Received: 28 October 2017 | Revised: 5 December 2017 | Accepted: 5 December 2017

DOI: 10.1111/jfpp.13592

ORIGINAL ARTICLE

Polyphenol antioxidants from cocoa pods: Extraction

optimization, effect of the optimized extract, and storage time
on the stability of palm olein during thermoxidation

Gires Boungo Teboukeu1 | Fabrice Tonfack Djikeng1,2 | Mathilde Julie Klang1 |

Serge Houketchang Ndomou1 | Mallampalli Sri Lakshmi Karuna3 |
Hilaire Macaire Womeni1

1
Department of Biochemistry, Faculty of
Science, University of Dschang, PO BOX 67,
Dschang, Cameroon
2
School of Agriculture and Natural
Resources, Catholic University Institute of
Buea, PO BOX 563, Buea, Cameroon
3
CSIR-Indian Institute of Chemical
Technology, Centre for Lipid Research,
Tarnaka, Hyderabad 500 007, India
Correspondence
Hilaire Macaire Womeni, Department of
Biochemistry, Faculty of Science, University
of Dschang, PO BOX 67, Dschang,
Cameroon.
Email: womeni@yahoo.fr
Abstract
This study was conducted in order to optimize the extraction process of phenolic antioxidants in
cocoa pods and to evaluate the effect of optimized extract and storage time on the oxidative sta-
bility of palm olein heated at 180 8C using response surface methodology (RSM). The optimization
of the extraction process of cocoa pods antioxidants was done and the results showed that the
total polyphenols of 153.51 mg GAE/g and scavenging activity of 100% were obtained under opti-
mal conditions. Vanillic acid was the phenolic antioxidant detected in the optimized extract using
HPLC-DAD (diode array detector). Rancimat test showed this extract was efficient in delaying
palm olein oxidation. To preserve quality of palm olein during heating at 180 8C, RSM concerning
iodine value and thiobarbituric acid value proposes to supplement the oil with 2,000 ppm of cocoa
pods extract during 1–7 days (03 hr heating cycle per day) of treatments.
Practical applications
To improve the oxidative stability and shelf-life of vegetable oils, only few natural sources who are
supposed to be safer than synthetic antioxidants have been authorized for industrial purpose (case
of rosemary). Therefore, there is a strong rational to continue the search of new natural sources of
antioxidants by screening plant materials. This study can be useful for the development of
industrial extraction process of cocoa pods and its application as an ingredient to delay lipid
oxidation in oils.

1 | INTRODUCTION
Thermoxidation is a process that subjects oils and fats to high tempera-
tures, similar to the frying process, but in the absence of food and
moisture. The temperature and oxygen availability are the factors that
determine oxidation rates (Ramalho & Jorge, 2008). During this pro-
cess, oils containing unsaturated fatty acids are susceptible to oxidize.
As a result, lipid oxidation leads to the formation of several secondary
oxidation products, obtained from the spontaneous decomposition of
hydroperoxides and which have been demonstrated to be potentially
toxic (Mc Clements & Decker, 2000). For these reasons, many strat-
egies have been developed in order to delay lipid oxidation. One of
these strategies involves the addition of antioxidants which have the
ability to donate their hydrogen atoms to lipids radicals, then delaying
their reactivity (Mc Clements & Decker, 2000). Synthetic antioxidants
usually supplemented to oils may be toxic (Maqsood & Benjakul, 2010)
and must be declared on the oil or fat labels, causing rejection of the
oil by consumers which generally associate the clean label with safety
(Mc Clements & Decker, 2000). Extensive research has identified
natural antioxidants in edible fruits, vegetables and plant materials.
However, using edible foods as a source of ingredients is conceptually
unethical and would raise prices of foods (Karen, 2011). Coupling these
concerns with recent demands for zero waste (reducing disposal vol-
umes by recycling natural materials) suggests the possibility of isolating
natural antioxidant from unused plant material such as peels, leaves,
and pulp.
Theobroma cacao is the name given to the cocoa tree, belonging to
the family Malvaceae (Jalil & Ismail, 2008). In the cocoa industry, fresh

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https://doi.org/10.1111/jfpp.13592
2 of 11 | BOUNGO TEBOUKEU
cocoa beans are fermented, dried and used for chocolate production
which has beneficial effects on nutrition and health (Mellor &
Naumovski, 2016). Other parts including the pods and shells are usually
discarded. Despite to be thrown, Karp, Wyrwisz, Kurek, and
Wierzbicka (2017) showed that cocoa shells contain fibers that can be
used in the formulation of low-calorie foods. Cocoa pods can be used
to remove pollutants from waters and wastewaters (Cruz et al., 2012).
The pods contain 45.6–46.4 mg gallic acid equivalent of soluble pheno-
lic (Vriesmann, Amboni, & Petkowicz, 2011), and Fapohunda and
Afolayan (2012) reported that cocoa pods contained phenolic acid
including caffeic acid. The extraction and valorization of phenolic
compounds of the cocoa pods as an ingredient in oil may contribute to
added-value product development. Extraction process is the first step
in investigating the phenolic compounds because extraction conditions
might impact the yields of phenolic antioxidant as well as their activity.
The choice of extraction solvents is critical for complex matrices
because the physicochemical properties of the solvents, particularly its
polarity, exert an influence on the amount and types of phenolic
compounds extracted (Balasundram, Sundram, & Samman, 2006). In
addition to the solvents used, other factors such as temperature and
extraction time affect extraction (Luthria, Mukhopadhyay, & Kwansa,
2006; Tomsone, Kruma, & Galoburda, 2012). Moreover, during treat-
ments at high temperature, the concentration of the extract and
storage time can influence the capacity of the extracts to limit lipids
oxidation (Maleki, Ariaii, & Fallah, 2016; Womeni, Tonfack, Anjaneyulu,
et al., 2016). In order to optimize the extraction process of phenolic
compounds in cocoa pods and evaluate the effect of optimized extract
and storage time on the stability of palm olein during storage at 180 8C,
response surface methodology (RSM) has been used.
2 | MATERIALS AND METHODS
2.1 | Materials
Refined, bleached, and deodorized palm olein (RBD Palm olein),
without additives was obtained from SCS/RAFCA Palm Oil Industry
Company Ltd (Bafoussam, West-Cameroon). Fresh cocoa pods were
collected at Bafang, Haut-Nkam Division, West-Cameroon, in January
2015. All the chemicals and reagents used were of analytical reagent
grade.
2.2 | Methods
2.2.1 | Optimization process of extraction
Experimental design
The optimum extraction conditions of polyphenols from T. cacao pods
were evaluated by central composite design (CCD). The real and coded
levels of the various parameters used are shown in Table 1. The inter-
vals of these independent variables were determined on the basis of
previous work. A total of 16 experimental runs with two replications at
the center point were completed and the total polyphenols (mg GAE/g)
and antioxidant activity (% inhibition) of cocoa pods expressed as the
dependent variables were determined (Table 2). This experimental
ET AL.
T AB LE 1 Coded and decoded levels of independent variables used
in the RSM design for the optimization process of extraction
Coded levels
–a (–1.73) –1 0 11 1a (1.73)
Independent variables Decoded levels
Temperature ( 8C), X1 25 31 40 49 55
Extraction time (hr), X2 6 9.35 15 20.35 24
Methanol 0 20 50 80 100
concentration (%), X3
design generates a second-degree polynomial model (Y) of the follow-
ing form:
Y 5 ax1 1bx2 1cx3 1dx21 1ex1 x2 1fx1 x3 1gx22 1hx2 x3 1ix23 1I
where Y represents the response variables (total polyphenols or antiox-
idant activity); x1, x2, and x3 are the levels of the independent variables;
a, b, and c are the linear terms; e, f, and h are the interaction terms; d, g,
and i are the quadratic terms; and I is a constant.
To confirm or validate the optimum conditions of polyphenol anti-
oxidants extraction, two experimental replicates were performed under
optimized conditions. The experimental and predicted values were
compared.
Extraction of phenolic compounds from cocoa pods
The extraction procedures were carried out randomly and in accord-
ance with the conditions set by the experimental design. Fresh T. cacao
pods were dried in an electric oven at 50 8C for 48 hr. The dried pods
were grounded to pass through a 1 mm sieve. Sample powder (7 g)
was blended with 150 mL of solvent (methanol/water) of concentration
specified by the experimental design (Table 2). The mixture was placed
in an electric oven and regularly subjected to shaking at the required
temperature and time specified by the full factorial design (Table 2).
The filtrate was concentrated on a rotary evaporator at 45 8C before
being stored at 4 8C for analysis.
Determination of total phenolic compounds (TPC)
The total phenolic of the extracts was evaluated using the Folin–
Ciocalteu colorimetric method as described by Gao, Ohlander,
€ rk, and Trajkovski (2000). Briefly, plant extract (20 lL)
Jeppsson, Bjo
was added in a test tube containing 2 mL of distilled water, 0.2 mL of
Folin–Ciocalteu reagent and incubated for 3 min at room temperature.
A total of 1 mL of 20% sodium carbonate was added to the mixture
and re-incubated for 20 min at 40 8C. The absorbance of the resulting
blue color was measured at 765 nm using a spectrophotometer. The
standard curve prepared from Gallic acid solution was used to express
the results as gallic acid equivalents (GAE) per gram of extract.
Determination of antioxidant activity
The 2.20 -diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity
was determined according to the modified method of Mensor et al.
(2001). A total of 900 mL of 0.3 mM methanolic solution of DPPH was
added to 100 mL of the samples containing extract at the concentration
BOUNGO TEBOUKEU ET AL. | 3 of 11

T AB LE 2 Experimental design and observed and predicted values for optimized extraction of phenolic antioxidant from cocoa pods extract

Experimental total Experimental Predicted

Methanol polyphenols Predicted total antioxidant antioxidant
Temperature (8C) Time (hr) (%) (mg GAE/g) polyphenols activity (% inhibition) activity
N X1 X2 X3 EY1 PY1 EY2 PY2

01 0(40) 0(15) 0(50) 57.97 6 1.58 55.00 64.82 6 1.07 71.17

02 0(40) 0(15) 0(50) 52.32 6 1.45 55.00 77.46 6 2.53 71.17

03 21(31) 21(9.35) 21(20) 8.69 6 1.23 21.10 48.24 6 2.87 51.69

04 11(49) 21(9.35) 21(20) 21.85 6 2.71 22.93 77.41 6 0.66 73.19

05 21(31) 21(9.35) 11(80) 92.60 6 1.49 97.95 86.88 6 1.07 89.22

06 11(49) 21(9.35) 11(80) 115.16 6 1.73 101.48 88.63 6 0.66 89.17

07 21(31) 11(20.35) 21(20) 15.16 6 0.77 27.69 59.61 6 3.44 59.35

08 11(49) 11(20.35) 21(20) 40.05 6 1.08 33.55 76.70 6 0.66 74.64

09 21(31) 11(20.35) 11(80) 78.14 6 0.78 75.92 84.23 6 1.32 88.72

10 11 (49) 11(20.35) 11(80) 52.61 6 3.75 61.27 85.65 6 1.32 82.47

11 –a(25) 0(15) 0(50) 41.17 6 1.12 37.14 78.83 6 2.55 73.00

12 1a(55) 0(15) 0(50) 39.39 6 1.28 45.04 80.39 6 2.74 85.83

13 0(40) 0(15) –a(0) 4.83 6 0.46 5.87 45.64 6 1.19 47.61

14 0(40) 0(15) 1a(100) 111.92 6 1.57 112.49 88.11 6 0.78 85.75

15 0(40) –a(06) 0(50) 52.76 6 2.96 62.34 81.96 6 1.89 80.84

16 0(40) 1a(24) 0(50) 60.72 6 3.86 52.75 80.91 6 1.48 81.64

of 100 mg/mL. The samples were kept in the dark at room temperature
and after 30 min, the absorbance was recorded at 517 nm. The absorb-
ance (Abs) of the samples, the control, and the blank was measured
against that of methanol. The results were expressed as percent
inhibition using the formula:
Akhtar, Zia-Ul-Haq, and Akbar (2008). The optimized extract was
separately added to preheated RBD palm olein (at 50 8C for 3 hr) at
concentrations of 500, 720, 1,250, 1,780, and 2,000 ppm. The efficacy
of natural antioxidants was evaluated by comparing their antioxidation
activity with those of butylated hydroxytoluene (BHT) employed at it

% inhibition 5ðAbscontrol 2Abssample Þ 3 100=Abscontrol legal limit of 200 ppm (Duh & Yen, 1997). Palm olein without additives
and prepared under the same conditions served as control.

2.2.2 | Phenolic compounds profile by HPLC-DAD
Phenolic compounds of cocoa pods were determined in the optimized
extract obtained under the optimal extraction conditions defined by
the experimental design about TPC. The extract was dissolved in meth-
anol (1 mg/mL). The analysis was carried out in an HPLC agilent system
1200 series equipped with a quaternary pump model G11311A and
Diode Array Detector (DAD) model G11315B. Data acquisition was
performed using Chemstation software. The column type was an RP-
C18 Lichrospher column, 5 mm, 4.0 mm internal diameter 3 250 mm.
Separations were done in the isocratic mode, using acetonitrile–1%
orthophosphoric acid in water (70:30, vol/vol) at a flow rate of 1 mL/
min; with an injection volume of 20 mL (sample and standards solution).
Identification of phenolic compounds (DAD detection at 280 nm) in
extract was achieved by comparing their retention time with those of
standards available.
2.2.3 | Rancimat test
Rancimat test is used for the evaluation of the antioxidant potential of
molecules to limit oxidation of oils and fats. Oil samples used for this
test were prepared according to the modified method of Iqbal, Haleem,
Induction periods (IP) of stabilized (oil containing the optimized
extract) and control oil samples were evaluated using an automated
Metrohm Rancimat model 892 as described by Womeni, Tonfack,
Anjaneyulu, et al. (2016). The time elapsed from the beginning until the
oil starts to become rancid (IP) was automatically recorded by the
instrument. The protection factor was calculated using the induction
time of oil with antioxidant (I) and the induction time of oil without
antioxidant (I0)
Protection factor 5 I=I0
2.2.4 | Effect of optimized extract concentration and
storage time on the oxidative stability of palm olein during
storage
Experimental design
The effects of process parameters (concentration of extract and stor-
age time) on the oxidative stability of palm olein were evaluated by
Central Composite Design (CCD). Real and coded levels of the
independent variables used are shown in Table 3 and the intervals
were determined on the basis of previous work and preliminary test.
4 of 11 | BOUNGO TEBOUKEU ET AL.

T AB LE 3 Coded and decoded levels of independent variables used Evaluation of oxidation parameters
in the RSM design for study effect of extract concentration and Iodine value and p-anisidine value were determined according to AOCS
storage time on the oxidative stability of palm olein
Official Method CD 18–90 (AOCS, 1999). TBA value was carried out
Coded levels according to the method described by Draper and Hadley (1990).
–a (–1.68) –1 0 11 1a (1.68)
Independent variables Decoded levels
2.3 | Statistical analyses
Extract concentration 500 720 1,250 1,780 2,000
(ppm), X1
STATGRAPHICS Plus 5.0 was used for the experimental design and
statistical analysis of the data. All responses were determined in dupli-
Storage time (days), X2 0 2 5 8 10
cate and the power of the model was determined by evaluating the
coefficient of determination (R2) obtained from the analysis of variance
(ANOVA). Statistical significance of the model variables was deter-
mined at the 5% probability level. Response surfaces and contour plots
were plotted using SigmaPlot v11.0 (c) systat.
The design consisted of 10 runs with two replicates at the center point The Rancimat test was performed in duplicate and results are rep-
and the iodine value, p-anisidine value, and thiobarbituric acid (TBA) resentatives of the mean 6 standard deviation. The Dunnett and
value expressed as the dependent variables were determined (Table 4). Student–Newmann–Keuls tests were used to compare means using
A full quadratic model was used for fitting of data: the software GraphPad-InStat, version 3.05 for Windows.
0
Y 5I1ax1 1bx2 1cx21 1dx22 1ex1 x2
0
where Y represents the response variables (iodine value, p-anisidine 3 | RESULTS AND DISCUSSION
value, and TBA value); x1 and x2 are the levels of the independent vari-
ables; a and b are the linear terms; e is the interaction term; c and d are
3.1 | Extraction optimization
the quadratic terms; and I is a constant. 3.1.1 | ANOVA and regression equations

Sample preparation
The optimized extract was dissolved and separately added to 50 g of
preheated RBD palm olein (at 50 8C for 3 hr) at concentrations indi-
cated by the full factorial design (Table 4). Stabilized oil samples were
placed in dark brown glass bottles with narrow necks and subjected to
accelerated storage in an electric hot air oven at 180 8C (03 hr heating
cycle per day) for 10 days. Samples were collected according to the
time (days) indicated by the experimental design and stored in the
refrigerator for further analysis. The oxidative deterioration level was
assessed by measuring oxidation parameters.
The antioxidant activity (% inhibition) and total polyphenols (mg GAE/
g) of cocoa pods extract obtained from the 16 experiments are pre-
sented in Table 2. The mean of experimental total phenolic content
and antioxidant activity varied from 4.83 to 115.16 mg GAE/g and
45.64 to 88.63% respectively. Karim, Azlan, Ismail, Hashim, and
Abdullah (2014) have reported that total polyphenols of cocoa pods
aqueous ethanolic (80%, vol/vol) extract oscillated from 19.59 to
49.54 mg GAE/g depending on the clones. The higher phenolic content
in our case could be the collection source of sample and particularly
the effect of methanolic solvent fraction used during extraction.

T AB LE 4 Experimental design, observed and predicted values of parameter effect on the oxidative stability of palm olein during
thermoxidation

Experimental Predicted Experimental Predicted Experimental Predicted

Storage iodine value Iodine value thiobarbituric thiobarbituric p-anisidine p-anisidine
Extract (ppm) time (days) (g I2/100 g) (g I2/100 g) acid values (ppm) acid values (ppm) value (ppm) value (ppm)
N X1 X2 EY1 PY1 EY2 PY2 EY3 PY3

01 0(1250) 0(5) 55.56 6 1.95 55.795 0.98 6 0.01 0.971 412.7 6 4.8 404.828

02 0(1250) 0(5) 56.03 6 1.34 55.795 0.9 6 0.12 0.971 396.9 6 10.5 404.828

03 1(1780) 1(8) 54.29 6 1.34 54.023 1.05 6 0.09 1.155 478.9 6 53.36 475.888

04 1(1780) 21(2) 57.15 6 1.57 58.667 0.84 6 0.03 0.765 147.3 6 21.2 124.836

05 21(720) 1(8) 53.18 6 2.01 52.810 1.18 6 0.05 1.307 557.4 6 7.3 553.84

06 21(720) 21(2) 55.08 6 0.00 56.493 0.88 6 0.03 0.830 173.7 6 14.9 150.723

07 a(2000) 0(5) 57.78 6 0.00 57.133 0.93 6 0.00 0.931 295.1 6 43.4 307.806

08 2a(500) 0(5) 55.24 6 0.89 54.739 1.12 6 0.06 1.084 367.8 6 5.2 381.231

09 0(1250) a(10) 51.43 6 0.22 52.117 1.47 6 0.12 1.327 575.4 6 4.7 574.765

10 0(1250) 2a(0) 59.84 6 0.22 58.005 0.61 6 0.01 0.714 14.6 6 5.12 41.486
BOUNGO TEBOUKEU ET AL. | 5 of 11

T AB LE 5 Regression coefficients (RC), p values, and coefficient of 95.1 and 91.8% of the results in the case of total phenolic content and
multiple determinations (R2) for total phenolic content and antioxy- antioxidant activity, respectively. The ANOVA also showed that solvent
dant activity of cocoa pods extract following CCD
(methanol/water mixture) is the only factor in the linear terms that sig-
Antioxidant nificantly affect (p < .05) the total phenolic content and antioxidant
Total polyphenols activity
activity. Other significant effects (p < .05) were provided by the inter-
RC p value RC p value
action of solvent with extraction time (case of the total polyphenols)
X1: temperature (8C) 7.4254 0.4840 20.9957 0.0609 and with extraction temperature (case of the antioxidant activity). The
X2: time (hr) 6.0700 0.4001 21.5476 0.8899 use of water in combination with other organic solvent led to the
creation of a moderately polar medium that ensures the extraction of
X3: methanol 2.7894 0.0001* 1.5345 0.0005*
fraction (%) polyphenols (Chirinos, Rogez, Campos, Pedreschi, & Larondelle, 2007;
Olivas-Aguirre et al., 2017). Additionally, Pinho and Macedo (2005)
X1 X1 20.0607 0.2457 0.0359 0.1972
observed that water/methanol mixture has a higher solvatation power.
X1 X2 20.0909 0.3119 20.0310 0.5005
The regression equations obtained for the independent and response
X1 X3 20.0189 0.2592 20.0199 0.0472* variables for total phenolic content (Y1) and antioxidant activity (Y2)
X2 X2 0.0292 0.8218 0.1155 0.1270 were:

X2 X3 20.0763 0.0214* 20.0122 0.3830 Y1 5 2 236:26 1 7:42X1 1 6:07X2 1 2:78X3 2 0:06X12 2 0:09X1 X2
X3 X3 0.0016 0.7125 20.0017 0.4597 2 0:01X1 X3 1 0:02X22 2 0:07X2 X3 1 0:001X32

Constant 2236.264 46.0158 Y2 5 46:01 2 0:99X1 2 1:54X2 1 1:53X3 1 0:03X12 2 0:03X1 X2

R2
0.951 0.918 2 0:01X1 X3 1 0:11X22 2 0:01X2 X3 2 0:001X32

R2 (adjusted) 0.879 0.796

*Independent variable that significantly (p < .05) affects the response.
The experimental data were used to determine the coefficients of
the second-order polynomial equation, to establish the coefficient of
determination (R2) and significant effect of independent variables
(Table 5). ANOVA showed that the second-order polynomial model
really represented the experimental data. The coefficient of determina-
2
tions (R ) for the responses of total phenolic content and antioxidant
activity of T. cacao pods extract being 0.951 and 0.918, respectively;
and are within the range of a good set (more than 0.75) (Joglekar &
May, 1987). This means that the observed model is able to explain
3.1.2 | Analysis of response surfaces
Draw response surfaces of the model is the best way to materialize the
effect of the independent variables on the response variables, by vary-
ing two independent variables within the experimental range. Figure 1
is a response surface plot showing the effect of temperature and sol-
vent fraction (a), extraction time, and solvent fraction (b) on the total
phenolic content of T. cacao pods extract. By increasing percentage of
methanol used during extraction, the total phenolic content also
increases. Many researchers (Guyot, Marnet, & Drilleau, 2001; Prior,
Lazarus, Cao, Muccitelli, & Hammerstone, 2001) reported that metha-
nol has been generally found to be more efficient in the extraction of
molecular polyphenols. The total phenolic content was also increasing

FIGURE 1 Response surfaces showing the effect of temperature and solvent fraction (a) and extraction time and solvent fraction (b) on
the total phenolic content of Theobroma cacao pods extract
6 of 11 | BOUNGO TEBOUKEU ET AL.

FIGURE 2 Response surfaces showing the effect of temperature and solvent fraction (a), and extraction time and solvent fraction (b) on
the antioxidant activity of Theobroma cacao pods extract

with extraction time ranged between 6 and 12 hr. For long extraction values for the same process include total polyphenolic content and
time, a decrease in the response was observed. In fact, long extraction antioxidant potential values of 150.89 6 0.47 mg GAE/g and
time facilitates the degradation of bioactive compounds by polymeriza- 97.56 6 1.86%. The experimental and predicted values did not vary
 et al., 2007). The same results were obtained by Jorge,
tion (Mane significantly at 5% level. These show that the models obtained can be
 (2013) who have
Heliodoro De La Garza, Alejandro, Ruth, and Noe used to prepare cocoa pods extract having an optimum phenolic
studied the optimization of phenolic compounds extraction from cactus content and high antioxidant activity.
pear skin in a reflux system using RSM.
The effects of the studied variables on the DPPH radical scaveng- 3.2 | Phenolic compound profile in cocoa pods extract
ing activity of T. cacao pods extract are illustrated by response surfaces by HPLC-DAD
in Figure 2. Results concerning solvent fraction are similar to those pre-
Detection of phenolic compounds was performed by HPLC-DAD in
viously obtained with total phenolic content. However, for extraction
the extract obtained by the improved conditions previously defined
temperature, the antioxidant activity is very interesting for low
(temperature: 47.48 8C; extraction time: 5.56 hr; methanol fraction:
(25–30 8C) and high (45–55 8C) temperatures. Increasing the tempera-
100%). The chromatogram (Figure 3) shows that vanillic acid (retention
ture during extraction process leads to an increase of diffusion phe-
time: 9.17 min; peak area: 31.10%) was the phenolic antioxidant
nomena that helps to extract polyphenols present in the plant, due to
detected. The presence of quercetine and caffeic acid in the cocoa
vibratory effects of the cell wall molecules which facilitates migration
pods extract has been previously reported by Karim, Azlan, Ismail,
of free compounds to the solvent (Moahamad, Ali, & Ahmad, 2010).
Hashim, Abd Gani, et al. (2014) and Fapohunda and Afolayan (2012),
respectively. This difference could be due to many factors such as
3.1.3 | Determination and experimental validation of the
environmental differences, extraction procedure, genetic characteristics
optimal conditions
of the plant, and its state of maturity (Ghumman, Singh, & Kaur, 2017;
The results in Table 6 present the optimal conditions for each individual
Womeni, Tonfack, Anjaneyulu, et al., 2016).
response. The optimum conditions for a high content of polyphenols
were obtained at 47.48 8C with incubation time of 5.56 hr using metha-
3.3 | Verification of antioxidation activity of cocoa
nol fraction of 100%. Optimum conditions for radical scavenging activ-
pods extract at high temperature (Rancimat test)
ity differ only at the extraction temperature (26.59 8C). Under these
conditions, the maximum total polyphenols and antioxidant activity The effects of different concentrations of the extract on the protection
were 153.51 mg GAE/g and 100%, respectively. The experimental factors and IP of palm olein in comparison with the oil stabilized with

T AB LE 6 Predicted and experimental values under optimum conditions for maximum total phenolic content and antioxidant activity

Responses Temperature (8C) Extraction time (hr) Methanol fraction (%) Predicted value Experimental value

Total polyphenols (mg GAE/g) 47.48 5.566 100.00 153.51 6 0.00 a

150.89 6 0.47a

Antioxidant activity (% inhibition) 26.595 5.566 100.00 100.00 6 0.00a 97.56 6 1.86a

Means within each row with same superscripts are not significantly (p < .05) different.
BOUNGO TEBOUKEU ET AL.
FIGURE 3 HPLC-DAD chromatogram of standards (a) (1 5 gallic
acid, 2 5 vanillic acid, 3 5 cafeic acid, 4 5 ferulic acid, and
5 5 ellagic acid) and Theobroma cacao pods extract (b) (2 5 vanillic
acid)
BHT (synthetic antioxidant) and control (palm olein free from additives)
are presented in Table 7. It was clearly demonstrated that the supple-
mented extract, at all concentrations, has significantly (p < .01)
increased the protection factors and prolonged the IP of oxidation,
compared to control and palm olein containing BHT (PO 1
BHT200 ppm). The activity of the extract was concentration dependent.
Long IP indicates higher resistance to lipid oxidation, good efficiency
and thermal stability of the added antioxidants. Similar results have
been reported by Womeni, Tonfack, Anjaneyulu, et al. (2016) in the
same oil system supplemented with soursop flowers extract at concen-
trations ranging between 200 and 1800 ppm.
3.4 | Effect of extract concentration and storage time
on the oxidative stability of palm olein during storage
at 180 8C
Experimental design was carried out to see the effects of extract
concentration and storage time on the oxidative stability of palm olein
| 7 of 11
by measuring the following parameters: iodine, TBA, and p-ansidine
values. These independent variables were taken into consideration
because previous results (Rancimat test) and several authors (Maleki
et al., 2016; Womeni, Tonfack, Anjaneyulu, et al., 2016; Womeni,
Tonfack, Iruku, et al., 2016) have demonstrated that the oxidative sta-
bility of oils at high temperatures strongly depends on the additive
added, its concentration, and the storage time. The iodine, TBA, and
anisidine values of oil samples obtained from the 10 experiments are
presented in Table 4. The initial physicochemical characteristics of
fresh RBD palm olein used in this study are given in experiment no 10.
The fresh RBD palm olein was of good quality, as shown by its low
anisidine value (<20), low TBA value (1 ppm) and its high iodine value
(56 g I2/100 g).
The coefficient of determination (R2), the coefficients of the
second-order polynomial equation and significant effect of independ-
ent variables presented in Table 8 were calculated using the experi-
mental data. The R2 statistics of the responses are within the range of
a good set (more than 0.75) (Joglekar & May, 1987), indicating that the
model is able to explain 81.9, 85.26, and 99.26% of the variability in
the iodine value, TBA value and anisidine value respectively. Giovanny
(1983) also reported that R2 > 0.75 is considered statistically accurate
for predicting changes in oil quality. From the significant tests given in
Table 8, storage time was found to be the most important factor influ-
encing (p < .05) all the chemical characteristics evaluated. Extract con-
centration in linear term and storage time in the second-order term had
significant (p < .05) effect on p-anisidine value. The mathematical mod-
els of relationship for iodine value (Y3), TBA value (Y4), and anisidine
value (Y5) with independent variables can be used to predict chemical
properties of palm olein during treatment at high temperature
(thermoxidation):
Y3 5 62:00810:001X1 20:099X2 12:47E27X12 20:0001X1 X2 20:04X22
Y4 5 0:8220:0001X1 10:06X2 16:48E28X12 20:00001X1 X2 10:002X22
Y5 5 2201:3910:26X1 1126:80X2 20:0001X12 20:008X1 X2 25:37X22
T AB LE 7 Induction times and protection factors of oil samples dur-
ing storage at 110 8C
Echantillon Induction time (hr) Protection factor
Control 23.19 6 0.12a 1.000 6 0.00a
PO 1 BHT 24.41 6 0.23b 1.052 6 0.007b
PO 1 Thca2,000 ppm 27.85 6 0.45e 1.200 6 0.008f
PO 1 Thca1,780 ppm 26.57 6 0.14d 1.145 6 0.004e
PO 1 Thca1,250 ppm 26.6 6 0.25d 1.147 6 0.0075e
PO 1 Thca720 ppm 25.56 6 0.46c 1.102 6 0.0045c
PO 1 Thca500 25.91 6 0.27cd 1.117 6 0.0026d
ppm
Values of columns with different letters differ significantly p < .05.
Control: palm olein without antioxidant; PO 1 BHT200 ppm: palm olein
containing BHT as antioxidant at concentration of 200 ppm;
PO 1 Thca200 ppm: palm olein supplemented with Theobroma cacao pods
extract at concentration 200 ppm.
8 of 11 | BOUNGO TEBOUKEU ET AL.

T AB LE 8 Regression coefficients (RC), p values, and coefficient of multiple determinations (R2) for iodine value, thiobarbituric acid (TBA)
value, and p-anisidine value following CCD

Iodine value TBA value p-Anisidine value

CR p value CR p value CR p value

X1: extract (ppm) 0.0017 0.1888 20.0001 0.3105 0.2603 0.0363*

X2: storage time (days) 20.0990 0.0176* 0.062 0.0098* 126.804 0.0000*

X1 X1 22.47E–7 0.9265 6.48E–8 0.7829 20.0001 0.0530

X1 X2 20.0001 0.7686 20.00001 0.7577 0.0081 0.3339

X2 X2 0.0407 0.6312 0.002 0.7101 25.3723 0.0121*

Constant 62.008 0.8224 2201.392

2
R (%) 0.819 0.852 0.992
2
R (adjusted) (%) 0.593 0.668 0.983

*Independent variable that significantly (p < .05) affects the response.

FIGURE 4 Contour plots showing the effect of storage time and extract concentration on the iodine value (a), thiobarbituric acid value (b),
and p-anisidine value (c) of palm olein during thermoxidation
BOUNGO TEBOUKEU ET AL.
Figure 4 is a contour plots showing the effect of the storage time
and extract concentration on the iodine value (a), TBA value (b), and
p-anisidine value (c) during storage at 180 8C of sample oils containing
cocoa pods extract at different concentrations.
Iodine value is a measure of the total number of unsaturated
double bonds present in oils (Jaswir, Yaakob, Che Man, & Kitts, 2000).
It is observed in Figure 4a that the iodine value decreases gradually
during storage. Nevertheless, for high concentrations of extracts, this
response decreases slightly. This decrement is generally attributed to
the destruction of fatty acid double bonds caused by oxidation process
(Tynek, Hazuka, Pawlowicz, & Dudek, 2001). The addition of the
extract at high concentration (>800 ppm) has significantly reduced the
alteration of fatty acids double bonds in palm olein and the protective
effect of the extract was observed. Similar results showing the capacity
of extract at high concentration to limit the alteration of fatty acids
double bonds have been reported by Jaswir et al. (2000) about
rosemary extract in palm olein during frying experiment. Codex
Alimentarius (2001) recommends an iodine value higher than or equal
to 56 g I2/100 g for good quality palm olein. Thus, it would be advisa-
ble to heat at 180 8C, palm olein enriched with cocoa pods extracts at
2,000 ppm for 1–7 days in order to preserve this quality.
The measurement of TBA value and anisidine value are intensively
used to assess secondary oxidation products like 2-alkenal,2,4-
alkadienal (anisidine value) and malonaldehyde (TBA value) (Anwar,
Qayum, Hussain, & Iqbal, 2010). Response surfaces (Figure 4b and c)
show that these responses increase with the storage time of the oil at
180 8C. However, the addition of the extract at high concentrations
(>1,600 ppm) in the case of TBA value slows down this oxidation.
Cocoa pods extract contains antioxidants who can donate their hydro-
gen atom to stabilize the free radicals present in oils and consequently
decrease the formation of peroxides (Gordon, 1990). By inhibiting pri-
mary oxidation products formation, cocoa pods extract reduce in the
same way the amount of secondary oxidation products in oil. In fact,
hydroperoxides are thermolabile molecules, which easily breakdown
into secondary oxidation products at high temperature. These results
are in agreement with the studies of Womeni, Tonfack, Iruku, et al.
(2016), which found that soursop extract delay the formation of malo-
naldehyde in oils. On the basis of the quality standards for oils with
respect to TBA value (1 ppm according to Patton & Kurtz, 1951), it
would be advisable to heat at 180 8C, palm olein enriched with cocoa
pods extracts at 2,000 ppm for 1–8 days in order to preserve its qual-
ity. The results concerning anisidine value show an increase of this
response beyond the standard (<20 according to Codex Alimentarius,
2015) at the first day of treatment.
4 | CONCLUSION
The RSM was successfully employed to optimize the extraction
conditions of cocoa (T. cacao) pods extract. The p-value indicated
that methanol concentration was the most significant factor affect-
ing total polyphenol content and antioxidant activity of the extract.
This extract under optimum conditions has a good thermal stability,
| 9 of 11
and the effect of it concentration and storage time during storage
of palm olein at 180 8C has revealed that cocoa pods have the
capacity to delay lipids oxidation and stabilize palm olein. Neverthe-
less, oxidative stability of palm olein during treatment at high tem-
perature depends significantly on the storage time. This study can
be useful for the development of industrial extraction process of
cocoa pods and its application as an ingredient to delay lipid oxida-
tion in oils.
AKNOWLEDG ME NT
The authors would like to thank the Council for Scientific and Indus-
trial Research-Indian Institute of Chemical Technology (CSIR-IICT),
India, where parts of this work were carried-out.
ORC ID
Fabrice Tonfack Djikeng http://orcid.org/0000-0003-4813-5759
RE FE RE NC ES
American Oil Chemists’ Society (AOCS). (1999). Official methods and
recommended practices of the American Oil Chemists’ Society.
Campaign: American Oil Chemists’ Society Press.
Anwar, F., Qayum, H. M. A., Hussain, A. L., & Iqbal, S. (2010). Antioxidant
activity of 100% and 80% methanol extracts from barley seeds
(Hordeum vulgare L.): Stabilization of sunflwer oil. Grasas Aceites, 61,
237–243. https://doi.org/10.3989/gya.087409
Balasundram, N., Sundram, K., & Samman, S. (2006). Phenolic compounds
in plants and agri-industrial by-products: Antioxidant activity, occur-
rence, and potential uses. Food Chemistry, 99, 191–203. https://
doi.org/10.1016/j.foodchem.2005.07.042
Chirinos, R., Rogez, H., Campos, D., Pedreschi, R., & Larondelle, Y.
(2007). Optimisation of extraction conditions of antioxidant phenolic
compounds from mashua (Tropaeolum tuberosum) tubers. Separation
and Purification Technology, 2(55), 217–225. https://doi.org/10.1016/
j.seppur.2006.12.005
Codex Alimentarius. (2001). Report of the seventeenth session of the Codex
Committee on fats and oils. London, UK.
Codex Alimentarius. (2015). Programme mixte FAO/OMS sur les normes
alimentaires. Commission du Codex Alimentarius. Rapport de la vingt-
quatrième session du comite du CODEX sur les graisses et huiles.
Melaka, Malaisie.
 n, L., Alvarenga, E., & Keiski,
Cruz, G., Pirilä, M., Huuhtanen, M., Carrio
R. L. (2012). Production of Activated Carbon from Cocoa (Theobroma
cacao) Pod Husk. Journal of Civil and Environmental Engineering, 2(2),
1–6. https://doi.org/10.4172/2165-784X.1000109
Draper, H. H., & Hadley, M. (1990). Malondialdehyde determination as
index of lipid peroxidation. Methods in Enzymology, 186, 421–431.
https://doi.org/10.1016/0076-6879(90)86135-I
Duh, P. D., & Yen, G. C. (1997). Antioxidant efficacy of methanolic
extracts of peanut hulls in soybean and peanut oils. Journal of the
American Oil Chemists’ Society, 74, 745–748. https://doi.org/
10.1007/s11746-997-0212-z
Fapohunda, S., & Afolayan, A. (2012). Fermentation of cocoa beans and
antimicrobial potentials of the pod husk phytochemicals. Journal of
Physiology and Pharmacology Advances, 2(3), 158–164. CiteWeb id:
20120814700.
€ rk, L., & Trajkovski, V. (2000).
Gao, X., Ohlander, M., Jeppsson, N., Bjo
Changes in antioxydant effects and their relationship to
10 of 11 | BOUNGO TEBOUKEU
phytonutrients in fruits of sea buckthorn (Hippophae rhamnoides L.)
during maturation. Journal of Agricultural and Food Chemistry, 48,
1485–1490. https://doi.org/10.1021/jf991072g
Ghumman, A., Singh, N., & Kaur, A. (2017). Chemical, nutritional and pheno-
lic composition of wheatgrass and pulse shoots. International Journal of
Food Science and Technology, 1–10. https://doi.org/10.1111/ijfs.13498
Giovanny, M. (1983). Response surface methodology and product optimi-
zation. Food Technology, 38, 41–45.
Gordon, M. H. (1990). The mechanisms of antioxidant action in vitro. In
B. J. F. Hudson (Ed.), Food antioxidants (pp. 1–18). London: Elsevier
Applied Science.
Guyot, S., Marnet, N., & Drilleau, J. (2001). Thiolysis-HPLC characteriza-
tion of apple procyanidins covering a large range of polymerization
states. Journal of Agricultural and Food Chemistry, 49, 14–20.
Iqbal, S., Haleem, S., Akhtar, M., Zia-Ul-Haq, M., & Akbar, J. (2008).
Efficiency of pomegranate peel extracts in stabilization if sunflower
oil under accelerated conditions. Food Research International, 45,
194–200. https://doi.org/10.1016/j.foodres.2007.11.005
Jalil, A. M. M., & Ismail, A. (2008). Polyphenols in cocoa and cocoa
products: Is there a link between antioxidant properties and health?
Molecules, 13, 2190–2219. https://doi.org/10.3390/molecules
13092190
Jaswir, I., Che Man, Y. B., & Kitts, D. D. (2000). Optimization of physico-
chemical changes of palm olein with phytochemical antioxidants
during deep-fat frying. Journal of the American Oil Chemists’ Society,
77, 1161–1168. https://doi.org/10.1007/s11746-000-0182-6
Joglekar, A. M., & May, A. I. (1987). Product excellence through design
of experiments. Cereal Food World, 32, 857–868.
Jorge, A. J., Heliodoro De La Garza, T., Alejandro, Z. C., Ruth, B. C., &
Noe, A. C. (2013). The optimization of phenolic compounds extrac-
tion from cactus pear (Opuntia ficus-indica) skin in a reflux system
using response surface methodology. Asian Pacific Journal of Tropical
Biomedicine, 3(6), 436–442. https://doi.org/10.1016/S2221-1691(13)
60093-3
Karen, C. C. (2011). Polyphenol antioxydants from potato peels: Extrac-
tion optimization and application to stabilizing lipid oxidation in
foods. In Proceedings of the national conferences on undergraduate
research, Ithaca College, New York, pp. 1995–2000.
Karim, A. A., Azlan, A., Ismail, A., Hashim, P., & Abdullah, N. A. (2014).
Antioxidant properties of cocoa pods and shells. Malaysian Cocoa
Journal, 8, 49–56.
Karim, A. A., Azlan, A., Ismail, A., Hashim, P., Abd Gani, S. S., Zainudin,
B. H., & Abdullah, N. A. (2014). Phenolic composition, antioxidant,
anti-wrinkles and tyrosinase inhibitory activities of cocoa pods
extract. BMC Complementary and Alternative Medicine, 14, 381–394.
https://doi.org/10.1186/1472-6882-14-381
Karp, S., Wyrwisz, J., Kurek, M. A., & Wierzbicka, A. (2017). Combined
use of cocoa dietary fibre and steviol glycosides in low-calorie muf-
fins production. International Journal of Food Science & Technology,
52, 944–953. https://doi.org/10.1111/ijfs.13358
Luthria, D. L., Mukhopadhyay, S., & Kwansa, A. L. (2006). A systematic
approach for extraction of phenolic compounds using parsley (Petro-
selinum crispum) flakes as a model substrate. Journal of the Science of
Food and Agriculture, 86, 1350–1358. https://doi.org/10.1002/
jsfa.2521
Maleki, M., Ariaii, P., & Fallah, H. (2016). Effects of celery extracts on
the oxidative stability of canola oil under thermal condition. Journal
of Food Processing and Preservation, 40, 531–540. https://doi.org/
10.1111/jfpp.12632
ET AL.
, C., Souquet, J. M., Olle
Mane , D., Verrie
s, C., Ve
ran, F., & Mazerolles, G.
(2007). Optimization of simultaneous flavanol, phenolic acid, and
anthocyanin extraction from grapes using an experimental design:
Application to the characterization of champagne grape varieties.
Journal of Agricultural and Food Chemistry, 55, 7224–7233. https://
doi.org/10.1021/jf071301w
Maqsood, S., & Benjakul, S. (2010). Comparative studies of four different
phenolic compounds on in vitro antioxidative activity and the preven-
tive effect on lipid oxidation of fish oil emulsion and fish mince. Food
Chemistry, 119, 123–132. https://doi.org/10.1016/j.foodchem.2009.
06.004
Mc Clements, D. J., & Decker, E. A. (2000). Lipid oxidation in oil-in-water
emulsions: Impact of molecular environment on chemical reactions in
heterogeneous food systems. Journal of Food Science, 65, 1270–1282.
https://doi.org/10.1111/j.1365-2621.2000.tb10596.x
Mellor, D. D., & Naumovski, N. (2016). Effect of cocoa in diabetes: The
potential of the pancreas and liver as key target organs, more than
an antioxidant effect? International Journal of Food Science & Technol-
ogy, 51, 829–841. https://doi.org/10.1111/ijfs.13075
Mensor, L. L., Menezez, F. S., Leitao, G. G., Reis, A. S., Dos Santos, T. C.,
Coube, C. S., & Leitao, S. G. (2001). Screening of Brazilian plant
extracts for antioxidant activity by the use of DPPH free radical
method. Phytotherapy Research, 15, 127–130. https://doi.org/
10.1002/ptr.687
Moahamad, M., Ali, M. W., & Ahmad, A. (2010). Modelling for extraction
of major phytochemical components from Eurycoma longifolia. Journal
of Applied Sciences, 21, 2572–2577. https://doi.org/10.3923/
jas.2010.2572.2577
Olivas-Aguirre, F. J., Gonz alez-Aguilar, G. A., Velderrain-Rodríguez,
G. R., Torres-Moreno, H., Robles-Zepeda, R. E., . . . Wall-Medrano,
A. (2017). Radical scavenging and anti-proliferative capacity of
three freeze-dried tropical fruits. International Journal of Food Sci-
ence and Technology, 52, 1699–1709. https://doi.org/10.1111/ijfs.
13408
Patton, S., & Kurtz, G. W. (1951). 2-Thiobarbituric acid as a reagent for
detecting milk fat oxidation. Journal of Dairy Science, 34, 69–74.
Pinho, S. P., & Macedo, E. A. (2005). Solubility of NaCl, NaBr, and KCl in
water, methanol, ethanol, and their mixed solvents. Journal of
Chemical & Engineering Data, 50, 29–32. https://doi.org/10.1021/
je049922y
Prior, R. L., Lazarus, S. A., Cao, G., Muccitelli, H., & Hammerstone, J. F.
(2001). Identification of procyanidins and anthocyanins in blueberries
and cranberries (Vaccinium spp.) using highperformance liquid chro-
matography/mass spectrometry. Journal of Agricultural and Food
Chemistry, 49, 1270–1276. https://doi.org/10.1021/jf001211q
Ramalho, V.C., & Jorge, N. (2008). Antioxidant action of rosemary extract
in soybean oil submitted to thermoxidation. Grasas Aceites, 59(2),
128–131. https://doi.org/10.3989/gya.2008.v59.i2.500
Tomsone, L., Kruma, Z., & Galoburda, R. (2012). Comparison of different
solvents and extraction methods for isolation of phenolic compounds
from horseradish roots. World Academy of Science, Engineering and
Technology, 64, 903–908.
Tynek, M., Hazuka, Z., Pawlowicz, R., & Dudek, M. (2001). Changes in
the frying medium during deep-frying of food rich in proteins and
carbohydrates. Journal of Food Lipids, 8, 251–261. https://doi.org/
10.1111/j.1745-4522.2001.tb00200.x
Vriesmann, L. C., Amboni, R. D. M. C., & Petkowicz, C. L. O. (2011).
Cacao pod husks (Theobroma cacao L.): Composition and hot-water-
soluble pectins. Industrial Crops and Products, 34, 1173–1181.
https://doi.org/10.1016/j.indcrop.2011.04.004
BOUNGO TEBOUKEU ET AL. | 11 of 11

Womeni, H. M., Tonfack, D. F., Anjaneyulu, B., Karuna, M. S. L., Prasad,

R. B. N., & Linder, M. (2016). Oxidative stabilization of RBD palm
olein under forced storage conditions by old Cameroonian green tea
leaves methanolic extract. NFS Journal, 3, 33–40. https://doi.org/
10.1016/j.nfs.2016.03.002
Womeni, H. M., Tonfack, D. F., Iruku, N. S. S. P., Karuna, M. S. L., Prasad,
R. N. B., & Linder, M. (2016). Valorization of soursop flowers (Annona
muricata L.) as potent source of natural antioxidants for stabilization
of palm olein during accelerated storage. Food Science & Nutrition, 3,
33–40. https://doi.org/10.1002/fsn3.349
How to cite this article: Boungo Teboukeu G, Tonfack Djikeng
F, Klang MJ, Houketchang Ndomou S, Karuna MSL, Womeni
HM. Polyphenol antioxidants from cocoa pods: Extraction opti-
mization, effect of the optimized extract, and storage time on
the stability of palm olein during thermoxidation. J Food Process
Preserv. 2018;e13592. https://doi.org/10.1111/jfpp.13592