentration of total an­
thocyanins was determined using equation (2.10).
Where, C: total anthocyanin concentration, A: absorbance at λ =
535nm, specific absorptivity E1%
1cm = 98.2.
Aλmáx
C (mg / 100g) = 1%
Eq. (2.10)
E1cm
2.11. Statistical analysis
Treatments were compared by analysis of variance and Tukey’s
multiple comparisons to determine statistical differences. The changes
in the contents of the bioactive compounds studied were obtained by
regression analysis; and the coefficients of determination and equations
of the models are presented. The statistical package SPSS V. 26 and
spreadsheet MS. Excel spreadsheet was used.
3. Results and discussion
3.1. Physicochemical analysis during fermentation
Given that fermentation processes are exothermic processes and as
expected (Díaz-Muñoz et al., 2023), in the fermentation of cocoa beans,
the temperature was progressively increased up to 41 ◦ C in 144 and 156
h for CCN-51 and Criollo varieties, respectively. Thereafter, the tem­
perature decreases slightly to different degrees for each variety. The
maximum temperature found for both cocoa types (>41 ◦ C) can be
considered suitable, considering it sufficient to inactivate the germina­
tion power of the seeds while generating the precursors of chocolate
aroma and flavour (Visintin et al., 2017).
On the other hand, pH decreased significantly during fermentation in
the two cocoa varieties, going from 6 to 3 and 4 at the end of the process,
values that agree with the previous reports (Nazaruddin et al., 2006).
Therefore, it may also be an indicator of the efficiency of the fermen­
tation process, since sugar degradation by microbial enzymes alters the
pH of the cocoa pulp and, consequently, the pH of seeds (Enrique and
Liévano, 1951). All these changes are typical and guarantee a successful
fermentation of the cocoa (Castro-Alayo et al., 2019). Furthermore, at
pH values close to 5, protease activity, which is important in the for­
mation of aromas in chocolate, is higher (Ho et al., 2014). Comple­
mentarily, titratable acidity increased inversely to pH during
fermentation.
Criollo beans needed less time to complete fermentation (156 h),
while CCN-51 variety had an additional time of 24 h. Although
fermentation rates above 1.6 indicate over-fermentation due to bean
colour changes values below 1 indicate insufficient fermentation (Ooi
et al., 2020). As it is displayed in Table 2, fermentation between 12 and
24 h of CCN-51 had high indices (1.76 and 1.66), which could be
erroneously attributed to over-fermented cocoa; however, because the
index is constructed by difference of absorbances, extracts from beans
coloured beans could influence the calculation. Consequently, in­
consistencies in the values found in the fermentation indices in the study
were due to the varying colour of the cocoa beans used (see Fig. 1).
D. Cortez et al. Current Research in Food Science 6 (2023) 100494

Table 2
Fermentation temperature, pH, titratable acidity, and fermentation rate of Criollo and CCN-51 cocoa.
Ferm. time (h) Fermentation temperature (◦ C) pH Total acidity (%) Fermentation index

Criollo CCN-51 Criollo CCN-51 Criollo CCN-51 Criollo CCN-51

0 31.00 ± 0.00k 31.67 ± 0.29h 6.67 ± 0.01a 6.72 ± 0.08a 0.01 ± 0.00p 0.11 ± 0.01hi 1.45 ± 0.08bc 1.55 ± 0.04ab
12 29.00 ± 0.00l 29.17 ± 0.29j 6.48 ± 0.01a 6.55 ± 0.00ab 0.02 ± 0.00o 0.10 ± 0.00i 1.44 ± 0.01bc 1.76 ± 0.02a
24 31.50 ± 0.00j 31.50 ± 0.00hi 6.33 ± 0.03ab 6.46 ± 0.04bc 0.03 ± 0.00n 0.10 ± 0.00i 1.32 ± 0.01d 1.66 ± 0.09ab
36 29.33 ± 0.29l 31.00 ± 0.00i 6.35 ± 0.02ab 6.44 ± 0.05bc 0.03 ± 0.00n 0.11 ± 0.01hi 1.39 ± 0.02cd 1.53 ± 0.36ab
48 32.50 ± 0.00i 33.33 ± 0.58g 6.28 ± 0.02ab 6.33 ± 0.06cd 0.04 ± 0.00m 0.11 ± 0.01hi 1.34 ± 0.02d 1.56 ± 0.03ab
60 34.50 ± 0.00e 33.00 ± 0.00g 6.14 ± 0.04abc 6.33 ± 0.13cd 0.05 ± 0.00l 0,12 ± 0,00h 1.44 ± 0.04bc 1.59 ± 0.00ab
72 36.50 ± 0.00b 38.00 ± 0.00d 5.89 ± 0.00bcd 6.31 ± 0.01cd 0.06 ± 0.00k 0,17 ± 0,01g 1.39 ± 0.04cd 1.51 ± 0.01ab
84 33.67 ± 0.29g 33.50 ± 0.00g 5.68 ± 0.30cd 6.24 ± 0.06d 0.06 ± 0.00k 0,18 ± 0,01fg 1.51 ± 0.00b 1.46 ± 0.03b
96 34.00 ± 0.00fg 35.50 ± 0.00f 5.81 ± 0.19bcd 6.17 ± 0.06d 0.07 ± 0.00j 0,20 ± 0,01f 1.44 ± 0.01bc 1.48 ± 0.00b
108 31.00 ± 0.00k 33.17 ± 0.29g 5.54 ± 0.17de 5.65 ± 0.02e 0.08 ± 0.00i 0,22 ± 0,01e 1.40 ± 0.01cd 1.46 ± 0.05b
120 35.23 ± 0.40d 35.00 ± 0.00f 5.39 ± 0.19de 5.63 ± 0.01e 0.09 ± 0.00h 0,23 ± 0,01e 1.52 ± 0.01b 1.39 ± 0.01b
132 33.00 ± 0.00h 38.00 ± 0.00d 5.09 ± 0.53ef 5.07 ± 0.06f 0.10 ± 0.01g 0,23 ± 0,01e 1.52 ± 0.04b 1.44 ± 0.01b
144 34.33 ± 0.29ef 41.00 ± 0.00a 5.08 ± 0.29ef 4.90 ± 0.01f 0.11 ± 0.00f 0,25 ± 0,01d 1.46 ± 0.04bc 1.51 ± 0.01ab
156 41.00 ± 0.00a 39.83 ± 0.29b 4.42 ± 0.03gh 4.45 ± 0.08g 0.12 ± 0.00e 0,52 ± 0,01c 1.63 ± 0.02a 1.51 ± 0.00ab
168 36.00 ± 0.00c 36.67 ± 0.29e 4.41 ± 0.01gh 4.23 ± 0.03h 0.14 ± 0.00d 0,53 ± 0,01c 1.63 ± 0.01a 1.48 ± 0.02b
180 33.00 ± 0.00h 35.00 ± 0.00f 4.61 ± 0.01fg 4.24 ± 0.02h 0.16 ± 0.00c 0.70 ± 0.01b 1.62 ± 0.01a 1.62 ± 0.00ab
192 35.00 ± 0.00d 39.00 ± 0.00c 4.00 ± 0.00h 4.28 ± 0.12gh 0.18 ± 0.00b 0.72 ± 0.00a 1.66 ± 0.03a 1.49 ± 0.03ab
204 32.50 ± 0.00i 33.50 ± 0.00g 3.00 ± 0.00i 4.14 ± 0.04h 0.20 ± 0.00a 0.74 ± 0.01a 1.68 ± 0.01a 1.47 ± 0.00b

Note: *Values expressed with standard deviation from the mean per g of sample on a dry basis (n = 3). Values with different letters within a column are significantly
different, according to the standard deviation Tukey test (p < 0.05).

3.2. Change in major phenolic compounds during cocoa fermentation
Fig. 2 shows that the four phenolic compounds quantified by liquid
chromatography are affected during cocoa fermentation. The tendencies
to decrease are more defined in Criollo cocoa than in CCN-51; in the
latter, contents of caffeic acid and epicatechin increase up to 75 h of
fermentation, and then there is decrease in their concentration. Impor­
tantly, the decrease in the content of phenolic compounds is not always
due to the degradation of these compounds, but rather to their reaction
with other more complex compounds. For example, gallic acid esterifies
and binds to a hydroxyl group of a polyol to form gallotannins (Badui,
2006).
We also found an inverse relationship between catechin and epi­
catechin contents (Fig. 2c and d). Criollo cocoa has higher catechin
content and lower epicatechin content than CCN-51.
Although the general trend is a reduction in phenolic content during
fermentation, the best models indicate a slight increase in some cases at
the end of the fermentation process, mainly in gallic acid, caffeic acid,
and epicatechin in Criollo cocoa. This could be due to the variety’s own
composition (Segura, 2019), which has an impact on changes in pH and
temperature, facilitateing the reduction of flavonoids and enzymatic
action of polyphenoloxidase (Badui, 2006; Febrianto and Zhu, 2020).
3.3. Change in methylxanthines during cocoa fermentation
Methylxanthines were also affected by the fermentation process
(Fig. 3). When parallel curves are obtained, it is evidence that the effect
of fermentation is similar for both varieties of cocoa studied, and the
differences lie mostly in the initial composition of the bean.
Unsurprisingly, the predominant methylxanthine was theobromine
(Fig. 3a), 5 to 7 times more than caffeine (Rojo-Poveda et al., 2020),
showing higher concentration in Criollo cocoa, in line with the reports of
Hernández-Hernández et al. (2018). The behaviour of theobromine in
the first hours of fermentation had no significant reduction, it may be
due to the structure of the bean that has not yet been altered and there
are no alkaloids that can penetrate through the testa (Bartella et al.,
2019). After 60 h of fermentation, there was a decrease of the content
until the end of fermentation, agreeing with the report made by

Fig. 1. Graphical abstract.

4
D. Cortez et al.
Fig. 2. Behaviour of phenolic compounds during fermentation of criollo and CCN-51 cocoa beans.
Ramos-Escudero et al. (2021).
Similar to the other compounds, temperature increase during
fermentation is critical for changes in the composition of methylxan­
thines such as theophylline in cocoa. Slight increases at the onset of
fermentation may be due to the extraction technique being less efficient
for raw cocoa beans (Bucheli et al., 2001) or that osmotic translocation
has already started (Brunetto et al., 2007).
As Luna et al. (2002) have demonstrated, the caffeine content was
also considerably reduced during cocoa fermentation. Although cocoa is
not the main source of caffeine in the human diet, its availability pro­
vides apart from many properties, the stimulating capacity in derived
foods.
The importance of this phenomenon lies in the fact that the reduction
of methylxanthines significantly improves the sensory properties of
cocoa products, due to the reduction of bitterness and astringency
(Febrianto and Zhu, 2020; Sandhya et al., 2016); however, their removal
would detract from the bioactivity of the food product, as these com­
pounds have been shown to help stimulate the release of serotonin in the
cerebral cortex, the brain (Badui, 2006) and are active principles asso­
ciated with bronchial relaxation (James, 2004). Therefore, cocoa
fermentation processes must be designed and executed in such a way as
to preserve concentrations of methylxanthines that benefit palatability
and preserve the bioactivity of the cocoa bean that will transferred to
delivered product such as chocolate (James, 2004).
3.4. Total polyphenols, antioxidant capacity and total anthocyanins
The behaviour of phenolic compounds and antioxidant capacity
decreased during the fermentation process in the two varieties evalu­
ated. Criollo cocoa had the highest values in both trials. Urbańska y
Kowalska. (2019) reported that the phenolic content in cocoa beans
differs between cultivars and geographical origin, which has been
corroborated by this research.
The trend patterns of total phenolic content and antioxidant capacity
of fermenting cocoa are very similar (Fig. 4a and b). In fact, flavonoids
5
Current Research in Food Science 6 (2023) 100494
have been shown to be the most important antioxidant compounds with
high bioactivity (Betin, 2022). Nitrogen compounds (alkaloids) have
also been shown to significantly delay or inhibit oxidation in the human
organism (Segura, 2019); therefore, methylxanthines may also be
responsible for the antioxidant capacity of cocoa.
Interesting, the anthocyanin content increased during the last hours
of fermentation to a greater extent for CCN-51 than for Criollo cocoa.
Notably, anthocyanins change colour when they form complexes with
other phenolic compounds (catechins, tannins, or flavonoids) or with
some polysaccharides, as it favours a shift of absorption to longer
wavelengths (Badui, 2006). In addition, anthocyanins are known to
contribute to food colour (Segura, 2019), are stimulated by light, and
influenced by temperature.
This research complements the knowledge on the change of bioactive
compounds reported by researchers such as Caporaso et al. (2018), who
mentioned that the origin and fermentation process of cocoa influence
the content of bioactive compounds in the bean. Also, the variety is
another factor that determines the biochemical composition of the cocoa
bean (Brito et al., 2017; Calvo et al., 2021).
As demonstrated in this research, Brunetto et al. (2007) found a
slight reduction of methylxanthines in Criollo and Forastero cocoa, also,
Febrianto and Zhu (2020) reported a reduction of catechins up to 15% at
240 h of fermentation.
In addition to the factors already described such as the genetic factor
(Barbin et al., 2018), biochemical changes could be associated with the
action of microorganisms (Viesser et al., 2021), taking into account that
microbial diversity, as demonstrated by Serra et al. (2019), depends on
geo-environmental conditions.
4. Conclusions
During fermentation, the phenolic content of cocoa is reduced and
these changes depend on the initial composition of the bean by variety.
Consequently, the bioactivity of the bean is also affected. Principal
cocoa methylxanthines are theobromine, caffeine, and theophylline,
D. Cortez et al. Current Research in Food Science 6 (2023) 100494

Fig. 3. Behaviour of methylxanthines during fermentation of criollo and CCN-51 cocoa beans.

6
D. Cortez et al. Current Research in Food Science 6 (2023) 100494

Fig. 4. Behaviour of total polyphenols, antioxidant capacity and total anthocyanins during fermentation of cocoa beans in the Criollo and CCN-51 varieties.

7
D. Cortez et al.
which are also influenced by the post-harvest fermentation process of
the bean.
Total polyphenols and antioxidant capacity decrease during
fermentation. However, the anthocyanin content increases slightly
during cocoa fermentation, though more specific studies are needed.
Finally, fermentation, indeed, affected the bioactive compounds of
cocoa beans to varying degrees depending on the variety.
Gratitude
We would like to express our gratitude to Gladis Marlon for her help
with the traduction of paper and colleagues of FISIOVEG department the
Instituto de Investigación para el Desarrollo Sostenible de Ceja de Selva
(INDES-CES) of the Universidad Nacional Toribio Rodríguez de Men­
doza in Amazonas.
Financing
This work was supported by National Council of Science, Technology
and Technological Innovation, Project N◦ 008-2020-FONDECYT-BM,
METACACAO.
CRediT authorship contribution statement
Denny Cortez: Conceptualization, Investigation, Data curation. Luz
Quispe-Sanchez: Investigation. Marilu Mestanza: Investigation.
Manuel Oliva-Cruz: Conceptualization, Methodology, Resources,
Writing – original draft, and, Writing – review & editing, Visualization,
and, Funding acquisition. Ives Yoplac: Investigation. Cesar Torres:
Methodology. Segundo G. Chavez: Conceptualization, Methodology,
Resources, Data curation, Writing – original draft, and, Writing – review
& editing, Visualization, and, Funding acquisition, All authors have read
and agreed to the published version of the manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
No data was used for the research described in the article.
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