Journal of Food Processing and Preservation ISSN 1745-4549

TOTAL PHENOLICS AND ANTIOXIDANT CAPACITY

OF COCOA PULP: PROCESSING AND STORAGE STUDY†
VIDYA ENDRAIYANI1, RICHARD D. LUDESCHER1, RONG DI2 and MUKUND V. KARWE1,3
1
Department of Food Science, Rutgers University, New Brunswick, NJ
2
Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ

3
Corresponding author.
TEL: 11-848-932-5487;
FAX: 11-732-932-6776;
EMAIL: karwe@aesop.rutgers.edu
†
Vidya Endraiyani conducted the
experiments. R.D. Ludescher supervised
ORAC experiments. R. Di provided
laboratory facility and supervised CAA
experiments. M.V. Karwe defined the goals
of the project and supervised the execution.
Received for Publication December 15, 2015
Accepted for Publication March 14, 2016
doi:10.1111/jfpp.13029
ABSTRACT
Oxygen radical absorption capacity (ORAC) and cellular antioxidant activity
(CAA) of cocoa pulp, as affected by single and double pasteurization treatments
(both at 85C for 60 s), were studied to understand its potential health benefits.
Single and double pasteurization decreased total phenolic content by 25 and 38%,
respectively. ORAC values were not significantly different between unpasteurized,
single and double pasteurized pulp samples. However, significant increase in CAA
values of double pasteurized pulp was observed in comparison to unpasteurized
pulp. Stability of total phenolics and ORAC were monitored for 8 weeks at 4, 25
and 37C. Overall, single and double pasteurized pulps showed minimal changes
in total phenolic content and ORAC values during storage at 4 and 25C. However,
at 37C, single pasteurization resulted in 50% relative loss in total phenolics and
40% reduction in ORAC values, whereas double pasteurization resulted in less
total phenolic loss and ORAC value reduction.

PRACTICAL APPLICATIONS
Effect of thermal processing on potential health benefits of cocoa pulp was dem-
onstrated. Partial removal of cocoa pulp before fermentation and utilizing it for
making various products after pasteurization will increase marketability of cocoa
pulp as a usable by-product of the cocoa industry.

INTRODUCTION its favorable macro- and micronutrients composition, cocoa

Cocoa pulp is a sweet white mucilaginous layer around indi-
vidual seeds of Theobroma cacao fruit. It plays an important
role in the fermentation process of cocoa beans, the main
source of cocoa for chocolate and its derivatives. During fer-
mentation, microorganisms act upon the pulp and bring
about external physicochemical changes that initiate and
govern biochemical reactions between enzymes and sub-
strates within the beans. The products of these reactions are
distinct flavor and aroma precursors, which are fixed in the
subsequent drying and roasting steps (Lopez and Dimick
1991). Those characteristics ultimately determine the com-
mercial value of cocoa beans.
Cocoa pulp mainly consists of 82–87% water, 10–15%
sugars, 1–3% citric acids and 1–1.5% pectin depending on
the cultivar type, degree of ripeness as well as growing
regions (Roelofsen 1958). In contrast, dried cocoa bean is
composed of 54% fats, 31% carbohydrates, 11% proteins,
3% polyphenols and <1% minerals (Beckett 2008). Despite
pulp is a post-harvest residue of cocoa bean processing. By
the end of fermentation, it drains away as sweating, and is
discarded.
There are increasing efforts that focus on extracting and
utilizing cocoa pulp before fermentation for value-added
products, such as juices, jams, jellies and alcoholic beverages
(Dias et al. 2007). Furthermore, studies by Lopez (1979) and
Biehl et al. (1989) have shown that partial removal of pulp
before fermentation has resulted in better yield and quality
without disturbing successful fermentation and it did not
affect the flavor development of cocoa beans. Nevertheless,
little research has been done to understand the phytochemi-
cal content and potential health benefits of cocoa pulp to
accommodate its diverse utilization, unlike cocoa beans
(Adamson et al. 1999; Noe et al. 2004; Keen et al. 2005). The
objectives of this study were to quantify total phenolics, oxy-
gen radical absorption capacity (ORAC) and cellular antiox-
idant activity (CAA) in cocoa pulp, as indicators of dietary

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COCOA PULP PHENOLICS AND ANTIOXIDANT CAPACITY
antioxidant potential of cocoa pulp. The effect of pasteuriza-
tion and storage study on the stability of total phenolics and
ORAC and CAA values were also investigated.
MATERIALS AND METHODS
Samples
Cocoa pulp, unpasteurized (raw) and single pasteurized
(85C for 60 s) was obtained from Ecuador through iTi Trop-
icals, Inc. (Lawrenceville, NJ). The pulp was kept frozen
(220C) during transportation and storage until processing.
The pulp was kept frozen for less than a month.
Double Pasteurization
To simulate the practice followed in food industry, the pas-
teurized cocoa pulp was subjected to secondary thermal
processing. Single pasteurized cocoa pulp was portioned
and vacuum packed into pouches (10 cm 3 12 cm), each
weighing 30 g. Pouches were placed in between dividers in a
metal wire basket, which was then immersed in hot water
(85C) placed in a steam-jacketed kettle. The basket was
rotated continuously during thermal processing to allow
good heat distribution and minimize hot or cold pockets. A
pouch located at the geometric center of the basket had a
flexible thermocouple (C-4, Ecklund Technologies) inserted
into it. The thermocouple was connected to a data acquisi-
tion system consisting of a high speed USB carrier NI USB-
9162 (National Instruments, Austin, TX) connected to a lap-
top computer. Labview 7 (National Instruments, Austin,
TX) software was used to record time-temperature data and
display calculated integrated lethality (F value). Based on
the pH of cocoa pulp (pH 3.80), the reference temperature
used for this thermal processing was 93.33C and the z value
was 8.8C (Weddig 2007). Hence, with the heating media of
85C, the thermal process was stopped when the calculated F
value at 93.33C reached 6 s, which is equivalent to holding
the pouch at 85C for about 60 s. The cooling process was
initiated by rapidly discharging the hot water from the kettle
and pouring a mixture of cold water and ice into the kettle
at the same time. Basket was removed from the kettle when
the temperature reached 22C within 60 s.
Extraction
Extracts were obtained from the processed and unprocessed
cocoa pulp samples using an extracting solvent mixture of
acetone/water/acetic acid (70:29.5:0.5, v/v), as described by
Prior et al. (2003). Briefly, in triplicate, cocoa pulp was sub-
jected to a two-step extraction procedure using 1:3 (w/v)
and 1:1.5 (w/v) pulp to solvent ratios, respectively. In both
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V. ENDRAIYANI ET AL.
extraction steps, samples were stirred continuously at room
temperature (22C) for 10 min. Homogenates were then cen-
trifuged at 5,000 3 g for 10 min. The supernatants obtained
from the two extraction steps were combined and analyzed
for total phenolic content, and chemical and cellular antiox-
idant capacity measurements.
Determination of Total Phenolic Content
Total phenolic contents were determined using Folin–Ciocal-
teu method with gallic acid as standard as described by Dew-
anto et al. (2002), using a Cary 50 UV-vis spectrophotometer
(Varian, Palo Alto, CA). Due to the presence of other interfer-
ing compounds, such as sugars and ascorbic acid, which could
overestimate the measurement of the total phenolic content,
correction was done by binding extracts to polyvinylpolypyr-
rolidone (PVPP) (Makkar et al. 1993). PVPP is an adsorbent
and precipitant of phenolic compounds, thereby leaving inter-
fering compounds in the unadsorbed or unprecipitated por-
tions. Thus, the extracts post-PVPP binding, account for the
absorbance of the interfering compounds. Pooled extracts
were subjected to 4% (w/v) PVPP binding for 1 h at 22C and
centrifugation for 10 min. The corrected total phenolic values
were obtained by subtracting total phenolic values post-PVPP
binding from the initial total phenolic values (pre-PVPP bind-
ing). Total phenolic contents were expressed as mg gallic acid
equivalents (GAE) per 100 g of pulp.
Determination of Chemical
Antioxidant Activity
Chemical antioxidant capacity measurements for unpas-
teurized and pasteurized pulps were done using a modified
ORAC assay as described by Ou et al. (2001). The fluores-
cence was monitored kinetically using a Cary Eclipse fluo-
rescence spectrophotometer (Varian, Palo Alto, CA) every
30 s with excitation wavelength of 485 nm, excitation slit
width of 20 nm, emission wavelength of 540 nm and emis-
sion slit width of 20 nm. The reaction was stopped when the
fluorescence intensity was below 2% of the initial fluores-
cence signal. The ORAC values were then obtained by inter-
polating the net area under the curve (AUC) for extracts,
against a standard curve of net AUC at different Trolox con-
centrations. Trapezoidal method was used to determine
AUC. The ORAC values were calculated as lmol Trolox
equivalent/100 g of pulp (lmol TE/g).
Determination of Cellular
Antioxidant Activity
The cellular antioxidant activity assays were performed as
described by Wolfe and Liu (2007). In this method, querce-
tin was selected as the standard, 20 ,70 -dichlorofluorescin
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V. ENDRAIYANI ET AL. COCOA PULP PHENOLICS AND ANTIOXIDANT CAPACITY

diacetate (DCFH-DA) was used as a fluorescent probe to

indicate oxidation, and 2,20 -azobis (2-amidinopropane)
dihydrochloride (ABAP) was used as the oxidant source.
Pulp extracts (described earlier) were evaporated under vac-
uum at 50C. The residues were then reconstituted in
dimethyl sulfoxide (DMSO) and stored at 240C. Further
dilution of pure phytochemicals and reconstituted pulp
extract was done in the treatment medium Williams’
medium E (WME) supplemented with 2 mM L-glutamine
and 10 mM Hepes). HepG2 cells used in the study were
between passages 12 and 24.
Cytotoxicity of cocoa pulp extracts was measured by Cell-
Titer 96 Aqueous One Solution Cell Proliferation Assay
(Promega, Madison, WI), which is composed of solutions
of tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5- FIG. 1. TOTAL PHENOLICS CONTENT OF UNPASTEURIZED AND
PASTEURIZED COCOA PULP (MEAN 6 SE, n 5 3)
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazo-
Different letters indicate significant differences.
lium, inner salt; MTS] and an electron coupling reagent
phenazine methosulfate (PMS). MTS is bioreduced into a
cocoa pulp during storage. These temperature settings were
formazan product, which is soluble in tissue culture
selected based on common industry practice. The cocoa
medium, due to the dehydrogenase activity produced by
pulp was stored in sealed moisture barrier MRE pouches;
metabolically active cells. The quantity of formazan formed
as measured through its absorbance at 490 nm is directly hence, external relative humidity did not affect the storage
proportional to the number of living cells in culture. Briefly, study. Triplicate analyses were done bi-weekly and samples
HepG2 cells were seeded at 6 3 104/well on 96-well plates in were monitored for up to 8 weeks.
100 lL of growth medium and incubated for 24 h at 37C.
The growth medium was removed and the cells were washed Statistical Analyses
with phosphate-buffered saline (PBS). Treatment medium
(100 lL, WME with 2 mM L-glutamine and 10 mM Hepes) All results are presented as mean 6 SE. Statistical analyses
R

containing pure phytochemicals or pulp extracts were then
applied to the cells followed by incubation at 37C and 5%
CO2 for 1 h. After incubation, 20 lL of CellTiter 96 Aqueous
One Solution Reagent was added into each well of the 96-
well assay plate without removing the treatment medium.
The plate was incubated for 90 min at 37C and 5% CO2, and
measured for its absorbance at 490 nm. Concentrations of
pure phytochemicals or pulp extracts that decreased the
absorbance by 10% compared to the control wells (contains
only treatment medium) were considered to be cytotoxic.
SynergyTMHT Multi-Detection Microplate Reader
(BioTek Instruments, Inc., Winooski, VT) was used for
kinetic fluorescence measurements. The emission filter used
was 485 nm with 20 nm bandpass, and excitation filter of
528 nm with 20 nm bandpass. The plate reader was con-
trolled by KC4TM version 3.4 (revision 10) with a sensitivity
setting of 50. EC50 values were converted to CAA unit of
lmol of quercetin equivalents (QE)/100 g pulp, derived by
normalizing the EC50 of the pulp samples to the EC50 of
quercetin as a reference.
Storage Study
Three temperature values (4, 25 and 37C) were selected to
study the stability of total phenolics and ORAC values of
were performed using MATLABV 7.9.0.529 (R2009b) (The
MathWorks, Inc., Natick, MA). Differences between means
were detected by ANOVA, which is followed by multiple
comparisons using Turkey significant difference test. Results
were considered to be significant when the P value
was < 0.05.
RESULTS AND DISCUSSION
Effect of Pasteurization on Total Phenolic
Content, ORAC and CAA
The average total phenolic content of unprocessed and pas-
teurized cocoa pulp is shown in Fig. 1. All values are mean-
6 SE. The average total phenolic content of unprocessed
pulp was 103.76 6 4.79 mg GAE/100 g pulp. This level of
phenolic content was significantly lower than cocoa powder
with reported value of 5,240 mg GAE/100 g by Miller et al.
(2006), though comparatively it was similar to the value for
green grape with lower total phenolic content of 145 mg
GAE/100 g (Wu et al. 2004). Single and double pasteurized
pulps contained total phenolics of 77.88 6 0.65 and
64.21 6 0.32 mg GAE/100 g pulp, respectively. In other
words, there were correspondingly 25 and 38% reduction of
phenolics (P < 0.05) in comparison to unpasteurized pulp.

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enhanced their bioavailability (Williamson 2003). Nonethe-

less, further investigations are needed to support the above
propositions.

Stability Study of Single and Double

Pasteurized Cocoa Pulp
Changes in total phenolic contents during a storage study of
8 weeks for single pasteurized pulp are shown in Fig. 3a. The
influence of storage temperature and storage time as well as
their interaction had significant influence (P < 0.05) on the
total phenolics of cocoa pulp. The degradation rate of phe-
FIG. 2. ORAC AND CAA VALUES OF UNPASTEURIZED AND nolics was significantly lower (P < 0.05) when single pas-
PASTEURIZED PULP (MEAN 6 SE, n 5 9)
teurized pulp was stored at 4C, followed by 25 and 37C. By
Different letters indicate significant differences within the same group
of antioxidant measurement.
the end of 8 weeks, there was a decrease of 14, 26 and 50% in
total phenolic content at storage temperatures of 4, 25 and
This result is consistent with previously reported studies 37C, respectively. Furthermore, when comparing the total
with peaches, which showed that thermal treatment signifi- phenolic content values that were taken at the same storage
cantly decreased total phenolics in clingstone peaches period but stored at different temperature, total phenolics
(Asami 2003). Degradation of phenolic compounds may be contents of single pasteurized pulp stored at 37C were con-
caused by the adverse effects of heat, other chemicals present sistently and significantly (P < 0.05) the lowest. These
in the pulp matrix, light, or oxygen, which might lead to
oxidation and subsequent polymerization as reported by
Nicoli et al. (1999).
Cocoa pulp, unpasteurized and pasteurized, contained
measurable amount of chemical antioxidant activity as
shown in Fig. 2. The antioxidant capacity of unpasteurized
pulp was 1871 6 58 lmol TE/100 g pulp, a comparable value
to the ORAC value of green grape of 1,118 6 96 lmol TE/
100 g raw fruit (Wu et al. 2004) and it was significantly lower
than 80,933 lmol TE/100 g raw fruit for cocoa beans
reported in literature (Miller et al. 2006). Furthermore, the
antioxidant capacities of single and double pasteurized
pulps were not significantly different from unpasteurized
pulp (P > 0.05). The ORAC values of single and double pas-
teurized pulps were 1,835 6 41 lmol TE/100 g pulp and
1,681 6 157 lmol TE/100 g pulp, respectively.
The results from the CAA assay are also shown in Fig. 2.
Double pasteurized pulp was found to be the most effective
at inhibiting peroxyl radical induced DCFH oxidation in the
CAA assay. Single pasteurized and unpasteurized pulp had
median effective dose (EC50) of 34.13 6 5.31 lmol of QE/
100 g and 23.71 6 3.12 lmol of QE/100 g, respectively. In
contrast, double pasteurized pulp showed an EC50 value of
59.76 6 9.26 lmol of QE/100 g, which suggested that double
pasteurization increased CAA values. This observation may
be due to the higher response by smaller, more soluble phe-
nolic compounds that were released from insoluble matrices
and formed from the breakdown of larger hydrolyzable pol-
yphenols (Bravo 1998). Furthermore, cocoa pulp may con-
tain phenolics in the stable glycoside forms which could be FIG. 3. CHANGES OF TOTAL PHENOLICS (TP) IN (a) SINGLE
deglycosylated by heat into aglycones which might have PASTEURIZED PULP, (b) DOUBLE PASTEURIZED PULP, AT 4, 25 and 37C

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V. ENDRAIYANI ET AL.
FIG. 4. CHANGES OF ORAC VALUES IN (a) SINGLE PASTEURIZED PULP,
(b) DOUBLE PASTEURIZED PULP at 4, 25 and 37C
observations are consistent with the stability study of haw-
thorn fruit and drink stored at 4C in comparison to 23 and
40C as reported by Chang et al. (2006). Previous studies
suggested that some possible degradation pathways of the
phenolics involve oxidation (De Gaulejac et al. 2001),
hydrolysis (Yamada et al. 1997) or isomerization (Wang and
Helliwell 2000). Furthermore, certain enzymes may still be
active in the fruits and contribute to the degradation of the
active components (Tomas-Barberan and Espın 2001).
Antioxidant capacity of single pasteurized cocoa pulp
(Fig. 4a) was most preserved when stored at 4C. Addition-
ally, the stability pattern of ORAC values (Fig. 4a) in single
pasteurized pulp was similar to total phenolics (Fig. 3a).
Hence, the antioxidant activity could be mostly attributed
to the phenolics. The retention of ORAC values in single
pasteurized pulp by the end of 8 weeks stability study were
95, 85 and 60% at 4, 25 and 37C storage conditions,
respectively.
Total phenolic content of double pasteurized pulp is
shown in Fig. 3b. At 4C storage temperature, there was an
increase in the total phenolic content of double pasteurized
pulp, starting at week 2 and remained stable until week 8.
The observed slight increase of total phenolic content, how-
ever, might not be practically relevant. Similarly, total phe-
nolic content of double pasteurized pulp also remained
stable when stored at 25C. In contrast, at 37C storage tem-
perature, total phenolics of double pasteurized pulp
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COCOA PULP PHENOLICS AND ANTIOXIDANT CAPACITY
appeared to be the least stable as shown by a steeper drop in
the phenolic content by the end of the storage study when
compared to those stored at 4 and 25C. The retention of
phenolics was only 70% compared to 99–100% when stored
at 4 and 25C, respectively.
Chemical antioxidant capacity of double pasteurized
pulp during storage is shown in Fig. 4b. The antioxidant
capacity of double pasteurized pulp remained stable and
was very similar between 4 and 25C storage conditions and
lowest at 37C. This result was in accordance with the finding
of Miller et al. (1995), in which the antioxidant activity in
apple juice remained stable for 10 days when stored at 4C
and at room temperature. The retention of ORAC values for
double pasteurized pulp by the end of the shelf life study
were 100, 95 and 77%, respectively, at 4, 25 and 37C.
CONCLUSIONS
Cocoa pulp contains heat-labile phenolic compounds which
are present in a relatively low amount. The amount of those
phenolic compounds was adversely affected by pasteuriza-
tion. Single and double pasteurization significantly
decreased the total phenolic content of cocoa pulp. In con-
trast, the ORAC values of cocoa pulp, as a measure of chem-
ical antioxidant activity of cocoa pulp, were not affected by
pasteurization. Cellular antioxidant activity of both single
and double pasteurized cocoa pulp, however, increased after
single and double pasteurization. During an 8 week storage
study, single and double pasteurized pulps showed very little
changes in phenolics and ORAC values when stored at 4 and
25C. However, at 37C of storage temperature, up to 50% rel-
ative loss in phenolics and 40% relative loss in ORAC values
were observed in single pasteurized pulp, whereas double
pasteurization resulted in lesser relative loss. Further study
needs to be done to assess cocoa pulp and its health benefits
to support the marketability of cocoa pulp as by-products of
the cocoa industry.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Kasi Sundaresan and
Dr. Don Giampetro of iTi Tropicals (Lawrenceville, NJ) for
the financial support, cocoa pulp samples and insights that
made this research possible. Authors also thank Dr. Deepti
Salvi for help in preparing the manuscript and for useful
discussions.
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