foods
Article
Kinetics Crystallization and Polymorphism of Cocoa Butter
throughout the Spontaneous Fermentation Process
Efraín M. Castro-Alayo 1,2,3,* , Llisela Torrejón-Valqui 1, Marleni Medina-Mendoza 1, Ilse S. Cayo-Colca 4
and Fiorella P. Cárdenas-Toro 3
1 Instituto de Investigación, Innovación y Desarrollo para el Sector Agrario y Agroindustrial de la Región
Amazonas (IIDAA), Facultad de Ingeniería y Ciencias Agrarias, Universidad Nacional Toribio Rodríguez de
Mendoza de Amazonas, Calle Higos Urco 342-350-356, Chachapoyas 01001, Amazonas, Peru;
llisela.torrejon@untrm.edu.pe (L.T.-V.); marleni.medina.epg@untrm.edu.pe (M.M.-M.)
2 Programa de Doctorado en Ingeniería, Departamento de Ingeniería, Pontificia Universidad Católica del Perú,
Av. Universitaria 1801, San Miguel 15088, Lima 32, Peru
3 Sección de Ingeniería Industrial, Departamento de Ingeniería, Pontificia Universidad Católica del Perú,
Av. Universitaria 1801, San Miguel 15088, Lima 32, Peru; fcardenas@pucp.pe
4 Facultad de Ingeniería Zootecnista, Agronegocios y Biotecnología, Universidad Nacional Toribio Rodríguez
de Mendoza de Amazonas, Calle Higos Urco 342-350-356, Chachapoyas 01001, Amazonas, Peru;
icayo.fizab@untrm.edu.pe
* Correspondence: efrain.castro@untrm.edu.pe; Tel.: +51-986376463
Abstract: The spontaneous fermentation process of Criollo cocoa is studied for its importance in the
development of chocolate aroma precursors. This research supports the importance of spontaneous
fermentation, which was studied through the crystallization behavior and polymorphisms of cocoa
butter (CB), the most abundant component of chocolate that is responsible for its quality physical
properties. The k-means technique was used with the CB crystallization kinetics parameters to
Citation: Castro-Alayo, E.M.; observe the division of the process during the first stage (day 0–3). The experimental crystallization
Torrejón-Valqui, L.; Medina- time was 15.78 min and the second stage (day 4–7) was 17.88 min. The Avrami index (1.2–2.94)
Mendoza, M.; Cayo-Colca, I.S.; showed that the CB crystallizes in the form of a rod/needle/fiber or plate throughout the process.
Cárdenas-Toro, F.P. Kinetics CB produced metastable crystals of polyforms β′1 and β′2. Three days of fermentation are proposed to
Crystallization and Polymorphism of generate Criollo cocoa beans with acceptable CB crystallization times.
Cocoa Butter throughout the
Spontaneous Fermentation Process. Keywords: criollo cocoa; cocoa butter; polymorphism; crystallization; differential calorimetry scanning;
Foods 2022, 11, 1769. https://
Avrami model; kinetics
doi.org/10.3390/foods11121769
Academic Editor: Danyang Ying
Received: 5 May 2022
1. Introduction
Accepted: 13 June 2022
Published: 15 June 2022 Cocoa butter (CB) is a natural fat extracted from cocoa beans [1]. CB has a crystalline
structure that determines the final quality of chocolate [2,3]. CB has a crystal acylglyc-
Publisher’s Note: MDPI stays neutral erols (TAGs) [4,5] that define its crystallization properties [6–8]. CB is mainly composed
with regard to jurisdictional claims in of 23.2–29.3% 1,3-di-stearoyl-2-oleoyl-glycerol (SOS), 34.2–38.6% 1-palmitoyl-2-oleoyl-3-
published maps and institutional affil-
stearoyl-glycerol (POS), and 13.5–17.1% 1,3-dipalmitoyl-2-oleoylglycerol (POP) [9,10]. These
iations.
TAGs allow CB to crystallize in six polyforms, which are γ, α, β′ ′2 , β′1, β2, and β1, and are
identified according to their melting temperatures (17.3, 23.3, 25.5, 27.5, 33.8, and 36.3 ◦C;
respectively) [9,11–14] and stability. Although the contribution of POP is less than those of
Copyright: © 2022 by the authors. POS and SOS, its presence may influence the formation of metastable polymorphic forms in
Licensee MDPI, Basel, Switzerland. CB [15]. The polymorphism is related to the organoleptic and physical characteristics of the
This article is an open access article chocolate [16]. That means that CB crystals define the sensory characteristics of chocolate,
distributed under the terms and such as flavor release, mouthfeel, and melting properties [3]. Characterizing these crystalline
conditions of the Creative Commons forms is crucial to meeting market needs, optimizing processes, or developing new prod-
Attribution (CC BY) license (https:// ucts [17]. Therefore, CB crystallization is an important phenomenon that must be considered
creativecommons.org/licenses/by/ to obtain high-quality chocolates [18]. Obtaining the β2 polyform (form V) is very important
4.0/).
Foods 2022, 11, 1769. https://doi.org/10.3390/foods11121769 https://www.mdpi.com/journal/foods
Foods 2022, 11, 1769 2 of 18
for chocolate manufacturers [8,17,19–21] due to it giving the optimal characteristics of gloss,
snap, texture, melting, sensation in the mouth, and bloom resistance [4,20,22].
In the chocolate manufacturing chain, the fermentation of cocoa beans is essential for
the generation of aromatic compounds in chocolate [23–25] and other metabolites, such
as amino acids, amines [26], and polyphenols [27], generated by biochemical reactions
that are favored by temperature, pH, and time [28]. This process occurs spontaneously on
farmers’ farms, unlike other fermentation processes [29]. The fermentation methods used
and the action of indigenous microorganisms result in a very heterogeneous process [29].
Therefore, controlling the process variables is necessary to obtain cocoa of better quality
and homogeneity [28]. A widely studied fermentation process is acidification by acetic acid
(the corresponding pH is a variable), whose action produces biochemical modifications
related to chocolate flavor [30]. Although they could also be related to the crystallization
behavior of CB during fermentation, there is not enough evidence for it [5,31]. Moreover,
no studies have analyzed the crystallization of CB in its natural state during spontaneous
fermentation (when CB is under the influence of other chemical components of the cocoa
bean). We believe that the closest test to reality would be to study the crystallization of CB
inside the cocoa bean without being influenced by any extraction method.
The most used method to describe the behavior of cocoa butter is the Avrami model,
which describes the kinetics of crystallization and crystal growth [1,32,33]. Avrami’s equa-
tion describes an initial period where crystallization occurs slowly and is followed by
a rapid increase in the mass of the crystal formed. It is assumed that this process oc-
curs under isothermal conditions, and crystal growth is by random nucleation and linear
growth [34–36]. On the other hand, the analytical technique of X-ray diffraction (XRD) is
the most used to study the polymorphism of fat crystals. However, access to XRD equip-
ment is not easy in a research and development unit, so differential scanning calorimetry
(DSC) [12] is an alternative. MacNaughtan et al. [37] demonstrated that the DSC technique
could provide reproducible kinetic data on the tristearin-tripalmitin crystallization and
polymorphism. They also provided the identity of the polymorphs after the fusion of
the crystallized material. Similarly, Simoes et al. [38] used DSC to study the polymorphic
transition of CB from form β′1 to β2, in the presence of emulsifiers and sugar.
Optimizing the fermentation process to obtain good quality cocoa beans involves
studying the metabolites generated [39]. However, we also consider it necessary to describe
the fermentation process based on the quantitative crystallization kinetics and CB polymor-
phism. Therefore, the objective of this work was to describe the kinetics of crystallization
and polymorphism of CB throughout the spontaneous fermentation process.
2. Materials and Methods
2.1. Materials and Chemicals
Fresh Criollo cocoa beans and pure CB were provided by Cooperativa de Servi-
cios Múltiples Aprocam (Bagua–Amazonas–Peru). The 1,3-dipalmitoyl-2-oleoylglycerol
(POP) ≥99% standard was purchased from Sigma Aldrich.
2.2. Monitoring Fermentation
Considering the work carried out by Deus et al. [26], the spontaneous fermentation
of Criollo cocoa was carried out in the Aprocam cocoa processing center. The cocoa pods
were harvested and opened with a stainless-steel knife. The beans, surrounded by their
pulp, were placed in polypropylene bags, taken to the processing center, and placed in
50 × 50 × 50 cm wooden boxes (40 kg capacity). The boxes were covered with jute bags to
prevent the bean mass from losing heat. The bean mass was passed from one box to another
to allow aeration. During each day of fermentation, the temperature and pH parameters
were recorded. Cocoa bean samples (50 g) were extracted daily from the total mass, and we
recorded the start time (Day 0) and end time of fermentation (day 7). The samples were
packed in sterile polypropylene bags and stored in liquid nitrogen until they were taken to
the UNTRM laboratory for physicochemical analysis in duplicate.
Foods 2022, 11, 1769 3 of 18
2.3. Physico-Chemical Parameters
2.3.1. Titratable Acidity and pH of the Cocoa Beans
The titratable acidity and pH were determined according to the AOAC 942.15 and
970.21 methods [40].
2.3.2. Moisture Content
According to Elbl et al. [41], with modifications, the moisture content of cocoa beans
was determined using a halogen moisture analyzer (Mettler Toledo, Excellence plus HX204,
Greifensee, Switzerland) based on the gravimetric principle. One gram of sample was
weighed in an analyzer crucible and heated to 105 ◦C until a constant weight was ob-
tained. The difference in weight (initial and final) calculated by the analyzer gave the
moisture content.
2.3.3. Water Activity
According to [42], water activity was determined with a portable water activity analyzer
(Rotronic AG, HygroPalm-HP23-AW-A, Bassersdorf, Switzerland), and the sample container
was filled with cocoa beans up to 4/5 of its capacity. The Aw probe was placed immediately
above the container. After 5 min, the AwQuick mode’s Aw of the sample was recorded.
2.4. Crystallization and Melting Profiles of POP and CB Inside Cocoa Bean
According to Sonwai et al. [20] and Foubert et al. [43], with some modifications, the
crystallization and melting profiles of POP and CB inside the cocoa beans were determined.
A differential scanning calorimeter (DSC) (TA Instruments, Discovery DSC 2500, New
Castle, DE, USA) was used. A 10–15 mg sample of cocoa bean was placed in an aluminum
pan and hermetically sealed with the press. An empty pan was used as a reference.
The sample was heated from room temperature to 60 ◦C and held for 15 min to ensure
homogeneity and remove any crystal memory. The sample was cooled with a ramp of
5 ◦C/min to −20 ◦C and held for 15 min, and then heated at 5 ◦C/min to 60 ◦C. The Trios
software determined the Tonset, Tendset, crystallization temperature Tc, melting temperature
Tm, and enthalpy.
2.5. Isothermal Crystallization of Cocoa Butter Inside the Cocoa Bean
According to Toro-Vásquez et al. [44], Martini [31], and Ray et al. [11], from the Tc and
Tm identified in 2.4, the experimental Tc was chosen, considering that the crystallization
process occurs at low supercooling (0–4 ◦C below the Tm of the sample), so the Tc used
were: 15, 16, 17, 18, and 19 ◦C. The samples were cocoa beans collected on each day of
spontaneous fermentation. A sample of pure CB was established as a control. A 10–15 mg
sample of cocoa bean was placed in an aluminum pan and hermetically sealed. For the
isothermal crystallization analysis and to erase any crystal memory, the sample was heated
to 60 ◦C for 20 min. The sample was cooled with a ramp of 3 ◦C min−1 until reaching the
correspondent Tc. Then, it was kept for 90 min until the exotherm (peak) of crystallization
was completed. Next, the sample was heated at 5 ◦C min−1 up to 60 ◦C to obtain the melting
endotherm (peak) that served to identify the polymorphic forms of CB, which were found
by comparing the Tm of the peaks with the literature. According to MacNaughtan et al. [37],
reheating the sample does not influence its melting and crystallization behavior.
2.6. Kinetics Crystallization
The data from the isothermal crystallization were fitted to the Avrami equation
(Equation (1)) [31,37,45] to calculate the crystallization kinetic parameters. The package
Crystallization fit of Origin Pro Software created by Lorenzo et al. [46] was used.
1−Vc(t) = exp(−ktn) (1)
Foods 2022, 11, 1769 4 of 18
where Vc is the fraction of crystal formed at time t during crystallization, k is the crystalliza-
tion rate constant, and n is the Avrami index related to the crystallization mechanism [33].
Equation (1) is expressed in logarithmic form as:
ln[− ln(1−Vc(t))] = ln k + n ln t (2)
From the plot of ln[− ln(1−Vc(t))] vs. ln t, n was calculated as the slope of the graph.
This is the so-called Avrami plot. k is a function of the crystallization temperature and
considers the crystal’s nucleation and crystal growth rate [34]. n indicates the crystal
growth mechanism [33]; that is, it indicates whether nucleation is instantaneous (when the
nuclei appear all at once when the process begins) or sporadic (when the number of nuclei
increases linearly with time) and whether nuclei grow as rods, disks, or spheres (Table 1).
Fractional values of n indicate the simultaneous generation of two or more types of crystals
or similar crystals from different types of nuclei [34].
Table 1. Values for the Avrami index, n, for different types of nucleation and growth.
n Type of Crystal Growth and Nucleation Expected
3 + 1 = 4 Spherical crystals grow from sporadic nuclei
3 + 0 = 3 Spherical crystals grow from instantaneous nuclei
2 + 1 = 3 Crystals grow plate-like from sporadic nuclei
2 + 0 = 2 Crystals grow plate-like from instantaneous nuclei
1 + 1 = 2 Crystals grow as rod/needle/fiber from sporadic nuclei
1 + 0 = 1 Crystals grow as rod/needle/fiber from instantaneous nuclei.
In addition to k and n, t1/2 describes the time taken to achieve half of the overall
crystallization and is calculated using Equ(ation (3):)
0.69315 1/n
t1/2 = (3)
k
Another important parameter is the crystallization induction time t0. It is defined as
the time required for the exotherm to begin to form [31] and calculated from the isothermal
thermogram obtained by DSC. The t0 is the time that elapses from the start of the isothermal
process to the start of crystallization. This is the point of the thermogram where the sample’s
heat fluxes deviate significantly from the baseline [47].
2.7. Polymorphism
The method developed by Fernandes et al. [18] was used to study the CB polymor-
phism during spontaneous fermentation. The sample was heated at 5 ◦C min−1 for each
Tc up to 60 ◦C until the crystals were melted (formed at the corresponding Tc). The
polymorphic forms of CB were identified based on the Tm.
2.8. Statistical Analysis
Spontaneous fermentation samples were related to the BC crystallization behavior
inside the cocoa bean, and unsupervised pattern recognition theory was used to form
clusters using the k-means technique [48]. The isothermal crystallization kinetic parameters
obtained by fitting the Avrami equation and the polymorphism results were analyzed by
k-means cluster analysis using the RMarkdown software (Rstudio, version 2021.09.0+351,
Boston, MA, USA) to find CB crystallization patterns within cocoa beans throughout the
fermentation. As cluster analysis is an exploratory method, no replicates of the measure-
ments [48] were used to run the k-means.
Foods 2022, 11, 1769 5 of 18
3. Results
3.1. Monitoring Fermentation
According to Figure 1, the parameters of the spontaneous fermentation of the Criollo
cocoa beans showed variations concerning the days of fermentation. Figure 1a shows that
the temperature increased as the fermentation developed until day 4 (45.35 ◦C) and then
decreased towards the end of the process (39.2 ◦C). Similar behavior can be verified in cocoa
bean moisture. Figure 1b shows that the pH and titratable acidity showed the opposite
behavior. While the pH decreased with the days of fermentation (from 6.83 to 4.55), the
acidity increased (from 1.15 to 2.8 mEq NaOH 100 g−1). Regarding the Aw, this parameter
showed slight growth until the end of fermentation up to 0.95.
Figure 1. Evolution of spontaneous fermentation parameters of Criollo cocoa beans. (a) Evolution of
temperature and moisture, (b) Evolution of pH, titratable acidity and water activity.
3.2. Crystallization and Melting Profiles
Table 2 shows CB’s crystallization and melting profiles compared to one of its main
triglycerides, 1,3-dipalmitoyl-2-oleoylglycerol (POP). The CB started to crystallize at 15.45 ◦C,
and Tc was 12.93 ◦C. The POP began to crystallize at 12.07 ◦C, and Tc was 10.40 ◦C. Crys-
tallization enthalpies were 48.42 J/g for CB and 54.91 J/g for POP. The CB fusion range
was 15.05 ◦C (T ◦
onset) to 26.04 C (Tendset), and the peak was at 19.65 ◦C (Tm); this range was
lower for POP (12.75–19.34 ◦C), and its fusion peak was 15.75 ◦C. Tc and Tm of CB were
used to establish the experimental Tc for the kinetic crystallization, between 0 and 4 ◦C
below Tm, thereby ensuring supercooling during isothermal kinetic crystallization. It can be
Foods 2022, 11, 1769 6 of 18
seen that the melting enthalpy of CB (69.01 J/g) is lower than the melting enthalpy of POP
(76.49 J/g).
Table 2. Crystallization and melting profiles of CB and POP standard polymorphism at 5 ◦C min−1.
Crystallization Melting
Sample Polymorphic
Tonset Tc Tendset Enthalpy Tonset Tm Tendset Enthalpy
◦ Form
( C) (◦C) (◦C) (J/g) (◦C) (◦C) (◦C) (J/g)
CB 15.45 12.93 2.99 48.42 15.05 19.65 26.24 69.01 γ
POP 12.07 10.40 4.55 54.91 12.75 15.75 19.34 76.49 γ
3.3. Kinetics of Crystallization
Figure 2 shows the graphs resulting from fitting the CB isothermal crystallization
experimental data at Tc = 15 ◦C to the Avrami equation using the Crystallization fit package
of Origin Pro software (Version: 9.8, Origin Lab, Northampton, MA, USA) 1 to 40% crystal
conversion range was used, and the data fit of R2 of 0.999 showed a good fit. The values of
n and k were 2.53 and 2.87 × 10−3 min−n, respectively. The experimental half time, t1/2,
was 8.75 min. Figure 1a corresponds to the experimental data obtained by DSC. Figure 1b
corresponds to the Avrami plot. Figure 2c shows the evolution of the untransformed
fraction, 1− Vc, as a function of time. POP and CB data crystallization at 15 ◦C can be
found in the Supplementary Materials (Tables S1 and S2, respectively).
The results in Table 3 describe the kinetics of the crystallization of CB and POP. The
CB n value increases as Tc increases from 2.53 to 3.73. The opposite occurs with the value of
k (from 2.87 × 10−3 to 8.54 × 10−5 min−n). The t1/2 increase means that the CB will need
more time to crystallize at a higher crystallization temperature. The same crystallization
behavior has the POP. All data have a good fit, determined by the high values of R2.
Table 3. Kinetics crystallization of CB and POP standard at different Tc.
Crystallization −n 2
Temperature (◦C) Sample n k (min ) t0 (min) t1/2 theo (min) t1/2 exp (min) R
CB 2.53 2.87 × 10−3 13.19 8.77 8.75 0.9990
15 POP 3.07 2.33 × 10−5 5.89 28.54 28.57 0.9996
CB 2.91 5.97 × 10−4 14.59 11.30 11.42 0.9993
16 POP 2.94 1.94 × 10−5 11.56 35.36 35.05 0.9920
CB 2.99 3.01 × 10−4 16.20 13.28 13.29 0.9992
17 POP 2.84 2.56 × 10−5 14.33 36.18 36.70 0.9999
CB 3.42 9.70 × 10−5 15.61 13.36 13.74 0.9997
18 POP 2.35 6.04 × 10−5 19.27 53.29 52.98 0.9998
CB 3.73 8.54 × 10−5 17.85 15.90 16.35 0.9993
19 POP 2.61 1.06 × 10−5 35.27 70.13 72.17 0.9989
Table 4 shows the parameters of the Avrami equation at different Tc for the study of
the kinetics of crystallization of CB inside the cocoa bean during spontaneous fermentation.
All the data have a good fit due to the high value of R2. For each Tc, it is shown that a
lower k generates higher theoretical and experimental t1/2. Therefore, slower crystallization
happens when crystallization occurred at 17, 18, and 19 ◦C and from day 4 of spontaneous
fermentation. The values of n range from 1.2 to 2.94, indicating that the crystals grow in
rod/needle/fiber or plate-like types.
Foods 2022, 11, 1769 7 of 18
Figure 2. Results of fitting the CB isothermal crystallization data at 15 ◦C using the package Crystal-
lization fit from Origin Pro. (a) Exothermal crystallization peak, (b) Avrami fitted range, (c) relative
amorphous fraction.
Foods 2022, 11, 1769 8 of 18
Table 4. Kinetics of crystallization parameters of CB inside cocoa beans during spontaneous fermentation.
Crystallization −n 2
Temperature (◦C) Sample n k (min ) t1/2 theo (min) t1/2 exp (min) R
Non-fermented 2.13 1.11 × 10−2 6.99 6.55 0.9992
Day 0 2.09 8.68 × 10−3 8.09 8.31 0.9984
Day 1 2.66 1.04 ×10−3 11.55 12.33 0.9993
Day 2 2.58 1.53 × 10−3 10.73 11.26 0.9994
15 Day 3 1.76 2.44 × 10−2 6.69 7.10 0.9918
Day 4 2.51 1.75 × 10−3 10.81 10.52 0.9995
Day 5 2.76 8.06 × 10−4 11.57 11.90 0.9998
Day 6 2.04 3.02 × 10−3 14.34 12.70 0.9999
Day 7 2.34 3.32 × 10−3 9.83 9.75 0.9994
Non-fermented 1.51 2.56 × 10−2 8.93 7.77 0.9921
Day 0 1.78 1.59 × 10−2 8.31 8.05 0.9973
Day 1 2.13 2.38 × 10−3 14.54 14.98 0.9991
Day 2 2.49 1.65 × 10−3 11.25 11.36 0.9998
16 Day 3 2.00 1.42 × 10−2 6.96 6.93 0.9992
Day 4 2.67 1.10 × 10−3 11.14 10.80 0.9977
Day 5 2.27 1.09 × 10−3 17.24 16.43 0.9929
Day 6 1.67 7.13 × 10−3 15.43 14.27 0.9903
Day 7 2.80 6.37 × 10−4 11.14 12.60 0.9992
Non-fermented 1.92 6.82 × 10−3 11.14 10.58 0.9965
Day 0 2.08 7.22 × 10−3 8.97 9.17 0.9991
Day 1 1.82 3.10 × 10−3 15.64 12.35 0.9924
Day 2 2.54 9.54 × 10−4 13.41 13.30 0.9986
17 Day 3 2.43 2.93 × 10−3 9.51 9.93 0.9998
Day 4 2.94 5.68 × 10−4 11.20 11.43 0.9999
Day 5 2.47 1.77 × 10−3 11.24 11.90 0.9985
Day 6 2.45 9.05 × 10−4 15.07 15.19 0.9990
Day 7 2.35 1.49 × 10−3 13.61 13.87 0.9999
Non-fermented 2.10 3.61 × 10−3 12.23 11.73 0.9952
Day 0 1.79 6.78 × 10−3 13.21 13.48 0.9936
Day 1 1.73 8.77 × 10−3 12.56 13.28 0.9984
Day 2 2.94 2.51 × 10−4 14.84 15.78 0.9994
18 Day 3 2.55 1.98 × 10−3 9.91 10.14 0.9999
Day 4 2.82 6.48 × 10−4 11.89 12.33 0.9998
Day 5 2.55 8.59 × 10−4 13.74 14.24 0.9993
Day 6 2.59 5.78 × 10−4 15.40 15.43 0.9993
Day 7 1.98 3.45 × 10−3 14.63 13.94 0.9964
Non-fermented 2.17 3.01 × 10−3 12.29 12.28 0.9997
Day 0 1.60 8.14 × 10−3 16.01 14.08 0.9862
Day 1 1.20 9.38 × 10−3 12.02 12.24 0.9991
Day 2 2.66 4.20 × 10−4 16.12 17.18 0.9989
19 Day 3 2.09 2.75 × 10−3 14.09 15.68 0.9985
Day 4 2.94 1.72 × 10−4 16.90 17.88 0.9981
Day 5 2.29 1.66 × 10−3 13.89 14.55 0.9995
Day 6 2.57 4.73 × 10−4 17.12 17.85 0.9996
Day 7 2.68 3.88 × 10−4 16.30 16.96 0.9986
Figure 3a shows the induction times, t0, for CB inside cocoa beans during spontaneous
fermentation. The relationship of the t0 is direct; that is, as the Tc increases, the t0 also
increases. The highest t0 corresponds to day 7 of fermentation, which means that the
crystallization process started later on day seven than in the first days. Regarding the
crystallization enthalpy, according to Figure 3b, this parameter shows a trend of increasing
with Tc, generating the highest values at higher Tc. The opposite can be seen for the melting
Foods 2022, 11, 1769 9 of 18
enthalpy (Figure 3c), which shows a decreasing trend with increasing Tc, presenting the
lowest values on day 1 of fermentation.
Figure 3. Induction times (a), crystallization enthalpy (b), and melting enthalpy (c) of the CB inside
cocoa beans as a function of Tc during spontaneous fermentation.
Foods 2022, 11, 1769 10 of 18
Figure 4a shows the evolution of the induction time, crystallization, and melting en-
thalpies from the point of view of the spontaneous fermentation process. The crystallization
induction times of non-fermented cocoa beans were lower than those of fermented ones in
all Tc and increased each day of fermentation. The crystallization enthalpy reached its high-
est value on day 5 at 19 ◦C and had an increasing trend at each Tc. Non-fermented beans
had the lowest values. Contrary to the enthalpy of crystallization, the melting enthalpy
decreased with Tc, and had its highest values on day 6. The crystallization enthalpies were
lower than the melting enthalpies during all days of fermentation.
Figure 4. Induction times (a), crystallization enthalpy (b), and melting enthalpy (c) of the CB inside
cocoa beans as a function of spontaneous fermentation days.
Foods 2022, 11, 1769 11 of 18
Figure 5 shows the results of the k-means analysis: the ratio between the between-
cluster sum of squares and the total sum of squares is 31.1%. In Figure 5a, the samples
with a fermentation day higher than 4 make up cluster 1, and the samples with less than
four days of fermentation and the non-fermented samples make up cluster 2. In Figure 5b,
each cluster shows the means of the kinetics of crystallization and polymorphic behavior
parameters. Cluster 1 is formed by the samples with the highest n values and the lowest
k values related to the highest values of t1/2. These samples also have higher melting
enthalpies and lower Tm values. The opposite occurs with cluster 2. These results mean
that cluster 1 groups samples that crystallize more slowly with less stable polyforms and
cluster 2 groups samples that crystallize faster with more stable polyforms. According
to n, in cluster 1 are the samples whose crystals grew spherically and formed sporadic
or instantaneous nuclei. Cluster 2 groups samples that crystallized as needles or fibers
instantly or sporadically.
Figure 5. Clusters of kinetics parameters and polymorphic behavior of CB inside Criollo cocoa beans
during spontaneous fermentation. (a) Division of the fermentation process into two stages (cluster),
(b) Mean values of the crystallization kinetic parameters and polymorphism in each cluster.
Foods 2022, 11, 1769 12 of 18
Table 5 shows the polymorphic behavior of CB and POP crystals at different Tc. The
Tm of all formed CB crystals increased directly with Tc, and the melting enthalpy decreased
from 89.53 to 69.73 J/g. The melting range also increased. The Tc used in the experiment
produced a single polyform β′1 in the CB. In the case of the POP, the Tm, enthalpy and the
melting interval had the same trend as the CB but with higher values, and the polyforms
generated were of type α and β′ ′2 .
Table 5. Polymorphisms of CB and POP standard at different Tc.
Crystallization Sample Enthalpy (J/g) T (◦C) T (◦ ◦
Temperature (◦C) onset m C) Tendset ( C) Polymorphic Form
CB 89.53 19.82 25.80 28.59 β′
15 1
POP 105.92 20.88 24.70 27.34 α
CB 87.16 20.48 26.27 28.80 β′
16 1
POP 105.06 21.38 25.92 27.71 β′2
CB 84.06 20.79 26.69 28.93 β′
17 1
POP 104.67 23.18 26.66 28.29 β′2
CB 75.80 21.42 27.01 29.04 β′
18 1
POP 103.20 23.46 26.17 27.87 β′2
CB 69.73 22.14 27.42 29.30 β′
19 1
POP 80.23 24.25 26.56 28.09 β′2
Table 6 shows the polymorphism of CB inside cocoa beans throughout fermentation.
The CB polymorphic forms were identified according to the sample Tm after crystallization
at a specific Tc. In all cases, the polymorphic forms identified were β′1 and β′2, which are
thermally less stable. The melting ranges were between 19.06 and 30.35 ◦C. The melting
enthalpies were higher when the crystallization was carried out at 15 ◦C than when it was
carried out at 19 ◦C.
Table 6. Polymorphism of CB inside cocoa beans during spontaneous fermentation.
Crystallization
Temperature (◦C) Sample Enthalpy (J/g) T ◦
onset ( C) T (◦m C) T ◦
endset ( C) Polymorphic Form
Non-fermented 23.42 20.31 26.19 29.29 β′1
Day 0 33.81 20.66 26.32 30.22 β′1
Day 1 17.53 19.14 24.72 28.17 β′2
Day 2 34.89 20.26 25.73 28.76 β′1
15 Day 3 33.33 19.55 24.84 28.17 β′2
Day 4 32.55 19.62 25.42 29.04 β′1
Day 5 32.94 20.65 26.14 29.59 β′1
Day 6 36.58 20.81 26.96 30.01 β′1
Day 7 22.88 19.06 24.44 27.08 β′2
Non-fermented 18.75 21.07 26.41 29.54 β′1
Day 0 17.69 22.38 26.48 28.81 β′1
Day 1 14.92 20.02 24.90 28.22 β′2
Day 2 33.92 20.78 25.79 28.67 β′1
16 Day 3 27.16 19.90 25.42 28.18 β′2
Day 4 30.88 20.14 25.69 29.07 β′1
Day 5 32.27 20.77 26.42 29.63 β′
Day 6 31.99 21.90 27.10 30.35 β′
1
1
Day 7 21.21 19.81 24.64 27.18 β′2
Foods 2022, 11, 1769 13 of 18
Table 6. Cont.
Crystallization
Temperature (◦C) Sample Enthalpy (J/g) T ◦
onset ( C) Tm (◦C) T ◦
endset ( C) Polymorphic Form
Non-fermented 16.19 21.40 26.69 29.86 β′1
Day 0 12.38 22.05 26.56 28.94 β′1
Day 1 11.61 20.83 25.28 28.28 β′2
Day 2 33.19 21.25 25.97 28.70 β′1
17 Day 3 22.61 20.81 25.82 28.34 β′1
Day 4 28.87 20.89 26.03 29.34 β′1
Day 5 31.42 21.22 26.54 29.63 β′1
Day 6 30.35 21.17 25.78 28.49 β′1
Day 7 18.95 20.53 24.97 27.16 β′2
Non-fermented 14.17 22.20 27.15 30.06 β′1
Day 0 9.16 22.57 26.79 29.31 β′1
Day 1 7.53 21.66 25.77 28.37 β′1
Day 2 28.42 22.11 26.26 28.90 β′1
18 Day 3 18.28 21.83 26.10 28.44 β′1
Day 4 26.78 21.73 26.38 29.63 β′1
Day 5 30.53 21.78 26.79 29.64 β′1
Day 6 29.60 21.85 26.20 28.62 β′1
Day 7 16.14 21.27 25.30 27.29 β′2
Non-fermented 11.21 23.19 27.63 30.30 β′1
Day 0 6.25 23.26 27.06 29.52 β′1
Day 1 4.01 22.42 26.42 28.65 β′1
Day 2 23.94 22.46 26.55 28.97 β′1
19 Day 3 13.86 22.48 26.48 28.69 β′1
Day 4 23.45 22.12 26.83 29.66 β′
Day 5 26.83 22.48 27.22 29.78 β′
1
1
Day 6 27.27 22.35 26.62 28.82 β′1
Day 7 12.12 21.82 25.98 27.61 β′1
4. Discussion
4.1. Monitoring Fermentation
There was a slight decrease in temperature throughout the spontaneous fermentation
of Criollo cocoa beans, from 45.35 ◦C on day 4 to 39.20 ◦C at the end of fermentation
(Figure 1a). It could have been caused by the existing climatic conditions in the cocoa
processing center of Aprocam. This behavior of the fermentation process is normal, if
we compare it with the results obtained by Deus et al. [26] and Chagas Junior et al. [49].
Chagas Junior et al. [49] observed similar behavior in cocoa fermentation carried out in
50 kg boxes, as the temperature reached 37 ◦C after 7 days of spontaneous fermentation.
This behavior coincides with the results reported by Visintin et al. [50]. Moisture also had
the same behavior as temperature, reaching values of 24.6% until the end of fermentation
(Figure 1a). In the spontaneous fermentation carried out by Deus et al. [26], decreasing
of pH and increasing of the titratable acidity were observed, demonstrating that these
behaviors are characteristic of a well-executed fermentation process [26,50–52]. Similar
behavior was obtained during the spontaneous fermentation studied in this work. The Aw
increased slightly throughout the fermentation (Figure 1b).
4.2. Crystallization and Melting Profiles of CB and POP
The CB crystallization profile showed that its crystallization begins at a Tonset of
15.45 ◦C and the peak is at Tc of 12.93 ◦C (Table 2); these results that agree with those
reported by Bayés-García et al. [53] (Tonset = 16.6 ◦C and T ◦
c = 13.5 C) and Aumpai [54]
(T ◦ ◦
onset = 15.6 C and Tc = 10.6 C). Since CB is a mixture of TAGs, we must talk about
melting range instead of melting temperature [55]. Bayés-García et al. [56] reported the
melting behavior at 2 ◦C/min of bulk CB and unfermented cocoa beans, finding forms α
Foods 2022, 11, 1769 14 of 18
and β′2 (Tonset and Tendset of ∼17.5 and 28.6 ◦C, respectively), values close to those reported
in Table 2. The difference in peak temperature is attributed to a different heating rate [57].
The POP crystallization results are according to Smith et al. [58]. The Tc and Tm of the POP
are lower than those of the CB; however, their corresponding enthalpies are higher. The Tm
of CB and POP indicate the presence of unstable polyforms of type γ. Based on what was
established by Sasaki et al. [15] and Ghazani [8], this polymorphic behavior of CB would
be influenced mainly by POP.
4.3. Kinetics Crystallization
Lorenzo et al. [46] suggested that the isothermal crystallization data with an R2 of
0.999 should be considered well adjusted, considering a conversion range according to
the analyzed material. Fernandes et al. [18] used a conversion range of 1 to 40% for
the crystallization kinetics of chocolate. This same range was used in the present work,
resulting in a good fit of the CB crystallization data that exceeded the value of 0.999, and
the graphs that characterize the Avrami equation were obtained (Figure 2). Figure 2a
shows CB crystallization’s exothermic peak (exotherm); half of the crystallization was
reached in 8.75 min. From this time, the secondary crystallization commenced. The initial
descent in Figure 2a corresponds to the thermal stabilization time of the DSC, and t0 was
calculated from the stabilization time until t = 0. The induction time is understood as the
time necessary to initiate crystallization [46]. Figure 2b shows a linear fit of the experimental
data, from which the slope and the intercept have been determined to obtain the parameters
of the Avrami equation (Equation (1)). Considering a conversion range for Vc from 1 to
40% [18], an R2 of 0.999 has been obtained, which shows a good data fit. Figure 2c compares
the experimental and predicted values of the relative untransformed fraction as functions
of time. The data show an excellent fit throughout the crystallization process, even beyond
the primary crystallization.
The Avrami index n describes the forms in which crystal growth occurs (Table 1) [45,59].
According to Table 1, n can take values from 1 to 4. The CB’s values of n increased from
2.53 at 15 ◦C to 3.73 at 19 ◦C. The crystal nuclei can grow from plate-like to spherical as Tc
increases. POP’s n values were reduced from 3.07 to 2.61 as the Tc increased, indicating
that its crystal nuclei’s growth behaves oppositely to that of CB.
On the other hand, decimal values of n indicate that both forms of crystal growth can
co-occur, either sporadically or instantaneously. In the study carried out by Badu et al. [1],
small values of n were obtained for Allanblackia seed oil and shea nut oil, which indicates
their rapid nucleation and crystallization mechanism. The crystallization rate constant, k,
of CB was higher than that of POP and increased with Tc; therefore, its crystallization is
faster. This is demonstrated by the low t1/2 values of the CB concerning the t1/2 values of
the POP.
Non-fermented cocoa beans show n values ranging from 2.13 at 15 ◦C to 2.17 at 19 ◦C
(Table 4), and within them, CB crystals grow as rod/needle/fiber and plate. As fermentation
began, the n values of the fermented cocoa beans changed every day from 1.20 to 2.94.
However, they did not reach values similar to those of the CB (n = 3.73, spherical nuclei),
indicating that the crystals grew the same as those of non-fermented beans. Crystallization
kinetics experiments also showed that the parameters k, t1/2 exp, and t1/2 theo changed
throughout the fermentation process for all temperatures tested; i.e., the rate constant k
was reduced, causing t1/2 to increase, hence a slower CB crystallization process as the
fermentation progressed. This behavior is due to the chemical composition of the cocoa
bean, which changes throughout fermentation by the generation of other components that
influence the crystallization behavior of CB [11,60,61].
The induction time, t0, is a function of the Tc [11,47,55]. Figure 3a shows an increasing
trend of t ◦
0 with Tc, being higher on day 7 at 19 C (Figure 4a), which indicates that the
crystallization of CB begins later in the last days of fermentation. This same trend was
reported by Martini et al. [62] in the study carried out on anhydrous milk fat crystallization.
Additionally, in this work, a decreasing trend of crystallization enthalpy concerning Tc was
Foods 2022, 11, 1769 15 of 18
reported [62], contrary to what can be observed in Figure 3a, which shows an increasing
trend, and whose highest values occur in the intermediate days of fermentation (Figure 4b).
The unfermented cocoa beans had the lowest crystallization enthalpy values during all the
days of fermentation. Figure 3c shows a decrease in the melting enthalpies of crystallized
CB within the cocoa beans at different temperatures, and the lowest values occurred on day
1 of fermentation (Figure 4c). This happened because of the driving force (supercooling)
decreases with increasing crystallization temperatures [63].
Deus et al. [26] used cluster analysis to divide the spontaneous fermentation of cocoa
into two stages: the first grouped the first days of fermentation, characterized by high
levels of pH and free amino acids; the second stage grouped the last days of fermentation,
characterized by high temperatures, high total titratable acidity, and high levels of free
bioactive amines. The unsupervised k-means classification method [64] applied to the
CB crystallization kinetics parameters divided the fermentation into two stages (cluster)
(Figure 5a). The first corresponds to day 0 to day 3 and the second from day 4 to day 7. These
findings reinforce the statements of Castro-Alayo et al. [51] and Cevallos-Cevallos et al. [65],
who mention that Criollo cocoa requires fermentation for 2 or 3 days. In this work, we
established that a fermentation process that exceeds 4 days will produce cocoa beans in
which the CB will crystallize more slowly later than in the first 3 days. In addition to that,
such a fermentation will produce less stable polyforms of type β′1 and β′2.
4.4. Polymorphism
It is crucial to study the polymorphic transitions of the CB because they influence the
properties of chocolate [61]. The stability and melting temperatures (Tm) of the CB poly-
forms increase when they are transformed from γ to β1. This irreversible transformation of
phases depends on time and temperature, from the least stable to the most stable [18]. As
mobility within the system increases due to a higher Tc, more stable polyforms are created
without starting from the initial polyforms [55]. Crystals of form β′1 and β′2 are considered
metastable [66]. In the case of CB, for all the Tc, only β′1 polyforms were formed (Table 5).
The increase in T (15–19 ◦c C) was insufficient to generate polymorphism from the less
stable forms to the more stable ones. To achieve this, more considerable increases in Tc are
necessary, according to what was reported by Fernandes et al. [18]. Similar behavior was
noted in the POP for Tc values from 17 to 19 ◦C, where only the β′1 polyform was generated.
However, at 15 ◦C, polyform α was generated due to its low melting point. The melting
enthalpies of the crystallized polyforms of CB and POP decreased with Tc. For any Tc, the
melting enthalpy of the POP is always greater than that corresponding to CB.
In the same way as the previous results, there was no evidence of any change in the
CB polymorphism towards more stable polyforms as fermentation went on (Table 6). This
means that, during fermentation and at any Tc, only metastable CB polyforms (β′1 and β′2)
are formed within the cocoa beans. The melting enthalpies of these polymorphic crystals
are lower than those corresponding to the enthalpies of pure CB throughout the entire
fermentation process. The results of Tables 4 and 6 agree with what was established by
Garbolino et al. [67], who state that the different results of the crystallization kinetics do
not always produce changes in the polymorphism.
5. Conclusions
The present work allowed us to confirm the importance of the spontaneous fermen-
tation process of Criollo cocoa analyzed from the point of view of the crystallization of
cocoa butter. The parameters describing the kinetic crystallization of cocoa butter inside
the Criollo cocoa bean show that only metastable crystals corresponding to polyforms β′1
and β′2 can be formed during spontaneous fermentation. The growth of CB crystals occurs
in the form of rod/needle/fiber and plate, just like non-fermented beans. The results allow
the fermentation process to be divided into two stages: the first fermentation stage until day
3 and the second stage from day 4 to day 7. Low crystallization rates are obtained in this
Foods 2022, 11, 1769 16 of 18
last stage, so the cocoa butter crystallizes more slowly. This happens due to the formation
of other chemical components within the cocoa bean due to the fermentation process.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/
10.3390/foods11121769/s1, Tables S1 and S2 shows the POP and CB isothermal crystallization
data, respectively.
Author Contributions: Conceptualization, E.M.C.-A. and F.P.C.-T.; methodology, E.M.C.-A., F.P.C.-T.
and L.T.-V.; software, E.M.C.-A., M.M.-M. and L.T.-V.; validation, E.M.C.-A.; formal analysis, E.M.C.-A.,
F.P.C.-T. and I.S.C.-C.; investigation, E.M.C.-A. and M.M.-M.; resources, I.S.C.-C.; data curation,
E.M.C.-A.; writing—original draft preparation, E.M.C.-A., F.P.C.-T. and I.S.C.-C.; writing—review
and editing, E.M.C.-A., F.P.C.-T. and I.S.C.-C.; visualization, E.M.C.-A. and I.S.C.-C.; supervision,
E.M.C.-A. and F.P.C.-T.; project administration, I.S.C.-C.; funding acquisition, I.S.C.-C. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Consejo Nacional de Ciencia, Tecnología e Innovación
Tecnológica-Concytec (project: Equipamiento Científico 2018-01/E044-2018-01-BM, Contrato N◦
012-2018-Fondecyt/BM) of the Peruvian Government, The World Bank Group, Proyecto SNIP N◦
381743-Creación de los Servicios de Investigación en Ingeniería de Alimentos y Poscosecha of the
Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas, and Pontificia Universidad
Católica del Perú. The APC was funded by the Consejo Nacional de Ciencia, Tecnología e Innovación
Tecnológica-Concytec.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in Supplementary Material.
Acknowledgments: The authors thank the Cooperativa de Servicios Múltiples APROCAM for the
facilities provided during the execution of this work.
Conflicts of Interest: The authors declare no conflict of interest.
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