Heliyon 7 (2021) e06154Contents lists available at ScienceDirect
Heliyon
journal homepage: www.cell.com/heliyonResearch articleRheological, bioactive properties and sensory preferences of dark chocolates
with partial incorporation of Sacha Inchi (Plukenetia volubilis L.) oil
Marleni Medina-Mendoza a, Roxana J. Rodriguez-P
erez a, Elizabeth Rojas-Ocampo a,
Llisela Torrejo
n-Valqui b, Armstrong B. Fern
andez-Jeri b, Guillermo Idrogo-V
asquez b,
Ilse S. Cayo-Colca c, Efraín M. Castro-Alayo b,*
a Programa Acad
emico de Ingeniería Agroindustrial, Facultad de Ingeniería y Ciencias Agrarias, Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas, Calle
Higos Urco 342-350-356, Chachapoyas, Amazonas, Peru
b Instituto de Investigaci
on, Innovaci
on y Desarrollo para el Sector Agrario y Agroindustrial de la Regio
n Amazonas (IIDAA), Facultad de Ingeniería y Ciencias Agrarias,
Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas, Calle Higos Urco 342-350-356, Chachapoyas, Amazonas, Peru
c Facultad de Ingeniería Zootecnista, Agronegocios y Biotecnología, Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas, Calle Higos Urco 342-350-356,
Chachapoyas, Amazonas, PeruA R T I C L E I N F O
Keywords:
Sacha Inchi oil
Dark chocolate
Criollo cocoa beans
Rheology
Sensory preferences
Functional food* Corresponding author.
E-mail address: efrain.castro@untrm.edu.pe (E.M
https://doi.org/10.1016/j.heliyon.2021.e06154
Received 2 September 2020; Received in revised fo
2405-8440/© 2021 The Author(s). Published by Els
nc-nd/4.0/).A B S T R A C T
We studied the effect of substituting partially, cocoa butter (CB) with Sacha Inchi (Plukenetia volubilis L.) oil (SIO)
on rheology, bioactive properties, and sensory preferences in potentially functional chocolate. For this 70% dark
chocolates were prepared and the CB was substituted with 1.5%, 3%, and 4.5% of SIO. Hardness and viscosity of
the SIO-chocolates were significantly reduced compared to the control (5451 
 658 g; 17.01 
 0.94 Pa s,
respectively). Total phenolic content remained constant while the antioxidant capacity increased up to IC50 of
2.48 
 0.10 as the content of SIO increased. The Casson yield stress and Casson plastic viscosity decreased as the
amount of SIO increased. Chocolates with 4.5% SIO had a similar color, better glossiness, preferable snap attri-
butes, and were more accepted (7.50 
 0.08) compared to the control (p < 0.05), measured with a hedonic scale.
Then, SIO can improve the bioactive properties of dark chocolates obtaining a potentially functional food with
acceptable physicochemical characteristics. SIO can be considered as a new cocoa butter equivalent.1. Introduction
The raw material for the manufacture of dark chocolates are cocoa
beans (Theobroma cacao L.), valuable for their essential nutrients and
functional properties (Z_yz_elewicz et al., 2018). Among others, the “Cri-
ollo” cocoa variety is considered as the finest variety. It is characterized
as an aromatic, mild-tasting with low bitterness seed (Castro-Alayo et al.,
2019; Ascrizzi et al.,2017; Qin et al., 2016). Due to its high cocoa content
(>35%), dark chocolate it is considered the product derived from cocoa
that has the highest content of polyphenols (Toker et al., 2018) corre-
lated with their catechin, epicatechin, and procyanidin contents. Then,
dark chocolate is recognized as an alternative antioxidant in the human
diet (Todorovic et al., 2015).
Dark chocolate is a mixture of cocoa liquor and other components,
surrounded by cocoa butter (CB) (Toker et al., 2018). It is a highly caloric
product that contains many carbohydrates and fats, mainly CB, as well as
a unique taste and texture. CB is expensive and its price increases due to. Castro-Alayo).
rm 13 October 2020; Accepted 2
evier Ltd. This is an open access athe low productivity of cocoa beans and its high industrial demand
(Watanabe et al., 2021). CB is the ingredient that gives chocolate its
proper gloss, snap, taste (Rodriguez Furla
n et al., 2017) and texture, are
considered as its appearance attributes (Ostrowska-Ligęza et al., 2019).
These properties are influenced by the rheology of chocolate, a science
that studies quality parameters such as viscosity, Casson yield stress and
Casson plastic viscosity (Glicerina et al., 2013; Beckett, 2009; Bahari and
Akoh, 2018).
In order to improve the production of chocolates (economically and
technologically), various types of chocolate are currently being pro-
duced, incorporating various proportions of CB and other vegetable oils:
illipe, palm, sal, shea, kokum gurgi, and mango kernel (Talbot, 2009);
whichmodify the physical characteristics of the final product (Silva et al.,
2017). These vegetable fats are the so-called cocoa butter equivalents
(CBE). A good CBE must be compatible with CB and not alter its physical
properties when used in the manufacture of chocolate (Bahari and Akoh,
2018). Therefore, CBE must contribute with a desirable texture7 January 2021
rticle under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
M. Medina-Mendoza et al. Heliyon 7 (2021) e06154(Gregersen et al., 2015). Researchers in food and chocolate manufac-
turers are using CBE to reduce production costs, improve physical
properties and reduce health risks for consumers (Watanabe et al., 2021).
Different types of CBE have been used, including enzymatically modified
vegetable oils (Watanabe et al., 2021; Zhang et al., 2020; Bahari and
Akoh, 2018; Kadivar et al., 2016), considering the maximum permitted
level (5%) required by the European regulation (Abdul Halim et al.,
2019). In this regard, Abdul Halim et al.,. (2019) suggest that substituting
4.5% coconut oil (CNO) for CB improves the chocolate's appearance.
In the Peruvian Amazon, Sacha inchi (Plukenetia volubilis L.) is grown,
known as Inca peanut, an important source of phenolic compounds and
high antioxidant capacity (Chirinos et al., 2013). So, its use as an
ingredient in food processing can improve the healthy properties of
these. Given the growing demand for natural CBE, we believe it is
necessary to propose alternative local sources to those already existing.
Furthermore, as there are few CBE in the Peruvian market for the
manufacture of chocolates, it is necessary to use other vegetable oils from
local raw materials. The objective of this study is to evaluate the rheo-
logical, bioactive properties and sensory preferences of Criollo
cocoa-dark chocolates with partial substitution of CB with Sacha Inchi oil
(SIO).
2. Materials and methods
2.1. Materials
Pure SIO and sugar were purchased from a local market. Ingredients
such as CB and Criollo cocoa beans were provided by the Cooperativa de
Servicios Múltiples Aprocam (Bagua-Amazonas-Perú) and sent to the
Agroindustrial Biotechnology Laboratory (LBA) from the Universidad
Nacional Toribio Rodríguez de Mendoza de Amazonas (UNTRM).2.2. Chemicals
All chemicals and standards: Folin-Ciocalteu's phenol, gallic acid,
sodium carbonate (-)-epicatechin  98% (HPLC); (-)-catechin  97%
(HPLC) from green tea, and petroleum ether  90%, 2,2-diphenyl-1-pic-
rylhydrazyl (DPPH) were purchased from Sigma-Aldrich (Diessenhofen,
Germany), except Methanol HPLC grade (JT Baker, Deventer, The
Netherlands).2.3. Dark chocolate making process
Dark chocolates were made according to the recipe of Aprocam,
considering the formula in Table 1, developed by Abdul Halim et al.,.
(2019). In brief, Criollo cocoa beans of uniform size were selected. The
roasting process was carried out in a tray dryer (Fischer Agro, Peru) at
110 C, for 2 h. Cocoa nibs were obtained during the winnowing (Imsa,
DC-C, Peru) from roasted beans. Next, the nibs were ground with an
industrial stone roller (Prosol SAC, Tritur-50, Peru) to obtain the cocoa
liquor. To carry out the conching process, all the ingredients were added
to a two-roller refiner (Premier, PG508, India) for 12 h. Then, the SIOwas
added to the mixture in different percentages according to Table 1 and
left for a remaining 1 h. The blend was tempered between 28 to 34 C.
Next, 45 g of the blend was put to the chocolate bar mold and kept for 15Table 1. Formula of dark chocolates containing different percentages of SIO.
Ingredient (%) Chocolate
Control Chocol
Criollo cocoa liquor 70 70
Sugar 25 25
CB 5 3.5
SIO 0 1.5
2
min at 18 C. Finally, it was wrapped in aluminum foil and stored at 8 C
until later physicochemical and sensory analysis.2.4. Chemical analysis
2.4.1. Phenolic extracts
Dark chocolates were defatted using the Soxhlet extraction according
to Zhou et al.,. (2015) with some modifications. In brief, 5 g of dark
chocolates were defatted using petroleum ether 90% in a fat extractor
(JP-Selecta, Det-Grasa N, Spain). Then, 1 g of defatted chocolate was
mixed with 10 mL of an 80% methanolic solution, after vortexing for 1
min, the sample was taken to an ultrasound bath for 10 min at 30 C and
centrifuged at 3000 rpm for 10 min (Gültekin-O€zgüven et al., 2016). The
supernatant was placed in vials and kept refrigerated.
2.4.2. Antioxidant capacity
The antioxidant capacity was determined according to Z_yz_elewicz
et al.,. (2018), Z_yz_elewicz et al.,. (2016), Scherer and Godoy (2009), and
Brand-Williams et al.,. (1995) with some modifications. A methanolic
solution (80%) of DPPH radical (solution B) was prepared to weigh 0.005
g of DPPH radical in 100 mL of this methanolic solution. Additionally,
another 80% methanolic solution was prepared for the dilutions of the
extract (solution C). Subsequently, dilutions of extract þ methanolic
solution C were prepared in the concentrations of 1:1, 1:2, 1:5, 1:10 and
1:20 (solution A). Seven test tubes containing 0.1 mL of solution A and
3.9 mL of solution B were prepared. The control was 0.1 mL of solution C
and 3.9 mL of solution B. The absorbance of the samples was measured at
517 nm in a spectrophotometer (Unico, S2100, United States), after 30
min of reaction in the dark. The percentage of DPPH radical inhibition (I
%) was calculated from Eq. (1), where Abs0and Abs1were the control
absorbance and the absorbance of the sample; respectively. The extract
concentration that produced 50% inhibition (IC50) of the DPPH radical
was calculated from the graph of percent inhibition versus extract con-
centration (Oke et al., 2009).

 
¼ Abs0  Abs
I% 1 *100 (1)
Abs0
2.4.3. Total phenolic content
Total phenolic content (TPC) in extracts was determined according to
the Folin–Ciocalteu's procedure of Singleton et al.,. (1999) and Pantelidis
et al.,. (2007), 0.05 mL of diluted extract and 0.45 mL water were mixed
with 2.5 mL of 1:10 diluted Folin–Ciocalteu's phenol reagent, followed by
2 mL of 7.5% (w/v) sodium carbonate. After 5 min at 50 C, absorbance
was measured at 760 nm (Unico, S2100, United States). TPC was esti-
mated from a standard curve of gallic acid and results were expressed as
mg gallic acid equivalents/g of sample (mgGAE/g). The calibration curve
(y ¼ 0:1073þ 0:0009x) was prepared from the dilutions of gallic acid in
a range of 0–2500 ppm (R2 ¼ 0:9952)
2.4.4. Quantification of epicatechin and catechin by UHPLC
Quantification of epicatechin and catechin in dark chocolate samples
was performed according to Coklar and Akbulut (2017) and Demir et al.,.
(2014) with some modifications. For that, an Agilent 1290 Infinity Series
UHPLC system equipped with a G7167B mutisampler, G7104A flexibleate A Chocolate B Chocolate C
70 70
25 25
2 0.5
3 4.5
M. Medina-Mendoza et al. Heliyon 7 (2021) e06154pump, G7116B column oven, and a G7117B diode array detector was
used. Before the injection, the extracts were filtered through a 0.45 μm
pore size x 33 mm syringe filter (Merck, Millex, Germany). The separa-
tion was achieved using a reversed-phase C18 column (5 μm, 250  4.6
mm i.d.). The mobile phase was (A) water/acetic acid (98:2) and (B)
water/acetonitrile/acetic acid (78:20:2). The flow rate was 0.75 mL/min
and the gradient was as follows: 10–14% B (5 min), 14–23% B (11 min),
23–35% B (5 min), 35–40% B (14 min), 40–100% B (3 min), 100% B
isocratic (3 min), 100–10% B (3 min) and 10% B isocratic (4 min). The
detector was set to 280 nm. The column temperature was 40 C. The
epicatechin and catechin were quantified by comparison with peak areas
of each standard. The data was analyzed using the Chemstation software.2.5. Physical analysis
2.5.1. Texture
Texture (hardness) was determined using a Texture Analyzer
(Brookfield, CT3 Texture Analyzer, United States) equipped with a load
cell of 25 kg and a TA2/1000 stainless steel probe. All measures were
kept at room temperature (20 C) for 1 h beforemeasuring. The following
parameters were used: pre-test speed was set at 0.5 mm/s, test speed at
2.0 mm/s, product vol at 0.7 mm  55 mm x 50mm, penetration depth
3mm, time set at 1–2 min, test speed at 50 mm/s, load cell at 25 kg, and
trigger force at 20 g. The hardness (grams) was reported. All measure-
ments were conducted at room temperature (18 
 2 C).
2.5.2. Rheology
The rheological properties of each sample were measured using a
rheometer (Anton Paar, Model MCR 92, Austria) equipped with a
concentric cylinder. Samples were first melted at 50 C for 60 min and
put into a cup. Rheological measurements were taken at 40 C. The
measurement cycle started with a preconditioning at 40 C for 60 s. The
measurements were processed with the equipment software (Rheo-
Compass) which is based on Casson Model (Equation 2), where σ(Pa) is
the yield stress, σ0is Casson yield stress (Pa), K1is Casson plastic viscosity
(Pa.s) and γ(s-1) is the shear rate (Abdul Halim et al., 2019).
σ0:5 ¼ðσ Þ0:5 þ K ðγÞ0:50 1 (2)
2.6. Sensory preferences
According to Abdul Halim et al., (2019), the sensory profile of the
chocolate was carried out to determine the consumer preference towards
all SIO-chocolates compared to the control. About 50 students from the
Facultad de Ingeniería y Ciencias Agrarias (FICA) were selected as pan-
elists for the sensory test (30 males and 20 females). The range of age
from the panelists was 20–30 years old. The chocolate samples were kept
at room temperature (18
 2 C) for 1 h before the evaluation. In order to
obtain the consent of the panelists to participate in the sensory tests, they
were informed about the research objectives and the composition of the
chocolate through an induction talk conducted before the test. Subse-
quently, they were given the questionnaire and they wrote their name as
a sign of their consent to participate. Then each panelist tasted all the
enriched chocolates including the control. For that, they were given four
types of chocolate samples (2.5  1.3  0.7 cm, each) with three repe-
titions, randomly distributed. Each panelist filled a questionnaire to score
from 1 (“extremely dislike”) to 9 (“extremely like”) using hedonic scales
for attributes like taste, color, snap, gloss, and acceptability.2.7. Statistical analysis
Data were analyzed by one-way analysis of variance (ANOVA) using a
Minitab Ver 17 Software (USA). Tukey's test was used to determine the
significant differences at a level of p < 0.05. All experimental data were
obtained in triplicates.3
3. Results and discussion
3.1. Total phenolic content and antioxidant capacity
The total phenolic content (TPC) reported by Toker et al., (2018) in
dark chocolate ranged between 12 and 15 mgGAE/g. In the same way,
Calva-Estrada et al., (2020) reported a range of 8.94–21.17 mg GAE/g in
Latin American chocolates. In our study, the range of the TPC was 16.53

 0.91–18.76
 0.15 mg GAE/g for the control and chocolates (Table 2),
the analysis of the groups formed by the Tuckey test, shows that these
values were similar for all the chocolates and the control. Then, the
partial substitution of CB with SIO (chocolate A, B, and C) did have a
significant increase in TPC (p < 0.05). Fern
andez-Romero et al., (2020)
reported a reduction in the content of TPC in 46.88%, during the roasting
of Criollo cocoa at 110 C for 50 min. Therefore, we believe that although
roasting may have reduced the TPC of the chocolates, they remained
within a considerable range compared to Toker et al., (2018) and Cal-
va-Estrada et al., (2020).
The IC50 parameter represents the concentration of chocolate extract
capable of reducing the initial concentration of DPPH radical by 50%
(Bordiga et al., 2015); therefore, small IC50 values mean a high antioxi-
dant capacity. The antioxidant capacity of chocolates samples increased
as the SIO content increased (p< 0.05). In a previous study performed by
Summa et al. (2006), the effect of roasting on the antioxidant capacity of
Ghanaian cocoa was evaluated, it was shown that the roasting process
had a significant effect on melanoidins (anti-radical components of mo-
lecular weight between 5 and 10 kDa), increasing the concentration of
anti-radical components. Chirinos et al., (2013) affirm that the range
value of TPC for the sixteen Sacha Inchi Peruvian cultivars was within the
0.65–0.80 mg GAE/g seed. Then, due to the possible loss of TPC and
antioxidant capacity during processing conditions, we tried to increase or
at least minimize the loss using SIO as a substitute for CB. In our
experiment, the antioxidant capacity were improved, probably due to the
incorporation of SIO. We think that the roasting could have produced
melanoidins and the SIO could have contributed compounds that are not
TPC but that have antioxidant capacity; then, this hypothesis must be
confirmed with more in-depth studies.
Several studies have confirmed the important role of dark chocolate
polyphenols in human health, highlighting their beneficial effects in the
treatment of cardiovascular diseases (Fanton et al., 2020; Z_yz_elewicz
et al., 2018; Greenberg, 2015; Kwok et al., 2015), but there are still few
studies that report the phenolic profile of chocolates (Martini et al.,
2018). In the study carried out by Nascimento et al., (2020) in fine and
artisan chocolates (from 28 to 100% of cocoa content), catechin and
epicatechin contents were found ranging from 0.047-0.60 mg/g and
0.048–1.68 mg/g, respectively; concluding that chocolates with a high
cocoa content are a good source of antioxidant compounds. In the present
study, the catechin and epicatechin content of the chocolates ranged
from 0.40 
 0.01–0.43 
 0.01 mg/g and 3.31 
 0.01–3.74 
 0.02 mg/g,
respectively (Table 2), this results were higher than those found by
Nascimento et al., (2020), Calva-Estrada et al., (2020) (0.45–1.03 mg/g
of epicatechin, 0.06–0.25 mg/g of to catechin) and Martini et al., (2018)
(2.03 mg/g of epicatechin and 0.66 mg/g of catechin). So we consider
that our chocolates represent an excellent source of antioxidants. These
chocolates could be considered products with functional potential,
despite the imminent degradation of catechin and epicatechin as a
consequence of roasting as reported by Fern
andez-Romero et al., (2020)
(epicatechin 72.77% and catechin 24%).
3.2. Texture and rheological properties of dark chocolates
Vegetable oils known as CBE have a chemical composition and
physical properties similar to CB (Talbot, 2012). This explains why
some studies focus on the use of CBE to improve other properties of
chocolates such as its rheological properties, texture, fat bloom sta-
bility, particle size distribution, thermal behavior and sensory profile
M. Medina-Mendoza et al. Heliyon 7 (2021) e06154
Table 2. TPC and antioxidant capacity of dark chocolates.
Chocolate TPC (mg GAE/g) Catechin (mg/g) Epicatechin (mg/g) Antioxidant capacity (IC50)
Control 18.76 
 0.15a 0.43 
 0.01a 3.74 
 0.02a 5.67 
 0.23a
Chocolate A 17.06 
 1.03ab 0.42 
 0.01ab 3.36 
 0.01b 4.38 
 0.07b
Chocolate B 16.53 
 0.91b 0.40 
 0.01b 3.31 
 0.01c 3.34 
 0.18c
Chocolate C 17.71 
 0.25ab 0.43 
 0.01a 3.74 
 0.02a 2.48 
 0.10d
Values are mean 
 SD (n ¼ 3).
Same column with different subscripts are significantly different (p < 0.05).
Table 3. Rheological properties of dark chocolates.
Chocolate Hardness (g) Casson plastic viscosity (Pa.s) Casson yield stress (Pa)
Control 5451 
 658a 17.01 
 0.94a 2.08 
 0.02a
Chocolate A 3503 
 844b 14.65 
 0.82b 1.78 
 0.04b
Chocolate B 5562 
 422a 17.89 
 0.27a 1.81 
 0.02b
Chocolate C 4739 
 587ab 16.08 
 1.20ab 1.60 
 0.02c
Values are mean 
 SD (n ¼ 3).
Same column with different subscripts are significantly different (p < 0.05).(Watanabe et al., 2021; Zhang et al., 2020; Abdul Halim et al., 2019;
Bahari et al., 2018; Biswas et al., 2017; Kadivar et al., 2016). Texture
determines the physical structure of a material and its mechanical and
surface properties. It is measured through the hardness of the final
product, understood as the force necessary to achieve a certain
deformation (Ostrowska-Ligęza et al., 2019; Torbica et al., 2016).
Table 3 represents the impact of the partial substitution with SIO for
CB on the texture of the chocolates. In our study, the partial incor-
poration of SIO reduced the hardness of the chocolates A and C. The
study carried out by Bahari and Akoh (2018) suggests that illipe butter
and the middle palm fraction can be used to make dark chocolate with
the same rheological properties and texture of a chocolates made with
only CB. The opposite occurs with the use of SIO, since chocolates
with lower hardness are produced with this substitute. However, our
results agree with Torbica et al. (2016) and Kadivar et al. (2016), who
found that the use of CBE has a softening effect on CB and therefore
on chocolate, we think it is possible to use SIO as CBE, without
altering the basic stander properties of the chocolate.
From a rheological point of view, dark chocolate has a complex
behavior, that is, it has an apparent elastic limit and a plastic viscosity
that are strictly dependent on the manufacturing process (Glicerina et al.,
2013). The addition of SIO also decreased significantly the values of
Casson yield stress and Casson plastic viscosity of all chocolates’ samples
(p< 0.05), these results agree with those obtained by Abdul Halim et al.,.
(2019). According to Sasaki et al.,. (2012), large β0 crystals disturb the
fine crystal network formed by βV crystals when both crystallize, which
affects rheological properties and reduces viscosity. Then, the decreasing
of Casson yield stress and Casson plastic viscosity was due to the for-
mation of β0 in SIO-chocolates.Table 4. Sensory preference test.
Chocolate Taste Color
Control 7.17 
 0.04b 7.34 
 0.09a
Chocolate A 6.56 
 0.19c 6.83 
 0.09b
Chocolate B 7.32 
 0.04ab 7.47 
 0.15a
Chocolate C 7.54 
 0.05a 7.51 
 0.01a
Values are mean 
 SD (n ¼ 3).
Same column with different subscripts are significantly different (p < 0.05).
Each panelist was asked to score from 1 (“extremely dislike”) to 9 (“extremely like”)
4
3.3. Sensory preference
The texture and appearance of chocolate are key attributes in the
choice and acceptability of the consumer (Ostrowska-Ligęza et al., 2019),
it is acceptable to use a maximum of 5% CBE on the total weight of the
chocolate (Talbot, 2009). A good dark chocolate is dark brown in color
and gloss (Afoakwa, 2010). Table 4 shows the results of the sensory
analysis of the chocolates containing three percentages of SIO (1.5%,
3.0%, and 4.5% SIO). In our study, chocolates B and C had similar color
appearance (p> 0.05) compared to the control (7.47
 0.15, 7.51
 0.01
and 7.34 
 0.09, respectively). Which means that the SIO substitution
did not affect the color of the chocolate. In contrast, there was a signif-
icant difference (p < 0.05) in gloss between the control and the choco-
lates containing SIO. Chocolates B and C scored the highest glosss
compared to the control (7.52 
 0.04, 7.60 
 0.07, and 7.21 
 0.15,
respectively). For snap attributes, there was a significant difference (p <
0.05) between the control and the chocolate C (7.21 
 0.10 and 7.57 

0.08, respectively). Most of the panelists considered that the snap of
chocolate C was the best one among the other chocolates. According to
Afoakwa et al. (2008), the tempering process during the production of
chocolates determines the gloss and snap, then, we think this charac-
teristic was improved by the SIO. In the same way, chocolate C was
significantly preferable (p< 0.05) than the control and chocolate A (7.54

 0.05, 7.17 
 0.04, and 6.56 
 0.19, respectively). The chocolate taste
is influenced by its ingredients composition. Therefore we think that SIO
increased the taste of the chocolates. Finally, chocolates B and C were
more accepted compared to the control and chocolate A (7.41
 0.15 and
7.50 
 0.08 vs 7.17 
 0.11 and 6.65 
 0.08, respectively). These results
agree with those reported by Abdul Halim et al. (2019), who used CNO asSnap Gloss Acceptability
7.21 
 0.10b 7.21 
 0.15b 7.17 
 0.11b
6.43 
 0.08c 6.91 
 0.10c 6.65 
 0.08c
7.28 
 0.09b 7.52 
 0.04a 7.41 
 0.15ab
7.57 
 0.08a 7.60 
 0.07a 7.50 
 0.08a
using hedonic scales.
M. Medina-Mendoza et al. Heliyon 7 (2021) e06154CBE, suggesting that the replacement of CNO at 4.5% improves the
appearance and rheological behavior of chocolate.
4. Conclusion
In our study, we found that the partial substitution of the cocoa
butter with SIO increased the antioxidant capacity of the chocolates,
though the levels added did not influence the amount of TPC
compared to the control. Nonetheless, the rheological behavior and
the texture of SIO-chocolates were minor compared to the control,
they were within the acceptable parameters for dark chocolates.
Finally, the partial substitution of CB with SIO in chocolates improved
consumer preferences towards sensory analysis. Chocolates containing
4.5% of SIO, rated the highest scores of color, snap, gloss, and
acceptability. Therefore, it can be said that SIO can be considered as
an acceptable CBE. We consider that this work will contribute to the
search for new CBE that, in addition to improving the rheological
properties of chocolate, will also improve its bioactive properties to be
considered a chocolate with functional potential.
Declarations
Author contribution statement
Marleni Medina-Mendoza: Performed the experiments; Analyzed and
interpreted the data; Wrote the paper.
Roxana J. Rodriguez-P
erez, Elizabeth Rojas-Ocampo: Performed the
experiments.
Llisela Torrejo
n-Valqui, Guillermo Idrogo-Va
squez: Analyzed and
interpreted the data.
Armstrong B. Ferna
ndez-Jeri: Contributed reagents, materials, anal-
ysis tools or data.
Ilse S. Cayo-Colca: Contributed reagents, materials, analysis tools or
data; Wrote the paper.
Efraín M. Castro-Alayo: Conceived and designed the experiments;
Analyzed and interpreted the data; Wrote the paper.Funding statement
This work was supported by Fondo Nacional de Desarrollo Científico,
Tecnolo
gico y de Innovacio
n Tecnolo
gica; World Bank Group; and Uni-
versidad Nacional Toribio Rodríguez de Mendoza de Amazonas (Con-
trato N 012-2018-Fondecyt/BM).Data availability statement
Data will be made available on request.Declaration of interests statement
The authors declare no conflict of interest.Additional information
No additional information is available for this paper.
Acknowledgements
A special acknowledgment to Cooperativa de Servicios Múltiples
Aprocam (Bagua-Amazonas-Perú) for providing us with their Criollo
cocoa beans for the realization of this work.5
References
Abdul Halim, H.S., Selamat, J., Mirhosseini, S.H., Hussain, N., 2019. Sensory preference
and bloom stability of chocolate containing cocoa butter substitute from coconut oil.
Journal of the Saudi Society of Agricultural Sciences 18, 443–448.
Afoakwa, E.O., 2010. Chocolate Science and Technology. Wiley-Blackwell, Chichester,
U.K., pp. 35–57
Afoakwa, E.O., Paterson, A., Fowler, M., Ryan, A., 2008. Flavor formation and character
in cocoa and chocolate: a critical review. Crit. Rev. Food Sci. Nutr. 48, 840–857.
Ascrizzi, R., Flamini, G., Tessieri, C., Pistelli, L., 2017. From the raw seed to chocolate:
volatile profile of Blanco de Criollo in different phases of the processing chain.
Microchem. J. 133, 474–479.
Bahari, A., Akoh, C.C., 2018. Texture, rheology, and fat bloom study of ‘chocolates’ made
from cocoa butter equivalent synthesized from illipe butter and palm mid-fraction.
LWT - Food Sci. Technol. (Lebensmittel-Wissenschaft -Technol.) 97, 349–354.
Beckett, S.T., 2009. Industrial Chocolate Manufacture and Use, fourth ed. Wiley-
Blackwell, Chichester, U.K., pp. 224–245
Biswas, N., Cheow, Y.L., Tan, C.P., Siow, L.F., 2017. Physical, rheological, and sensorial
properties, and bloom formation of dark chocolate made with cocoa butter substitute
(CBS). LWT - Food Sci. Technol. (Lebensmittel-Wissenschaft -Technol.) 82, 420–428.
Bordiga, M., Locatelli, M., Travaglia, F., Coïsson, J.D., Mazza, G., Arlorio, M., 2015.
Evaluation of the effect of processing on cocoa polyphenols: antiradical activity,
anthocyanins and procyanidins profiling from raw beans to chocolate. Int. J. Food
Sci. Technol. 50, 840–848.
Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to
evaluate antioxidant activity. LWT - Food Sci. Technol. (Lebensmittel-Wissenschaft
-Technol.) 28, 25–30.
Calva-Estrada, S.J., Utrilla-V
azquez, M., Vallejo-Cardona, A., Roblero-P
erez, D.B., Lugo-
Cervantes, E., 2020. Thermal properties and volatile compounds profile of
commercial dark-chocolates from different genotypes of cocoa beans (Theobroma
cacao L.) from Latin America. Food Res. Int. 136, 109594.
Castro-Alayo, E.M., Idrogo-V
asquez, G., Siche, R., Cardenas-Toro, F.P., 2019. Formation
of aromatic compounds precursors during fermentation of Criollo and Forastero
cocoa. Heliyon 5, e01157.
Chirinos, R., Zuloeta, G., Pedreschi, R., Mignolet, E., Larondelle, Y., Campos, D., 2013.
Sacha inchi (Plukenetia volubilis): a seed source of polyunsaturated fatty acids,
tocopherols, phytosterols, phenolic compounds and antioxidant capacity. Food Chem.
141, 1732–1739.
Coklar, H., Akbulut, M., 2017. Anthocyanins and phenolic compounds of Mahonia
aquifolium berries and their contributions to antioxidant activity. Journal of
Functional Foods 35, 166–174.
Demir, N., Yildiz, O., Alpaslan, M., Hayaloglu, A.A., 2014. Evaluation of volatiles,
phenolic compounds and antioxidant activities of rose hip (Rosa L.) fruits in Turkey.
LWT - Food Sci. Technol. (Lebensmittel-Wissenschaft -Technol.) 57, 126–133.
Fanton, S., Cardozo, L.F.M.F., Combet, E., Shiels, P.G., Stenvinkel, P., Vieira, I.O.,
Narciso, H.R., Schmitz, J., Mafra, D., 2020. The sweet side of dark chocolate for
chronic kidney disease patients. Clin. Nutr. In press.
Fern
andez-Romero, E., Chavez-Quintana, S.G., Siche, R., Castro-Alayo, E.M., Cardenas-
Toro, F.P., 2020. The kinetics of total phenolic content and monomeric flavan-3-ols
during the roasting process of Criollo cocoa. Antioxidants 9, 146.
Glicerina, V., Balestra, F., Rosa, M.D., Romani, S., 2013. Rheological, textural and
calorimetric modifications of dark chocolate during process. J. Food Eng. 119,
173–179.
Greenberg, J.A., 2015. Chocolate intake and diabetes risk. Clin. Nutr. 34, 129–133.
Gregersen, S.B., Miller, R.L., Hammershøj, M., Andersen, M.D., Wiking, L., 2015. Texture
and microstructure of cocoa butter replacers: influence of composition and cooling
rate. Food Struct. 4, 2–15.
Gültekin-O€zgüven, M., Berktaş, I_., O€zçelik, B., 2016. Influence of processing conditions on
procyanidin profiles and antioxidant capacity of chocolates: optimization of dark
chocolate manufacturing by response surface methodology. LWT - Food Sci. Technol.
(Lebensmittel-Wissenschaft -Technol.) 66, 252–259.
Kadivar, S., De Clercq, N., Mokbul, M., Dewettinck, K., 2016. Influence of enzymatically
produced sunflower oil based cocoa butter equivalents on the phase behavior of
cocoa butter and quality of dark chocolate. LWT - Food Sci. Technol. (Lebensmittel-
Wissenschaft -Technol.) 66, 48–55.
Kwok, C.S., Boekholdt, S.M., Lentjes, M.A.H., Loke, Y.K., Luben, R.N., Yeong, J.K.,
Wareham, N.J., Myint, P.K., Khaw, K.-T., 2015. Habitual chocolate consumption and
risk of cardiovascular disease among healthy men and women. Heart 101, 1279–1287.
Martini, S., Conte, A., Tagliazucchi, D., 2018. Comprehensive evaluation of phenolic
profile in dark chocolate and dark chocolate enriched with Sakura green tea leaves or
turmeric powder. Food Res. Int. 112, 1–16.
Nascimento, M.M., Santos, H.M., Coutinho, J.P., Lôbo, I.P., Silva Junior, A.L.S. da,
Santos, A.G., Jesus, R.M. de., 2020. Optimization of chromatographic separation and
classification of artisanal and fine chocolate based on its bioactive compound content
through multivariate statistical techniques. Microchem. J. 152, 104342.
Oke, F., Aslim, B., Ozturk, S., Altundag, S., 2009. Essential oil composition, antimicrobial
and antioxidant activities of Satureja cuneifolia Ten. Food Chem. 112, 874–879.
Ostrowska-Ligęza, E., Marzec, A., Go
rska, A., Wirkowska-Wojdyła, M., Bry
s, J., Rejch, A.,
Czarkowska, K., 2019. A comparative study of thermal and textural properties of
milk, white and dark chocolates. Thermochim. Acta 671, 60–69.
Pantelidis, G.E., Vasilakakis, M., Manganaris, G.A., Diamantidis, G., 2007. Antioxidant
capacity, phenol, anthocyanin and ascorbic acid contents in raspberries, blackberries,
red currants, gooseberries and Cornelian cherries. Food Chem. 102, 777–783.
M. Medina-Mendoza et al. Heliyon 7 (2021) e06154Qin, X.-W., Lai, J.-X., Tan, L.-H., Hao, C.-Y., Li, F.-P., He, S.-Z., Song, Y.-H., 2016.
Characterization of volatile compounds in Criollo, Forastero, and Trinitario cocoa
seeds (Theobroma cacao L.) in China. Int. J. Food Prop. 1–15.
Rodriguez Furl
an, L.T., Baracco, Y., Lecot, J., Zaritzky, N., Campderro
s, M.E., 2017.
Influence of hydrogenated oil as cocoa butter replacers in the development of sugar-
free compound chocolates: use of inulin as stabilizing agent. FoodChem. 217, 637–647.
Sasaki, M., Ueno, S., Sato, K., 2012. Polymorphism and mixing phase behavior of major
triacylglycerols of cocoa butter. In: Garti, N., Widlak, N.R. (Eds.), Cocoa Butter and
Related Compounds. United States of America: AOCS Press, pp. 151–172.
Scherer, R., Godoy, H.T., 2009. Antioxidant activity index (AAI) by the 2,2-diphenyl-1-
picrylhydrazyl method. Food Chem. 112, 654–658.
Silva, T.L.T. da, Grimaldi, R., Gonçalves, L.A.G., 2017. Temperature, time and fat
composition effect on fat bloom formation in dark chocolate. Food Struct. 14, 68–75.
Singleton, V., Orthofer, R., Lamuela-Raventos, R., 1999. Analysis of total phenols and
other oxidation substrates and antioxidants by means of Folin Ciocalteau reagent. In:
Pelosi, P., Knoll, W. (Eds.), Methods in Enzymology. Academic Press, pp. 152–178.
Summa, C., Raposo, F.C., McCourt, J., Scalzo, R.L., Wagner, K.-H., Elmadfa, I., Anklam, E.,
2006. Effect of roasting on the radical scavenging activity of cocoa beans. Eur. Food
Res. Technol. 222, 368–375.
Talbot, G., 2009. Vegetable fats. In: Beckett, S.T. (Ed.), Industrial Chocolate, Manufacture
and Use. Blackwell Publishing, York, UK, pp. 415–433.
Talbot, G., 2012. Chocolate and cocoa butter-structure and composition. In: Garti, N.,
Widlak, N.R. (Eds.), Cocoa Butter and Related Compounds, 1–34. United States of
America: AOCS Press.6
Todorovic, V., Redovnikovic, I.R., Todorovic, Z., Jankovic, G., Dodevska, M., Sobajic, S.,
2015. Polyphenols, methylxanthines, and antioxidant capacity of chocolates
produced in Serbia. J. Food Compos. Anal. 41, 137–143.
Toker, O.S., Konar, N., Palabiyik, I., Rasouli Pirouzian, H., Oba, S., Polat, D.G.,
Poyrazoglu, E.S., Sagdic, O., 2018. Formulation of dark chocolate as a carrier to
deliver eicosapentaenoic and docosahexaenoic acids: effects on product quality. Food
Chem. 254, 224–231.
Torbica, A., Jambrec, D., Tomi
c, J., Pajin, B., Petrovi
c, J., Kravi
c, S., Loncarevi
c, I., 2016.
Solid fat content, pre-crystallization conditions, and sensory quality of chocolate with
addition of cocoa butter analogues. Int. J. Food Prop. 19, 1029–1043.
Watanabe, S., Yoshikawa, S., Sato, K., 2021. Formation and properties of dark chocolate
prepared using fat mixtures of cocoa butter and symmetric/asymmetric stearic-oleic
mixed-acid triacylglycerols: impact of molecular compound crystals. Food Chem.
339, 127808.
Zhang, Z., Song, J., Lee, W.J., Xie, X., Wang, Y., 2020. Characterization of enzymatically
interesterified palm oil-based fats and its potential application as cocoa butter
substitute. Food Chem. 318, 126518.
Zhou, S., Seo, S., Alli, I., Chang, Y.-W., 2015. Interactions of caseins with phenolic acids
found in chocolate. Food Res. Int. 74, 177–184.
Z_ yz_elewicz, D., Budryn, G., Oracz, J., Antolak, H., Kręgiel, D., Kaczmarska, M., 2018. The
effect on bioactive components and characteristics of chocolate by functionalization
with raw cocoa beans. Food Res. Int. 113, 234–244.
Z_ yz_elewicz, D., Krysiak, W., Oracz, J., Sosnowska, D., Budryn, G., Nebesny, E., 2016. The
influence of the roasting process conditions on the polyphenol content in cocoa
beans, nibs and chocolates. Food Res. Int. 89, 918–929.