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OPEN Optimizing vacuum drying

process of polyphenols, flavanols
and DPPH radical scavenging assay
in pod husk and bean shell cocoa
Fernando Ramos‑Escudero 1,2*, Sandra Casimiro‑Gonzales 3, María de la Luz Cádiz‑Gurrea 4,
Keidy Cancino Chávez 1, Jaime Basilio‑Atencio 5, Elizabeth S. Ordoñez 5, Ana María Muñoz 1,3 &
Antonio Segura‑Carretero 4

The objective of this study was to optimize different vacuum drying conditions for cocoa pod husk
and cocoa bean shell in order to enhance these by-products for commercial applications. To carry out
the optimization, the response surface methodology was applied using a Box–Behnken experimental
design with 15 experiments for which different conditions of temperature ­(X1), drying time ­(X2) and
vacuum pressure ­(X3) were established. The response variables were the content of total polyphenols,
the content of flavanols and the radical scavenging activity evaluated in the extracts of the different
experiments. Temperature (50–70 °C), drying time (3–12 h) and vacuum pressure (50–150 mbar)
were considered as independent variables. The main factors affecting the response variables were
temperature, followed by vacuum pressure. For the content of polyphenols, the optimal response
values predicted for the cocoa pod husk was 11.17 mg GAE/g with a confidence limit (95%) of 9.05 to
13.28 mg GAE/g (optimal conditions: 65 °C, 8 h and 75 mbar), while for the cocoa bean shell cocoa was
29.61 mg GAE/g with a confidence limit (95%) of 26.95 to 32.26 mg GAE/g (optimal conditions: 50 °C,
5 h and 100 mbar). Therefore, results of this study suggest a high content of phenolic compounds
obtained from these by-products that show relevance as functional ingredients for application in the
food, nutraceutical, and cosmeceutical industries.

Cocoa (Theobroma cacao L.) is a plant resource of great economic importance for the main producing regions
in the world. Nibs, liquor, cocoa powder, and cocoa butter are obtained during primary processing, while the
by-products obtained during pre-processing and processing are cocoa pod husks and cocoa bean s­ hell1, 2. The
estimated production of cocoa beans (2020/2021) according to the International Cocoa Organization (ICCO)
is around 5240 thousand t­ onnes3. Of this production, only one tenth will be used for the production of liquor,
butter, cake or cocoa powder, while the remaining biomass (80 to 90%) is discarded as a by-product (including
cocoa pod husk, cocoa bean shell, mucilage and placenta)4. Cocoa bean shell that are generated during the
roasting process represent between 10 and 17% of the total weight of the cocoa b ­ ean5. The recovery of cocoa
by-products from the perspective of the circular economy is essential to promote the value chain and mitigate
environmental impacts. From this context, the promotion of innovative models using the cocoa pod husk,
and the cocoa bean shell for the production of bioactive components (carbohydrates, dietary fiber, proteins,
polysaccharides, polyphenols, minerals, etc.), as well as in the application in food products with high added
value (beverages, chocolates, jams, oils, sausages, etc.), and for the production of biofuels (biochar, bioethanol,
biogas, bio-oils, etc.) they are highly ­valued6–8.

1
Unidad de Investigación en Nutrición, Salud, Alimentos Funcionales y Nutraceúticos, Universidad San Ignacio
de Loyola (UNUSAN-USIL), Calle Toulon 310, 15024 Lima, Peru. 2Carrera de Nutrición y Dietética, Facultad
de Ciencias de la Salud, Universidad San Ignacio de Loyola, Av. La Fontana 550, 15024 Lima, Peru. 3Instituto de
Ciencias de los Alimentos y Nutrición, Universidad San Ignacio de Loyola (ICAN-USIL), Campus Pachacamac,
Sección B, Parcela 1, Fundo La Carolina, Pachacámac, 15823 Lima, Peru. 4Department of Analytical Chemistry,
Faculty of Science, University of Granada, Fuentenueva s/n, 18071 Granada, Spain. 5Facultad de Ingeniería
en Industrias Alimentarias, Universidad Nacional Agraria de la Selva, Carretera Central km. 1,2, Tingo María,
Peru. *email: [email protected]

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Several classes of polyphenols have been identified in cocoa pod and cocoa shell, including procyanidins,
flavanols, flavonols, phenolic ­acids9, 10. In cocoa shell the main classes of polyphenols are phenolic acids, including
gluconic acid, homovanillic acid, vanillic acid glycoside, etc.10. These compounds present in cocoa by-products
have shown various biological ­effects2. Among the bio-functionalities of the cocoa shell, it is postulated as an
antibacterial agent, inhibiting the activity against Streptococcus mutans11. Rossin et al.12 have reported a preventive
effect against damage associated with intestinal integrity from oxidative/inflammatory reactions. The results of
this study report that probably those responsible for the protection from the adverse effects is its high content of
phenolic compounds. Several authors have reported that cocoa shell and pod extracts have antioxidant activity
in vitro through DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2ʹ-azino-di-(3-ethylbenzothiazoline)) assays
-6-sulfonic acid) and FRAP (ferric reducing antioxidant power)13, 14. In addition, cocoa shell polyphenols are
capable of inhibiting the production of reactive oxygen species, protecting cells from oxidative stress by induction
of hydrogen peroxide in human umbilical vein endothelial c­ ells14. In the scientific literature, the use of cocoa
by-products obtained during pre-processing (cocoa pod) and processing (cocoa shell) has been reported. The
approach to the use of these by-products is based on strengthening value chain and the use of its bioactive
components as ingredients for functional foods, nutraceuticals, and c­ osmeceuticals15. A previous process for
the recovery of bioactive components is the raw material stabilization through different drying conditions such
as sun drying, forced air drying oven, vacuum drying, infrared drying, microwave drying, etc.13. By-product
drying methods present both advantages and disadvantages during the drying process. Water removal from
food matrices is a complex process that drastically affects the content of bioactive components, nutrients, and
sensory properties, especially the shape, color, aroma, and consistency of dehydrated p ­ roducts16, 17. Of most
drying methods, forced-air-drying is widely known and widely used as a low-cost method for the industrial
production of dehydrated foods from fruits, vegetables, seeds, nuts, and ­almonds18. In addition, the use of
conventional technologies during drying has a negative impact on the overall yield and affects the quality of
the finished ­product19. On the other hand, vacuum drying is considered an alternative technology compared to
conventional methods that use higher temperatures, therefore vacuum drying could promote the conservation
of bioactive components present in f­ ood20. For example, the impact of the drying process on anthocyanins and
uncolored phenols in winemaking by-products is highly variable, compared to freeze-drying, which is less drastic
for anthocyanins and uncolored ­phenols21. However, freeze-drying processes are not very profitable for the
food processing industry due to long times and high process ­costs22. For example, the results of vacuum drying
beetroot at 50 °C and 150 mbar on functional properties were comparable to freeze-drying22.
The response surface (MRS) methodology is a widely used tool for the optimization process. Some optimized
studies based on the vacuum drying process using design of experiments (DOEs) have focused on the evaluation
of the physicochemical, functional, antioxidant properties and biochemical changes in order to preserve the
bioactive compounds. Šumić et al.23 proposed vacuum drying for frozen sour cherries and this method was
optimized using a central composite rotatable design (CCRD) to observe the influence of factors (drying
temperature and vacuum pressure) on phytochemicals and textural characteristics. Almeida-Trasviña et al.24
dehydrated apple pomace in order to optimize the effect of vacuum drying using MRS with a central composite
design (CCD). Subsequently, Šumić et al.25 dehydrated fresh red currants applying MRS and a Box–Behnken
(BBD) experimental design. The optimized conditions for the response variables were established at a vacuum
pressure 39 mbar, temperature 70.2 °C and drying time 8 h. Osama et al.26 used MRS to maximize vitamin C
retention and minimize color degradation and a Box–Behnken design (BBD) was used for vacuum drying
experiments. The results of the optimized process after applying MRS were established for the following factors:
bed thickness 3.67 mm, vacuum pressure ~ 213.316 mbar, drying temperature 65 °C and blanching temperature
100 °C. The results of these studies are focused on evaluating the effect of vacuum drying to minimize the impact
of heat treatment on the response variables.
For this reason, vacuum drying becomes a benchmark for the food industry, its application in food drying is
easy and operating costs are lower compared to emerging dehydration technologies, being feasible for small and
medium scale processors. Therefore, the objective of the present study was to determine the effects of vacuum
drying conditions, such as temperature, drying time, and vacuum pressure, on the content of total polyphenols,
flavanols, and DPPH free radical scavenging of cocoa pod husk and cocoa bean shell, by applying a Box–Behnken
design (BBD). In addition, moisture, and chromatic properties (L*, a*, and b*) were evaluated.

Materials and methods

Plant material. Cocoa pods (CCN 51) were supplied by the small company Choco Bekita (in the small town
of Puerto Ángel, situated in Leoncio Prado province, Peru) in January 2022, while cocoa beans were provided
by Cooperativa Agroindustrial Cacao Alto Huallaga (from the Castillo Grande district, Leoncio Prado province,
Peru) in November 2021. For this study, the identification of the cocoa fruit (CCN 51 genotype) was not carried
out, since it is a genotype widely distributed in different cocoa-producing regions in Peru, as well as in Ecuador,
Colombia, and ­Brazil27. Harvest and post-harvest processing was carried out in accordance with the standards
established by the International Cocoa Organization (ICCO) (https://​www.​icco.​org/​harve​sting-​post-​harve​st-​
new/). The cocoa beans were previously roasted in a toaster oven (IMSA, model ERTC, Oxapampa, Peru). Cocoa
shells and nibs were obtained using dehusking and bean grinding machines with an independent outlet for the
shells and nibs (IMSA, Oxapampa, Peru) (Fig. 1).

Cocoa pod pretreatment and vacuum drying. Before the cocoa pod drying process, a cross-section
was made for extracting the kernel and placenta. The cocoa pod husk (CPH) was cut into small cubes, placed
in bags, and vacuum sealed. The dehydration process was conducted in a vacuum oven (VO 400, Memmert,
Schwabach, Germany), and drying processes were performed at different temperatures (50–70 °C), drying times

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Cocoa pods (CCN51) Julienne cut cocoa

Cocoa pod cross section pod husk

Fermentation and Vacuum drying of

Drying cocoa pod husks

Husking of roasted cocoa beans A

Cocoa Pod Husk

(CPH)
Output of cocoa bean
shells (CBS)
Cocoa bean roasting
B

Cocoa Bean Shell

Output of roasted cocoa
nibs of different caliber

(CBS)

Figure 1.  Obtaining cocoa by-products. Cocoa pod husk powder (A) and cocoa bean shell powder (B).

(3–12 h), and pressures (50–150 mbar) as per the Box–Behnken design (BBD) shown in Table 1. The temperature
and pressure conditions were the same for the cocoa pods and shells, whereas the drying times varied depending
on the sample, CBS (3–5 h), while CPH (8–12 h).

Moisture content. The moisture content was assessed using a forced air heater (UF160, Memmert,
Schwabach, Germany). Approximately 2 g of sample was placed in a Petri dish and dehydrated at 100 °C to
constant weight.

Chromatic properties. Before measuring the chromatic properties, the sample was pulverized using a
blade mill (GM 200, Retsch, Haan, Germany). Approximately 500 mg of sample was placed on a glass side
and then taken to the adapter base. For color measuring purposes, a Nix QC color sensor (D65 illuminant,

Levels
Factors Symbol Sample Low (− 1) Intermediate (0) High (1)
CPH 50 60 70
Temperature (°C) X1
CBS 50 60 70
CPH 8 10 12
Time (h) X2
CBS 3 4 5
CPH 50 100 150
Pressure (mbar) X3
CBS 50 100 150

Table 1.  Factors and levels for cocoa pod husk and cocoa bean shell.

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10° observer scan settings, 380–730 spectral range, and measurement geometry of 45°/0°) was used, controlled
through a mobile application. Information regarding chromaticity coordinates (L*, a*, and b*) was sent via
Bluetooth using the iOS app Nix QC™ installed in an iPhone 13 Pro Max device. The mean color was obtained
from 10 scans per sample. Color charts were obtained from https://​www.​nixse​nsor.​com/​free-​color-​conve​rter/.

Total polyphenol content (TPC). Despite Folin–Ciocalteu assay has been widely used to quantify total
polyphenols in plant extracts and other food ­matrices28, the mechanism associated of the reaction is not specific,
because can react with phenols and non-phenolic compounds generating an overestimating in TPC, despite
this evidence the AOAC official method 2017.13 considers it a first action ­method29. Taking into account the
previous description, the TPC was estimated using the Folin–Ciocalteu ­method30, with slight modifications.
The polar fraction was extracted using approximately 70 mg pod and 50 mg husk, which were placed in 2.0 mL
microcentrifuge tubes. Then, 1.0 mL of 50% ethanol solution was added to the mixture and subsequently vortex-
agitated (LP Mixer, Thermo Scientific™, South Korea) at 2000 rpm for 1 h. Next, the tubes were placed in an
ultrasonic bath (CPX5800HE, Bransonic, Danbury, CT, USA) at a frequency of 40 kHz for an extraction time of
30 min at 30 °C. Finally, the supernatant was obtained using a microcentrifuge (5425R, Eppendorf, Hamburg,
Germany) operated at 10,000 rpm for 5 min at 20 °C. Then, 40 μL of a 50% ethanol solution was added to an
aliquot of the supernatant (10 μL), followed by adding 375 μL of the Folin–Ciocalteau solution (0.2 N). After a
reaction time of 5 min, 375 μL of 7.5% sodium carbonate were added to the mixture. The reaction took 2 h under
dark conditions. The absorbance readings were taken in a glass cell for a path length of 2 mm at a wavelength of
765 nm using a UV–Vis spectrophotometer (V-770, Jasco, Tokyo, Japan). The TPC in the sample was expressed
in terms of mg of gallic acid equivalent per g of sample (mg GAE/g).

Flavanol content (TFC). The flavanol content was measured using the method described by Gallego
et al.31, modified by Ramos-Escudero et al.30. Thus, 200 μL of a 50% ethanol solution was added to an aliquot of
the preceding supernatant (25 μL), followed by adding a p-dimethylaminocinnamaldehyde solution, prepared
using 0.1% of ethanol solution containing 1 N of hydrochloric acid. The mixture was vortex-agitated for 30 s
before being allowed to react for 10 min at room temperature. The absorbance reading was measured at 640 nm
using a UV–Vis spectrophotometer (V-770, Jasco, Tokyo, Japan), and the flavanol content was expressed in mg
of catechin equivalent per g of sample (mg CE/g).

DPPH free radical scavenging activity (RSA). The DPPH test was measured following the method
previously described by Brand-Williams et al.32, with slight modifications. An aliquot of the previously diluted
supernatant (5.0 μL of the extract and 45 μL of 50% ethanol) was mixed with 1.0 mL of the 2.2-diphenyl-1-
picrylhydrazyl ([DPPH]; 100 μmol/L in 65% ethanol). The reaction was vortex-agitated for 30 s, after which it
was maintained for 10 min at room temperature. Absorbance values were recorded at 515 nm using a UV–Vis
spectrophotometer (V-770, Jasco, Tokyo, Japan). The RSA was expressed in mmol of Trolox equivalent per g of
sample (mmol TE/g).

Analysis of methylxanthines and catechin derivatives. Methylxanthines and catechin derivatives of

CPH and cocoa bean shell (CBS) were analyzed using a high-performance liquid chromatograph with diode array
detector (Hitachi Chromaster™, High-Technologies Corporation, Tokyo, Japan). The analytes were separated
using a LiChroCART®150 × 4.6 mm with Purospher® STAR RP-18 endcapped, 5 µm (Burlington, MA), at 30 °C
and a flow rate of 1.0 mL/min. Separations were performed in a gradient system using 0.1% orthophosphoric
acid in water (mobile phase A) and 0.1% orthophosphoric acid in methanol (mobile phase B)33. The gradient
program was as follows: 0 min, (20% B); 0–20 min, 40% (B); 20–30 min, 50% (B); 30–32 min, (20% B). The
concentration of each component was calculated from the retention times of the respective standards (green tea
catechin mix, namely, caffeine, (+)-catechin, (−)-catechin 3-gallate, (−)-epicatechin, (−)-epicatechin-3-gallate,
(−)-epigallocatechin 3-gallate, (−)-gallocatechin and (−)-gallocatechin 3-gallate).

Experimental design and statistical analysis. A BBD was used to optimize the vacuum drying
conditions through the response surface methodology (RSM). In this study, the drying condition parameters
were temperatures ­(X1, °C), time ­(X2, h), and pressure ­(X3, mbar) and the response variables were TPC, total
flavanol content (TFC), and antioxidant activity (AA). A 3-factor and 3-level (­ 3k) BBD was used to determine
the main influences, as well as the combined ones, for the vacuum drying conditions on the response variables
in cocoa pods, and cocoa shell. Furthermore, mathematical models were established between the dehydration
factors and response variables (TPC, TFC, and RSA). The design consisted of fifteen experiments, including
three focal points that were assigned according to the second-order B ­ BD34, with three independent variables.
The order in which experiments were conducted was completely random to protect against any unexplained
variation effects in the actual responses.
The responses to variables were adjusted to the second-order polynomial model, which describes the
interaction between the factors and response variables obtained through RSM, according to the following Eq. (1):

k 
k k−1 
 k
Y = β0 + βi xi + βii xi2 + βij xi xj , (1)
i=1 i=1 i=1 j=2

where Y is the predicted variable; β0 is a coefficient of the models; βi , βii , and βij are the coefficients of the
equations representing the effects of linear, quadratic, and interaction models, respectively; and xi xj are the

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independent variables that determine the change generated in the response v­ ariable35. Statistical analysis was
performed using STATISTICA version 8.0 (StatSoft, Inc., Tulsa, OK, USA). Analysis of variance (ANOVA)
was performed for each response, with a significance level of p < 0.05, followed by Tukey’s post hoc test. The
experiments were conducted in triplicate.

Results and discussion

Response surface analysis and CPH optimization. The experimental results of the response values for
moisture content, chromatic parameters (L*, a*, and b*), TPC, TFC, and RSA obtained through different vacuum
dehydration conditions (temperature, drying time, and pressure) under a BBD are summarized in Table 2.
Experiments were conducted after randomization, and each response variable corresponds to the mean value
of three repetitions. Before applying the experimental design analysis, the means of different experiments were
analyzed using ANOVA, followed by Tukey’s HSD test, to identify the means of experiments that are different.
The results showed that high temperatures may degrade phenol c­ ompounds36, with 70 °C being the highest
temperature both for the cocoa pod and husk. The TPC of the CPH varied from 1.63 to 13.37 mg GAE/g of
sample, whereas the RSA, as measured by the DPPH test, ranged from 0.003 to 0.12 mmol TE/g (Table 2). The
results showed that the temperature, drying time, and vacuum pressure parameters influenced the response
variables. These results were also observed by Vakula et al.37 during the vacuum drying of sweet cherry.
The ANOVA results are described in Table 3. The R ­ 2 value for the models of response variables was 0.9. TPC
(0.958), TFC (0.936), and RSA (0.950; as measured by the DPPH test) and the polynomial models applied were
obtained from experimental data. Conversely, the statistical significance values for the responses evaluated were
lower than 0.05 in all responses. These p-values of < 0.05 indicate that the mathematical models developed were
ideal for experimental data. Furthermore, Fisher’s test used to determine non adjustment (Fisher’s lack-of-fit
test) yielded p-values > 0.05, suggesting that the quadratic model properly fit the experimental data.
The regression data and their statistical significance for each response variable are summarized in Table 4. The
TPC shows four terms that demonstrate its statistical significance: temperature ­(X1), vacuum pressure ­(X3) with
p-values equal to 0.02, while the interaction between temperature and dehydration time (­ X1X2) provided p-values

Independent variable
T° T (h) mbar Color properties Responses
Runs X1 X2 X3 Moisture (%) L* a* b* View TPC TFC RSA
f d
1 60 10 100 13.13 58.09 10.64 22.49 10.53 ± 0.47 2.28 ± 0.10 0.11 ± 0.01b

2 70 8 100 12.32 61.17 11.05 24.29 11.73 ± 0.63c 2.49 ± 0.01b 0.12 ± 0.01a

3 50 10 50 11.97 59.96 12.72 26.27 8.25 ± 0.02i 1.69 ± 0.09i 0.08 ± 0.001e

4 50 10 150 30.91 35.45 10.03 11.42 2.75 ± 0.31k 0.35 ± 0.01l 0.01 ± 0.008h

5 50 12 100 12.69 58.84 13.11 27.40 8.80 ± 0.34g 1.76 ± 0.02h 0.08 ± 0.003e

6 60 8 150 24.75 40.92 13.55 18.20 4.94 ± 0.64j 0.81 ± 0.009k 0.04 ± 0.001g

7 60 12 50 9.80 64.18 9.66 23.63 11.01 ± 0.35d 2.21 ± 0.05e 0.10 ± 0.06b

8 50 8 100 44.12 33.05 9.17 8.60 1.63 ± 0.03l 0.25 ± 0.01m 0.003 ± 0.0008i

9 60 12 150 11.15 59.51 11.20 24.01 8.33 ± 0.26hi 1.69 ± 0.08i 0.08 ± 0.0001e

10 70 12 100 9.42 63.55 10.72 24.15 10.41 ± 0.10f 2.02 ± 0.01f 0.09 ± 0.001c

11 60 10 100 13.68 54.99 11.15 23.30 12.14 ± 0.05b 2.40 ± 0.07c 0.12 ± 0.07a

12 60 8 50 10.67 62.96 9.82 24.05 13.37 ± 0.11a 3.09 ± 0.21a 0.12 ± 0.01a

13 70 10 150 9.38 57.89 10.01 22.04 8.40 ± 0.04h 1.22 ± 0.08j 0.06 ± 0.007f

14 70 10 50 8.59 63.09 10.87 22.33 10.99 ± 0.08d 1.87 ± 0.01g 0.09 ± 0.003d

15 60 10 100 11.15 57.50 10.03 22.59 10.85 ± 0.04e 2.00 ± 0.01f 0.09 ± 0.001c

Table 2.  Box–Behnken matrix for cocoa pod husk. TPC (mg GAE/g) total polyphenols; TFC (mg CE/g) total
flavanols; RSA (mmol TE/g) by DPPH assay. Different letters by column indicate significant differences at
p < 0.05.

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ANOVA df SS MS F-value p-value

TPC
Model 11 469.43 42.67 12.68 0.010*
Residual 33 20.70 0.63
Lack of fit 27 16.34 0.61 0.83 0.664ns
Pure error 6 4.36 0.73
Cor. total 44 490.13
­R2 0.958
TFC
Model 11 24.68 2.24 8.16 0.020*
Residual 33 1.68 0.05
Lack of fit 27 1.43 0.05 1.29 0.404ns
Pure error 6 0.25 0.04
Cor. total 44 26.36
­R2 0.936
RSA
Model 11 0.05 0.00 10.57 0.010*
Residual 33 0.00 0.00
Lack of fit 27 0.00 0.00 0.42 0.943ns
Pure error 6 0.00 0.00
Cor. total 44 0.06

Table 3.  Analysis of variance (ANOVA) for the content of total polyphenols, total flavanols and the DPPH free
radical scavenging (RSA) of the cocoa pod husk. *Significant at p ≤ 0.05. ns Not significant at p > 0.05.

Term Total polyphenol content Flavanol content DPPH assay

Regression data Coeff. Std. err. p-value Coeff. Std. err. p-value Coeff. Std. err. p-value
Intercept 8.38 0.34 0.000* 1.61 0.09 0.000* 0.07 0.004 0.000*
Linear
­X1 2.51 0.42 0.002* 0.44 0.12 0.013* 0.02 0.005 0.006*
­X2 0.86 0.42 0.093ns 0.13 0.12 0.319ns 0.01 0.005 0.131ns
­X3 − 2.40 0.42 0.002* − 0.59 0.12 0.004* − 0.03 0.005 0.004*
Interaction
­X1X2 − 2.12 0.59 0.015* − 0.49 0.17 0.031* − 0.03 0.007 0.017*
­X1X3 0.73 0.59 0.269ns 0.17 0.17 0.349ns 0.01 0.007 0.231ns
­X2X3 1.44 0.59 0.057ns 0.44 0.17 0.046* 0.01 0.007 0.108ns
Quadratic
­X11 − 2.42 0.61 0.011* − 0.63 0.17 0.015* − 0.03 0.007 0.009*
­X22 − 0.61 0.61 0.365ns 0.03 0.17 0.856ns 0.00 0.007 0.546ns
­X33 − 1.15 0.61 0.117ns − 0.31 0.17 0.134ns − 0.02 0.007 0.052ns

Table 4.  Regression data of the fitted second-order polynomial models for response variables of the cocoa pod
husk (CPH). (X1): temperature, ­(X2): drying time, ­(X3): pressure. *Significant at p ≤ 0.05. ns Not significant at
p > 0.05.

of < 0.015. Furthermore, the temperature factor showed a quadratic effect (­ X11) (p < 0.011), which was found to
be considerable for the TPC. Similar results were reported by Almeida-Trasviña et al.24 for the coefficients of the
linear model ­X1, ­X2 as well as for interactions ­(X1X2) and ­(X2X3), while the temperature and vacuum pressure
factors in the quadratic model exhibited a significant effect on the polyphenol content.
As for the TFC, it could be observed that five terms were found to be statistically significant, namely,
temperature ­(X1) and vacuum pressure (­ X3), as their p-values were lower than 0.05 (p = 0.013 and p = 0.004).
Meanwhile, interaction was influenced by ­(X1X2) and ­(X2X3), with p-values of 0.031 and 0.046, respectively. The
quadratic factor effect ­(X11) was significantly influenced by vacuum pressure, followed by temperature, whereas
the dehydration time showed statistically nonsignificant results (p = 0.319). On the other hand, Rebollo-Hernanz
et al.38 reported that temperature is the main variable contributing to the retrieval of flavanols 37.7% (p < 0.001),
whereas dehydration time contributes just 0.1% (p > 0.05). Šumić et al.25 reported that the temperature (­ X1)

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showed significant differences (p < 0.05) during the red currants vacuum drying process for the content of
flavonoids and total polyphenols.
The RSA measured using the DPPH method showed that four terms are responsible for its statistical
significance. Similar to the TPC, factors ­X1 and X ­ 3 have a linear effect on the RSA, given their p-values were
lower than 0.01, while the interaction between temperature and time (­ X1X2) yielded a p-value of 0.017 and, in
quadratic terms, temperature ­(X11) yielded a p-value of 0.009; thus, both the interaction and the quadratic model
were found to be statistically significant. These results are similar to those found by Rebollo-Hernanz et al.38
in their study in terms of the temperature variables (­ X1) (p < 0.001) and (­ X11) (p < 0.01), while the interaction
between temperature ­(X1) and dehydration time ­(X2) was statistically nonsignificant, contributing just 2.5%. On
the other hand, Šumić et al.25 mentioned that the temperature ­(X1) and the vacuum pressure ­(X3) had a significant
influence on the inhibition coefficient at 50% (­ IC50) for the radical DPPH.
The regression equations (Eqs. 2, 3, 4) that describe the response variables for dehydration using vacuum
drying for CPH are as follows:
YTPC = −150.926 + 4.07236 × X1 − 0.0242137 × X1 × X1 + 8.39838 × X2 − 0.152049 × X2 × X2
− 0.186957 × X3 − 0.000461833 × X3 × X3 − 0.106118 × X1 × X2 (2)
+ 0.00145575 × X1 × X3 + 0.0143976 × X2 × X3 ,

YTFC = −31.4613 + 1.01619 × X1 − 0.00632153 × X1 × X1 + 0.945365 × X2 + 0.00832258 × X1

× X1 − 0.0517102 × X3 − 0.000124629 × X3 × X3 − 0.0247685 × X1 × X2 (3)
+ 0.00034494 × X1 × X3 + 0.00439692 × X2 × X3 ,

YRSA = −1.72726 + 0.0474875 × X1 − 0.000285559 × X1 × X1 + 0.0846286 × X2

− 0.000874837 × X2 × X1 − 0.00169076 × X3 − 7.3657e − 06 × X3 (4)
× X3 − 0.00128114 × X1 × X2 + 2.01257e − 05 × X1 × X3 + 0.000144524 × X2 × X3 .
The results for TPC found in this study are very similar to the value reported by Delgado-Ospina et al.39,
with a mean of 8.44 ± 0.04 mg GAE/g. Another study reported that the TPC ranged from 46 to 57 mg GAE/g,
although a low content of it can be found as well. Campos-Vega et al.40 reported that the content variation
depends on the geographical origin, genotype, and recovery system of bioactive compounds, in addition to
the sample’s dehydration method. Table 2 shows that experiment No. 12 had a higher TPC (13.37 mg GAE/g)
compared with other experiments. To achieve the maximum polyphenol content, the dehydration process was
conducted at 60 °C for 8 h with a vacuum pressure of 50 mbar. On the other hand, experiment No. 8 showed the
lowest content of polyphenols, with an average of 1.63 mg GAE/g, under dehydration parameters of 50 °C, 8 h,
and 100 mbar. Overall, an increase in the dehydration temperature and time improved the polyphenol content
if the vacuum pressure applied was reduced to 50 mbar (Fig. 2a–c). Experiments No. 5, 10, and 7 demonstrated
that the TPC increased to 8.80, 10.41, and 11.01 mg GAE/g when pressure decreased from 100 to 50 mbar. These
results are similar to those found by Šumić et al.25 and Almeida-Trasviña et al.24. Therefore, the optimal vacuum
drying conditions were the following: temperature (65 °C), drying time (8 h) and vacuum pressure (75 mbar)
to obtain a TPC of 11.17 mg GAE/g.
The TFC obtained in the different experiments showed less variation compared to the TPC. Delgado-Ospina
et al.39 reported that the TFC in CPH was 2.9 ± 0.1 mg rutin equivalent per gram of sample. Table 2 shows that
experiment No. 12 had a greater content of flavanols (3.09 mg CE/g), while a content of 0.25 mg CE/g was
obtained in experiment No. 8. Both TFC and TPC showed similar results for all experiments (1–15) (correlation
coefficient = 0.9415; p = 0.0000). The effect of temperature, drying time, and decreased vacuum pressure play
an important role in flavanol retrieval (Fig. 2d–f). Rebollo-Hernanz et al.38 also reported that flavanol retrieval
improved with an increase in temperature.
On the other hand, the RSA measured using the DPPH method (Fig. 2g–i) was similar to the mean reported
by Delgado-Ospina et al.39, with a value of ~ 0.058 mmol TE/g. When 5 categories are formed, the RSA is
distributed as follows: in 2 experiments ranging from 0.0035 to 0.0269 mmol TE/g, in 1 experiment ranging
from 0.0269 to 0.0503 mmol TE/g, in 1 experiment ranging from 0.0503 to 0.0738 mmol TE/g, in 6 experiments
ranging from 0.0738 to 0.0972 mmol TE/g, and in 5 experiments ranging from 0.0972 to 0.1206 mmol TE/g. The
first 4 correspond to experiments 4, 6, 8, and 13, with a mean of 0.03 mmol TE/g and an average temperature
and vacuum pressure of 57.5 °C and 137.5 mbar, respectively, whereas the 6-experiment group presented a
mean of 0.09 mmol TE/g. This group was characterized by an average temperature value of 60 °C and a vacuum
pressure of 91.7 mbar. Finally, the last group showed a mean of 0.11 mmol TE/g, with a temperature of 62 °C
and a vacuum pressure of 80 mbar. These results are consistent with those reported by Almeida-Trasviña et al.24,
who found that the best effect for RSA was achieved when the temperature was high, and the vacuum pressure
was low. The correlation of DPPH with TPC and with TFC was high: DPPH vs. TPC ­(r2 = 0.9531) and DPPH
vs. TFC (­ r2 = 0.9572; Fig. 3). CPH is an excellent source of antioxidants. Indrianingsih et al.41 reported that
this subproduct contains an important bioactive compound, which has been found to have antioxidant and
antidiabetic properties.

Response surface analysis and CBS optimization. The results obtained with the BBD for the CBS
(Table 5) showed that the TPC varied between 19.56 and 35.31 mg GAE/g of sample, the TFC ranged from 5.07
to 6.32 mg CE/g of sample, and the AA ranged from 0.21 to 0.24 mmol TE/g. These results indicate that the

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Figure 2.  Fitted surface graphs for cocoa pod husk (CPH), showing the combined effects of vacuum drying
process of the factor interactions: temperature (°C), drying time (h), and pressure (mbar) for TPC (mg GAE/g)
total polyphenols; TFC (mg CE/g) total flavanols; RSA (mmol TE/g) by DPPH assay.

polyphenol content was mostly influenced by temperature, drying time, and vacuum pressure factors, compared
to the TFC and RSA.
The ANOVA results are summarized in Table 6. As for the shell of cocoa beans, the variables TFC, and RSA
were statistically nonsignificant. The F-values for the variables were 0.45, and 0.36, while the p-values were 0.860,
and 0.911, respectively. The R­ 2 variables for the response variables were far from encouraging, with TPC, TFC,
and RSA values of 0.710, 0.449, and 0.393, respectively. Since TPC contribution was significant (p < 0.046), the
mathematical model was generated for this variable.
The regression equation (Eq. 5) describing the response variable for dehydration through vacuum drying for
cocoa bean husk is as follows:
YTPC = 55.4573 − 2.97554 × X1 + 0.0232052 × X1 × X1 + 14.8585 × X2
− 0.656262 × X2 × X2 − 0.2598 × X3 + 0.00181709 × X3 × X3 (5)
+ 0.00461714 × X1 × X2 + 0.000598781 × X1 × X3 − 0.0163812 × X2 × X3 .
The representation of the 3D response surface was generated for TPC (Fig. 4), while ANOVA results (Table 7)
revealed only one factor as statistically significant for polyphenol content—drying time (­ X2) (p = 0.043). On the
other hand, the flavanol content and RSA yielded nonsignificant values (p > 0.05). In addition, the mathematical

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Flavanols (mg CE/g)

Polyphenols (mg GAE/g)

r2 = 0.9415

DPPH (mmol TE/g)

r2 = 0.9572 r2 = 0.9531

Figure 3.  Correlation between response variables for cocoa pod husk: TPC (mg GAE/g) total polyphenols; TFC
(mg CE/g) total flavanols; RSA (mmol TE/g) by DPPH assay.

models generated were not fit to predict the responses. In fact, the lack of fitness was significant for the polyphenol
content variable (p < 0.05), while the p-value was 0.271 and 0.826 for the flavanol content and RSA responses,
respectively (Table 6). The factors selected did not show a great effect on the response variables but only a slight
influence of drying time on polyphenol content in the linear model. On the other hand, vacuum pressure had no
significant effect on the responses either. Rebollo-Hernanz et al.38 reported a high influence of the temperature
factor ­(X1) on polyphenol content, flavanol content, and RSA, with contributions ranging from 37 to 43%. The
temperature ranged from 30 to 100 °C during the study. Furthermore, it could be observed that the drying time
­(X2) did not have a significant influence, with a contribution of 0.1–0.5%. The interaction between temperature
and drying time ­(X1X2) for the three response variables was statistically nonsignificant. As for vacuum pressure
­(X3), Almeida-Trasviña et al.24 reported that the effect of X ­ 3 in the linear model was nonsignificant, both for
polyphenol content and RSA.
The TPC in CBS was quite similar to that reported by Rojo-Poveda et al.42, ranging from 3 to 43 mg GAE/g for
CBS of different origins—even Peruvian samples had a median of ~ 34.5 mg GAE/g. Conversely, Cádiz-Gurrea
et al.10 reported bean shell values between ~ 7 and ~ 22 mg GAE/g. These variations in polyphenol content may be
associated with geographic o ­ rigin43. Table 5 indicates that experiment No. 14 had a higher polyphenol content,
with factors ­(X1 = 50 °C, ­X2 = 5 h, and ­X3 = 100 mbar) statistically nonsignificant compared with experiment No.
13 ­(X1 = 60 °C, ­X2 = 5 h, and ­X3 = 150 mbar). Experiment No. 2 obtained the lowest polyphenol content (19.56 mg
GAE/g). Therefore, the optimal vacuum drying conditions were the following: temperature (50 °C), drying time
(5 h) and vacuum pressure (100 mbar) to obtain a TPC of 29.61 mg GAE/g.
As for the flavanol content, the experiments performed showed little variation—the lower and upper quartile
yielded values ranging from 5.12 to 6.08 mg CE/g, and the difference between the minimum and maximum
values was 1.25 mg CE/g of sample. The minimum and maximum values corresponded to experiments No.
9 ­(X1 = 70 °C, ­X2 = 3 h, and ­X3 = 100 mbar) and 6 ­(X1 = 50 °C, ­X2 = 4 h, and ­X3 = 50 mbar), respectively. The
mean value of the set of experiments was found in experiment No. 8 (5.68 mg CE/g) ­(X1 = 70 °C, ­X2 = 4 h and
­X3 = 150 mbar). The flavanol content in this study was lower compared to the results found by Cádiz-Gurrea
et al.10, who reported values from ~ 16 to ~ 36 mg of catechin equivalent per g of sample. These differences may
be attributed to geographic ­origin42, sample characteristics, extraction system, and analytical p ­ rocedure10.
The RSA in the experiments varied slightly, with a lower and upper quartile of 0.21 and 0.24 mmol TE/g
of sample, respectively. Delgado-Ospina et al.39 reported values of ~ 0.071 mmol TE/g, whereas Rojo-Poveda
et al.42 examined CBS samples of different geographic origins and reported a variation of ~ 0.03 to ~ 0.18 mmol
TE/g. Peruvian samples showed a mean of ~ 0.15 mmol TE/g, while the RSA values reported by Barbosa-Pereira

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Independent variable
T° t(h) mbar Color properties Responses
Runs X1 X2 X3 Moisture (%) L* a* b* View TPC TFC RSA
f c
1 60 5 50 1.48 35.41 11.30 10.07 23.33 ± 0.25 5.12 ± 0.10 0.22 ± 0.02c

2 60 3 50 1.89 31.43 9.05 6.14 19.56 ± 0.95g 6.27 ± 0.13a 0.23 ± 0.01a

3 70 5 100 1.02 34.39 11.13 8.55 31.74 ± 0.53b 5.56 ± 0.11b 0.23 ± 0.01abc

4 60 4 100 1.54 32.03 11.43 8.97 29.00 ± 0.30cd 6.08 ± 0.11a 0.24 ± 0.03a

5 60 4 100 1.21 33.73 11.31 9.20 30.84 ± 0.34b 5.60 ± 0.07b 0.22 ± 0.02abc

6 50 4 50 1.28 33.05 11.43 8.61 28.17 ± 0.25de 6.32 ± 0.12a 0.23 ± 0.01a

7 70 4 50 0.74 34.37 13.36 11.22 30.14 ± 0.51bcd 5.37 ± 0.08bc 0.22 ± 0.01c

8 70 4 150 1.43 37.22 8.77 8.04 27.31 ± 0.57e 5.68 ± 0.06b 0.23 ± 0.03a

9 70 3 100 1.31 33.44 8.32 6.55 24.15 ± 0.78f 5.07 ± 0.12c 0.22 ± abcd

10 60 3 150 1.40 33.78 11.36 10.13 23.46 ± 0.28f 6.10 ± 0.09a 0.24 ± 0.03a

11 50 4 150 1.59 39.69 15.08 16.78 24.47 ± 0.60f 5.08 ± 0.07c 0.23 ± 0.02a

12 50 3 100 2.00 31.91 11.95 8.76 27.60 ± 0.49de 5.09 ± 0.09c 0.22 ± 0.04abc

13 60 5 150 1.04 33.52 12.68 9.99 33.77 ± 0.58a 5.61 ± 0.16b 0.23 ± 0.02ab

14 50 5 100 1.74 33.56 12.56 9.42 35.31 ± 0.57a 5.77 ± 0.07b 0.23 ± 0.03a

15 60 4 100 1.40 36.57 8.96 8.30 28.98 ± 0.50de 5.34 ± 0.10bc 0.21 ± 0.03d

Table 5.  Box–Behnken matrix for cocoa bean shell. TPC (mg GAE/g) total polyphenols; TFC (mg CE/g) total
flavanols; RSA (mmol TE/g) by DPPH assay. Different letters by column indicate significant differences at
p < 0.05.

et al.43 for the CBS of Creole and Trinitarian genotype, native to Venezuela, ranged from ~ 0.017 to ~ 0.026 mmol
TE/g. The RSA values obtained in this study were slightly higher than those reported by other ­researchers42, 43.

Moisture content of CPH and CBS. When the set of experiments was assessed, the moisture content
for CPH varied from 8.59 to 44.12% (Table 2). Nguyen et al.13 reported moisture values of 9.22%, 10.61%, and
11.99%, with temperatures of 60 °C, 80 °C, and 100 °C for vacuum drying. Three groups can be identified
when considering a histogram. The first group, comprising experiments with a moisture range of 5–10%,
represented 26.67% of the experiments; the second group, comprising experiments with a moisture range of
10–15%, accounted for 53.33% of the total experiments; and the third group comprised experiments with
moisture values > 15%, representing 20% of the experiments. Experiments with low moisture percentages had a
mean temperature of 67.50 °C and a mean vacuum pressure of 87.50 mbar, whereas those with a high moisture
percentage yielded an average drying temperature value of 53.33 °C and a vacuum pressure of 133.33 mbar.
These results were similar to those obtained by Šumić et al.25, who reported that an increased vacuum pressure
results in slow drying and produces samples with high moisture content. In contrast, when vacuum pressure
decreases, the drying process is faster, producing samples with low moisture content. Furthermore, the drying
time influenced the moisture content—experiments with values ranging from 5 to 10% yielded an average of
11 h, while those with values ranging from 10 to 15% had a mean of 8.67 h. Moisture content also affected the
response variables—experiments with high drying temperatures and low vacuum pressure showed high TPC,
TFC, and RSA. Conversely, experiments (4, 6, and 8; Table 2) conducted at low temperature and high vacuum
pressure yielded lower values of polyphenol (3.11 mg GAE/g) and flavanol (0.47 mg CE/g) contents as well as an
RSA of 0.02 mmol TE/g. Similar results were reported by Almeida-Trasviña et al.24, with lower values for TPC
and RSA for temperatures ranging from 32 to 41 °C and a vacuum pressure ranging from ~ 420 to ~ 505 mbar.
In the case of CBS, the moisture content varied between 0.74 and 2.0%, which are similar to those published
by Delgado-Ospina et al.39, who reported a mean of 1.9%. In addition, the moisture content slightly varied as a
function of the experiment. However, those between 1.0 and 1.6% accounted for 73.33% of all experiments. This
group showed a mean of 53.33 °C, 3.67 h, and a vacuum pressure of 83.33 mbar. Conversely, experiments with
a lower moisture percentage showed the following factors: X­ 1 = 70 °C, ­X2 = 4 h, and ­X3 = 50 mbar. As in the case

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ANOVA df SS MS F-value p-value

TPC
Model 11 480.77 43.70 88.13 0.000*
Residual 33 298.13 9.03
Lack of fit 27 244.01 9.04 21.11 0.046*
Pure error 6 54.12 9.02
Cor. total 44 778.90
­R2 0.710
TFC
Model 11 6.85 0.23 0.45 0.860ns
Residual 33 1.37 0.04
Lack of fit 27 1.21 0.05 1.74 0.252ns
Pure error 6 0.16 0.25
Cor. total 44 8.22
­R2 0.449
RSA
Model 11 0.00 0.00 0.36 0.911ns
Residual 33 0.00 0.00
Lack of fit 27 0.00 0.00 0.09 0.999ns
Pure error 6 0.00 0.00
Cor. total 44 0.0027

Table 6.  Analysis of variance (ANOVA) for the content of total polyphenols, total flavanols and the DPPH free
radical scavenging (RSA) of the cocoa bean shell. *Significant at p ≤ 0.05. ns Not significant at p > 0.05.

of CPH, a decrease in the vacuum pressure provided fairly rapid drying kinetics. For this group of experiments,
the TPC was greater than that of experiments with a higher moisture percentage. Nevertheless, the flavanol
content and RSA showed no major changes. As for AA, Šumić et al.25 found that a higher temperature and
vacuum pressure yielded lower values of RSA; thus, high temperatures result in the inactivation of antioxidants.

Chromatic properties of CPH and CBS. The color parameters of the CPH used in different experiments
showed that clarity (L*) varied between 33.05 and 64.18 units (Table 2). Chromaticity coordinates, such as a*,
ranged from 9.17 to 13.55 units, while b* varied between 8.60 and 27.40. These chromatic values found for
the different experiments are quite similar to those obtained by Delgado-Ospina et al.39 and Delgado-Ospina
et al.44. The variations in color parameters can be attributed to the sample’s stabilization process, such as drying
by lyophilization and thermal treatment to inactivate enzymes, followed by lyophilization, sun-drying, vacuum
drying, infrared drying, and microwave drying, among ­others13. In this regard, the drying temperatures
influenced the chromatic parameters, and experiments that yielded low moisture content values (5–10%)
showed clearer L* values (mean: 62.18 units), while experiments with values ranging from 10 to 15% showed
slightly lower L* values (mean: 59.13 units). Conversely, much lower L* values were found in experiments with
a moisture content exceeding 15% (mean: 36.47 units). These results are very similar to those reported by other
authors where the increase in moisture content produces a decrease in the L* parameter. As for the chromaticity
parameter a*, no remarkable changes were observed despite the variation in moisture content. Delgado-Ospina
et al.44 observed similar changes in dehydrated (4.02 units) and hydrated (4.56 units) CPH samples. As for the
chromaticity coordinate b*, the different experiments showed considerable variations—treatments with < 15%
of moisture showed higher means (approximately 23.67 units), while those with > 15% of moisture yielded a
mean of 12.74 units. The contribution of coordinate b* (yellowness) to the color of CPH was more relevant,
probably due to its carotenoid content. Pico Hernández et al.45 reported a carotenoid content of 64.35 mg/g,
using a supercritical fluid extraction system. Taking the correlation values into consideration, parameters L* and
b* vs. moisture showed an negative relation (L* vs. moisture: r =  − 0.9512; p = 0.0000; R ­ 2 = 0.9049) and (b* vs.
moisture: r =  − 0.9238; p = 0.0000; ­R2 = 0.8535), while the chromaticity parameter a* vs. moisture showed little or
no correlation (a* vs. moisture: r =  − 0.1648; p = 0.5572; ­R2 = 0.0272).
With regard to the CBS, the clarity parameter (L*) ranged from 31.43 to 39.69 units (Table 5), whereas
chromaticity parameters for a* and b* varied between 8.32 and 15.08 units and 6.14 and 16.78 units, respectively.
As for the L* parameter, several authors reported a variation from 45 to 51 ­units46, 47. The variation in the
L* parameter may be associated with the effect of temperature and drying times as a result of the process of
caramelization of carbohydrates and amino acids, causing their browning and making their clarity decrease
toward the darkest side. The results of chromaticity parameters, both a* and b*, were slightly similar to the results
found in previous ­studies39, 46, 47.

Methylxanthines and catechin derivatives of CPH and CBS. The results obtained from the LC-DAD
analysis showing the content of theobromine, caffeine, and catechin derivatives are presented in Table 8. These

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Figure 4.  Fitted surface graphs for cocoa bean shell (CBS), showing the combined effects of vacuum drying
process of the factor interactions: temperature (°C), drying time (h), and pressure (mbar) for TPC (mg GAE/g).

compounds were previously identified in methanol extracts of the CPH and C ­ BS10, 13. The theobromine content
in CPH and CBS was 0.14 and 7.49 mg/g of sample, respectively. According to Nguyen and N ­ guyen48, the
theobromine concentration in CPH obtained with various extraction systems, such as 70% chloroform and
ethanol, was ~ 0.004 and ~ 0.02 mg/g, respectively, while under optimal extraction conditions with 70% ethanol
for 90 min, it was ~ 0.07 mg/g. On the other hand, the theobromine content in CBS was found to be within the
range established for toasted CBS of different geographic origins and genotypes, with values ranging from ~ 0.76
to ~ 9.03 mg/g42, although the values for CBS of different Mexican varieties ranged from 7.39 to 18.20 mg/g of
­sample49. The caffeine content in the CPH and CBS sample was 0.05 and 1.86 mg/g, respectively, which are lower
than those reported by Botella-Martínez et al.47, who found that the caffeine content according to a particle size
of 417–701 µm and < 417 µm was 11 mg/g and 6.13 mg/g, r­ espectively47. The total methylxantine content in CPH
and CBS was 0.19 and 9.35 mg/g per sample, respectively. Rojo-Poveda et al.42 reported similar values for CBS of
Peruvian origin, with values between 8.3 and 9.7 mg/g.
Regarding the catechin content of CPH and CBS, values ranging from 1.23 to 3.3 mg/g were found, with
the values for CPH being similar to those reported by Valadez-Carmona et al.50, who reported values ranging
between ~ 0.84 and ~ 2.28 mg/g for different dehydration systems, with microwave dehydration as the best
treatment. Although the CBS catechin content was found to be within the range established by Hernández-
Hernández et al.49, with values between 0.55 and 4.66 mg/g, the catechin content in toasted CBS of different
geographical origins and genotypes was found to be lower, ranging from 0.012 to 0.18 mg/g42. Moreover, Botella-
Martínez et al.47 reported a free catechin content ranging from 1.96 to 4.21 mg/g of sample.
Another bioactive component found in relevant amounts was epicatechin; in this study, CPH and CBS had
epicatechin amounts of approximately 2.23 and 5.64 mg/g of sample. Furthermore, an investigation performed

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Total polyphenol
Term content
Regression data
Intercept 27.42 1.11 0.000*
Linear
­X1 − 0.28 1.36 0.846ns
­X2 3.67 1.36 0.043*
­X3 0.98 1.36 0.506ns
Interaction
­X1X2 − 0.03 1.93 0.988ns
­X1X3 0.22 1.93 0.915ns
­X2X3 1.63 1.93 0.436ns
Quadratic
­X11 1.29 2.01 0.548ns
­X22 − 1.20 2.01 0.577ns
­X33 − 3.38 2.01 0.153ns

Table 7.  Regression data of the fitted second-order polynomial model for response variable (TPC) of the
cocoa bean shell (CBS). (X1): temperature, ­(X2): drying time, ­(X3): pressure. * Significant at p ≤ 0.05. ns Not
significant at p > 0.05.

Compounds Cocoa pod husk (mg/g) Cocoa bean shell (mg/g)

Theobromine 0.14 ± 0.00 7.49 ± 0.01
caffeine 0.05 ± 0.00 1.86 ± 0.05
(+)-Catechin 1.23 ± 0.03 3.13 ± 0.09
(−)-Catechin 3-gallate 0.03 ± 0.00 0.61 ± 0.01
(−)-Epicatechin 2.23 ± 0.09 5.64 ± 0.03
(−)-Epicatechin-3-gallate 0.08 ± 0.00 0.53 ± 0.00
(−)-Epigallocatechin 3-gallate 1.60 ± 0.00 1.55 ± 0.02
(−)-Gallocatechin n.d. 0.69 ± 0.03
(−)-Gallocatechin 3-gallate 0.17 ± 0.01 0.20 ± 0.01
Suma catechin + epicatechin 3.47 8.77
Suma catechin derivatives 1.87 3.58
Suma methylxanthines 0.19 9.35

Table 8.  Content of methylxanthines and catechin derivatives obtained by optimal conditions of vacuum
drying in cocoa pod husk and cocoa bean shell. Optimization conditions for vacuum drying: CPH (65 °C, 8 h
and 75 mbar), and CBS (50 °C, 5 h and 100 mbar).

to assess the effect of microwaves drying, hot-air drying, and lyophilization on the epicatechin content of CPH
resulted in values between 1.59 and 3.69 mg/g50. On the other hand, Rojo-Poveda et al.42 reported an epicatechin
content of 0.044–0.74 mg/g in toasted CBS. These values were lower than those reported in the present study,
although Hernández-Hernández et al.49 obtained much greater values, ranging from 4.40 to 26.68 mg/g of sample.

Conclusion
RSM was used for optimizing the vacuum drying process of the CPH and CBS. ANOVA results provided evidence
of the significance of the models for polyphenol and flavanol contents and RSA (p < 0.05). Although the lack-of-fit
was nonsignificant (p > 0.05) and the correlation coefficient was greater than 0.9 for CPH, the ANOVA results
proved that the models were nonsignificant (p > 0.05). The model for polyphenol content showed a lack-of-fit
value (p = 0.046) with a contribution of 71%; the model for flavanol content showed a lack-of-fit value (p = 0.271)
with a contribution of 44.9%; and that for RSA showed a lack-of-fit value (p = 0.826) with a contribution of 39.3%
for CBS. The mathematical models generated for CPH were fit for experimental data, contrary to those generated
for CBS, which showed nonfit values to predict responses.

Data availability
All data generated or analyzed during this study are included in this published article.

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Received: 6 April 2023; Accepted: 17 August 2023

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Acknowledgements
The authors would like to express our special thanks to Andres Nuñez and Juan Francisco Iman during the
process of roasting and shelling the cocoa beans.

Author contributions
F.R.-E.: conceptualization, methodology, software, data curation, and writing-original draft preparation. A.S.C.,
M.d.l.L.C.-G.: conceptualization, methodology, and software. S.C.-G., K.C.C.: investigation, data curation, and
writing. J.B.-A., E.S.O.: investigation, software, and data analysis. A.M.M.: supervision, resources and project
administration. All authors reviewed the manuscript.

Funding
This research was funded by Fondo Nacional de Desarrollo Científico, Tecnológico y de Innovación Tecnológica
of Perú, project number 184-2020-FONDECYT.

Competing interests
The authors declare no competing interests.

Additional information
Correspondence and requests for materials should be addressed to F.R.-E.
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