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Corresponding author. ABSTRACT
TEL: 11-848-932-5487;
FAX: 11-732-932-6776; Oxygen radical absorption capacity (ORAC) and cellular antioxidant activity
EMAIL: karwe@aesop.rutgers.edu (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.
†
Vidya Endraiyani conducted the Single and double pasteurization decreased total phenolic content by 25 and 38%,
experiments. R.D. Ludescher supervised
respectively. ORAC values were not significantly different between unpasteurized,
ORAC experiments. R. Di provided
single and double pasteurized pulp samples. However, significant increase in CAA
laboratory facility and supervised CAA
experiments. M.V. Karwe defined the goals values of double pasteurized pulp was observed in comparison to unpasteurized
of the project and supervised the execution. 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
Received for Publication December 15, 2015 in total phenolic content and ORAC values during storage at 4 and 25C. However,
Accepted for Publication March 14, 2016
at 37C, single pasteurization resulted in 50% relative loss in total phenolics and
40% reduction in ORAC values, whereas double pasteurization resulted in less
doi:10.1111/jfpp.13029
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.
antioxidant potential of cocoa pulp. The effect of pasteuriza- extraction steps, samples were stirred continuously at room
tion and storage study on the stability of total phenolics and temperature (22C) for 10 min. Homogenates were then cen-
ORAC and CAA values were also investigated. 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-
MATERIALS AND METHODS idant capacity measurements.
Samples
Determination of Total Phenolic Content
Cocoa pulp, unpasteurized (raw) and single pasteurized
(85C for 60 s) was obtained from Ecuador through iTi Trop- Total phenolic contents were determined using Folin–Ciocal-
icals, Inc. (Lawrenceville, NJ). The pulp was kept frozen teu method with gallic acid as standard as described by Dew-
(220C) during transportation and storage until processing. anto et al. (2002), using a Cary 50 UV-vis spectrophotometer
The pulp was kept frozen for less than a month. (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,
Double Pasteurization correction was done by binding extracts to polyvinylpolypyr-
To simulate the practice followed in food industry, the pas- rolidone (PVPP) (Makkar et al. 1993). PVPP is an adsorbent
teurized cocoa pulp was subjected to secondary thermal and precipitant of phenolic compounds, thereby leaving inter-
processing. Single pasteurized cocoa pulp was portioned fering compounds in the unadsorbed or unprecipitated por-
and vacuum packed into pouches (10 cm 3 12 cm), each tions. Thus, the extracts post-PVPP binding, account for the
weighing 30 g. Pouches were placed in between dividers in a absorbance of the interfering compounds. Pooled extracts
metal wire basket, which was then immersed in hot water were subjected to 4% (w/v) PVPP binding for 1 h at 22C and
(85C) placed in a steam-jacketed kettle. The basket was centrifugation for 10 min. The corrected total phenolic values
rotated continuously during thermal processing to allow were obtained by subtracting total phenolic values post-PVPP
good heat distribution and minimize hot or cold pockets. A binding from the initial total phenolic values (pre-PVPP bind-
ing). Total phenolic contents were expressed as mg gallic acid
pouch located at the geometric center of the basket had a
equivalents (GAE) per 100 g of pulp.
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- Determination of Chemical
9162 (National Instruments, Austin, TX) connected to a lap- Antioxidant Activity
top computer. Labview 7 (National Instruments, Austin,
Chemical antioxidant capacity measurements for unpas-
TX) software was used to record time-temperature data and
teurized and pasteurized pulps were done using a modified
display calculated integrated lethality (F value). Based on
ORAC assay as described by Ou et al. (2001). The fluores-
the pH of cocoa pulp (pH 3.80), the reference temperature cence was monitored kinetically using a Cary Eclipse fluo-
used for this thermal processing was 93.33C and the z value rescence spectrophotometer (Varian, Palo Alto, CA) every
was 8.8C (Weddig 2007). Hence, with the heating media of 30 s with excitation wavelength of 485 nm, excitation slit
85C, the thermal process was stopped when the calculated F width of 20 nm, emission wavelength of 540 nm and emis-
value at 93.33C reached 6 s, which is equivalent to holding sion slit width of 20 nm. The reaction was stopped when the
the pouch at 85C for about 60 s. The cooling process was fluorescence intensity was below 2% of the initial fluores-
initiated by rapidly discharging the hot water from the kettle cence signal. The ORAC values were then obtained by inter-
and pouring a mixture of cold water and ice into the kettle polating the net area under the curve (AUC) for extracts,
at the same time. Basket was removed from the kettle when against a standard curve of net AUC at different Trolox con-
the temperature reached 22C within 60 s. centrations. Trapezoidal method was used to determine
AUC. The ORAC values were calculated as lmol Trolox
Extraction equivalent/100 g of pulp (lmol TE/g).
containing pure phytochemicals or pulp extracts were then were performed using MATLABV 7.9.0.529 (R2009b) (The
applied to the cells followed by incubation at 37C and 5% MathWorks, Inc., Natick, MA). Differences between means
CO2 for 1 h. After incubation, 20 lL of CellTiter 96 Aqueous were detected by ANOVA, which is followed by multiple
One Solution Reagent was added into each well of the 96- comparisons using Turkey significant difference test. Results
well assay plate without removing the treatment medium. were considered to be significant when the P value
The plate was incubated for 90 min at 37C and 5% CO2, and was < 0.05.
measured for its absorbance at 490 nm. Concentrations of
pure phytochemicals or pulp extracts that decreased the RESULTS AND DISCUSSION
absorbance by 10% compared to the control wells (contains
only treatment medium) were considered to be cytotoxic. Effect of Pasteurization on Total Phenolic
SynergyTMHT Multi-Detection Microplate Reader Content, ORAC and CAA
(BioTek Instruments, Inc., Winooski, VT) was used for
kinetic fluorescence measurements. The emission filter used The average total phenolic content of unprocessed and pas-
was 485 nm with 20 nm bandpass, and excitation filter of teurized cocoa pulp is shown in Fig. 1. All values are mean-
528 nm with 20 nm bandpass. The plate reader was con- 6 SE. The average total phenolic content of unprocessed
trolled by KC4TM version 3.4 (revision 10) with a sensitivity pulp was 103.76 6 4.79 mg GAE/100 g pulp. This level of
setting of 50. EC50 values were converted to CAA unit of phenolic content was significantly lower than cocoa powder
lmol of quercetin equivalents (QE)/100 g pulp, derived by with reported value of 5,240 mg GAE/100 g by Miller et al.
normalizing the EC50 of the pulp samples to the EC50 of (2006), though comparatively it was similar to the value for
quercetin as a reference. 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
Storage Study
64.21 6 0.32 mg GAE/100 g pulp, respectively. In other
Three temperature values (4, 25 and 37C) were selected to words, there were correspondingly 25 and 38% reduction of
study the stability of total phenolics and ORAC values of phenolics (P < 0.05) in comparison to unpasteurized pulp.
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-
FIG. 4. CHANGES OF ORAC VALUES IN (a) SINGLE PASTEURIZED PULP, trast, the ORAC values of cocoa pulp, as a measure of chem-
(b) DOUBLE PASTEURIZED PULP at 4, 25 and 37C ical antioxidant activity of cocoa pulp, were not affected by
pasteurization. Cellular antioxidant activity of both single
observations are consistent with the stability study of haw- and double pasteurized cocoa pulp, however, increased after
thorn fruit and drink stored at 4C in comparison to 23 and single and double pasteurization. During an 8 week storage
40C as reported by Chang et al. (2006). Previous studies study, single and double pasteurized pulps showed very little
suggested that some possible degradation pathways of the changes in phenolics and ORAC values when stored at 4 and
phenolics involve oxidation (De Gaulejac et al. 2001), 25C. However, at 37C of storage temperature, up to 50% rel-
hydrolysis (Yamada et al. 1997) or isomerization (Wang and ative loss in phenolics and 40% relative loss in ORAC values
Helliwell 2000). Furthermore, certain enzymes may still be were observed in single pasteurized pulp, whereas double
active in the fruits and contribute to the degradation of the pasteurization resulted in lesser relative loss. Further study
active components (Tomas-Barberan and Espın 2001). needs to be done to assess cocoa pulp and its health benefits
Antioxidant capacity of single pasteurized cocoa pulp to support the marketability of cocoa pulp as by-products of
(Fig. 4a) was most preserved when stored at 4C. Addition- the cocoa industry.
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 ACKNOWLEDGMENTS
to the phenolics. The retention of ORAC values in single The authors would like to thank Dr. Kasi Sundaresan and
pasteurized pulp by the end of 8 weeks stability study were Dr. Don Giampetro of iTi Tropicals (Lawrenceville, NJ) for
95, 85 and 60% at 4, 25 and 37C storage conditions, the financial support, cocoa pulp samples and insights that
respectively. made this research possible. Authors also thank Dr. Deepti
Total phenolic content of double pasteurized pulp is Salvi for help in preparing the manuscript and for useful
shown in Fig. 3b. At 4C storage temperature, there was an discussions.
increase in the total phenolic content of double pasteurized
pulp, starting at week 2 and remained stable until week 8.
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