Research Article

Received: 24 March 2013 Revised: 8 August 2013 Accepted article published: 21 August 2013 Published online in Wiley Online Library: 9 October 2013

(wileyonlinelibrary.com) DOI 10.1002/jsfa.6361

Thermal stability of liquid antioxidative

extracts from pomegranate peel†
Wenjuan Qu,a∗ Pingping Li,b Jihua Hong,a Zhiling Liu,a Yufang Chen,a
Andrew P Breksa IIIc and Zhongli Pana,d∗

Abstract
BACKGROUND: Liquid extracts from pomegranate peel have the potential for use as natural antioxidant products. This study
investigates the quality changes of liquid extracts before and after thermal treatment during sterilization and storage. Liquid
pomegranate peel extracts were prepared, sterilized under ultra-high temperature (UHT) at 121 ◦ C for 10 s and then stored
at three temperatures (4, 25 and 37 ◦ C) for up to 180 days. The industrial, color, UV-visible spectrum profile and antioxidant
(phenolics) characteristics were measured.

RESULTS: Thermal sterilization treatment had no negative effects on the industrial, color, spectral and antioxidant characteristics
of the extracts. After 180 days, the extracts stored at 4 ◦ C retained 67% of the initial total soluble phenolic content and 58% of
the original scavenging activity. The major antioxidant components in the extracts (stored at 4 ◦ C for 180 days) were gallic acid,
punicalagin A, punicalagin B and ellagic acid having concentrations of 19.3, 197.2, 221.1 and 92.4 mg L−1 , respectively.

CONCLUSION: The results show that liquid pomegranate peel extracts had acceptable thermal stability after sterilization and
storage. The recommended storage condition of this product was low temperature.

c 2013 Society of Chemical Industry

Keywords: pomegranate peel extracts; antioxidants; sterilization stability; storage stability; phenolics

INTRODUCTION characteristics,16,18,19 industrial characteristics,20 – 22 and content,

Pomegranate (Punica granatum) is one of the fruits containing
important bioactive phenolic ingredients,1 – 3 wherein they are
widely used as botanical ingredients in herbal medicines and
dietary supplements.4 Pomegranate is increasingly consumed as
various processed products, such as juices, wines, jams, jellies and
extracts. In pomegranate juice processing, 1 ton of fresh fruit gen-
erates 669 kg by-product – pomegranate marc – containing 78%
peel and 22% seeds, based on our previous study.5 Researchers
have investigated various extraction methods for producing
phenolics from pomegranate peel as natural antioxidants.6 – 9 We
also found that the peel of pomegranate marc had a high level of
phenolics (content of 201 g kg−1 based on dry weight).5 Therefore
pomegranate peel has great potential for use in producing natural
antioxidants (10.2% yield based on dry weight) when water
extraction is used as an environmentally friendly method.10
There is a considerable interest in utilizing natural antioxidant
products. Such products could be in a liquid form and require
thermal sterilization and storage treatments to ensure high safety
and long shelf life. It is ideal that natural antioxidative products are
stable for thermal sterilization and storage at high temperature.
However, there is little information on the quality stability of liquid
antioxidative extracts from pomegranate peel during thermal
sterilization and storage, which needs to be studied.
It has been reported that the quality stability of liquid antioxida-
tive products could be affected by thermal sterilization treatment
activity and composition of bioactive ingredients.23,24 It is reported
that pomegranate peel is rich in antioxidative phenolics, mainly
punicalagins with A and B (α and β) anomers, gallic and ellagic
acids.1 – 3 Therefore, punicalagin A, punicalagin B, gallic acid
and ellagic acid are important antioxidative indicators of liquid
pomegranate peel extracts.
This study was aimed at determining the thermal stability
of liquid antioxidative extracts isolated from pomegranate peel
under temperature-related conditions, including sterilization
∗
Correspondence to: Wenjuan Qu, College of Food and Biological Engineering,
Jiangsu University, 301 Xuefu Road Zhenjiang, Jiangsu 212013, China. E-mail:
quwenjuan2005@yahoo.com.cn
†
This research was conducted at the University of California, Davis, and
USDA-ARS Western Regional Research Center.
a College of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road
Zhenjiang, Jiangsu 212013, China
b Key Laboratory of Modern Agricultural Equipment and Technology, Ministry of
Education (Jiangsu University), Jiangsu Provincial Key Laboratory of Modern
Agricultural Equipment and Technology, Jiangsu University, 301 Xuefu Road
Zhenjiang, Jiangsu 212013, China
c Processed Foods Research Unit, USDA-ARS Western Regional Research Center,
800 Buchanan Street Albany, CA 94710, USA

and storage conditions.11 – 15 The quality of such products is nor- d Department of Biological and Agricultural Engineering, University of California,
1005

mally evaluated by color or pigment characteristics,16 – 18 spectral Davis, One Shields Avenue, Davis, CA 95616, USA

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and storage. Thus the quality indicators of liquid extracts
were measured before and after ultra-high-temperature (UHT)
sterilization (121 ◦ C for 10 s) and at three storage temperatures
(4, 25 and 37 ◦ C) for various time periods (0–180 days). The
studied quality categories included the industrial, color, UV-visible
spectrum profile and antioxidant (phenolics) characteristics.
Preparation of liquid pomegranate peel extracts
The liquid antioxidative extracts from pomegranate peel were
produced by the following extraction conditions: temperature,
25 ◦ C; water-to-peel ratio, 50:1 (w/w); and time, 2 min. The
procedure was established and described in detail in our previous
publication.10

Sterilization and storage experiments

MATERIALS AND METHODS
Chemicals and materials The liquid extract samples after centrifugation were individually
sealed in 50 mL screw-cap centrifuge tubes (Sarstedt Inc., Newton,
Folin–Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH),
NC, USA) and then sterilized at 121 ◦ C for 10 s according to
and tannic, gallic and ellagic acids (high-performance liquid
a modified UHT sterilization condition in an autoclave (small
chromatography (HPLC) grade) were purchased from Sigma-
rectangular sterilizer, Consolidated Stills & Sterilizers, Boston,
Aldrich Co. (St Louis, MO, USA). A mixture of punicalagins A
MA, USA).26 The unsterilized liquid extract samples were used
and B (HPLC grade) was purchased from ChromaDex Co. (Irvine,
as the control for the sterilization study. Following thermal
CA, USA). Methanol (HPLC grade), acetonitrile (HPLC grade), o-
sterilization, the samples in transparent tubes were cooled to
phosphoric acid (H3 PO4 , 85%), and sodium carbonate (Na2 CO3 )
ambient temperature using water rinsing, centrifuged and stored
were purchased from Fisher Scientific Inc. (Fair Lawn, NJ, USA).
in dark containers at one of three temperatures (4, 25 and 37 ◦ C)
Water used in the HPLC analysis was deionized to ≥18.1 M cm−1
for up to 180 days. Individually sterilized samples were evaluated
resistance using a Barnstead NANOpure Deionization System
at days 0, 1, 5, 10, 30, 60, 90 and 180 days. The control was not
(Dubuque, IA, USA) and filtered through a 0.45 µm type HA
included in the storage study because sterilization is a prerequisite
membrane filter prior to use.
for producing safe products with a reasonable shelf life.
Pomegranate marc, which is the residue of pomegranate fruit
after juice extraction, was obtained from POM Wonderful LLC
(Del Rey, CA, USA). The extract samples were prepared by the Determination of industrial characteristics
following procedures. The pomegranate marc was dried at 40 ◦ C Industrial characteristics were evaluated by measuring pH value,
using hot air in a cabinet drier. The seeds were manually separated total soluble solids (TSS) content and clarity. Measurement of
from the peels and the dried peels ground into 40-mesh powder pH value was done with a pH meter (AR 20, Accumet Research,
using a mill were used as the raw material in this research. The Fisher Scientific Inc., Pittsburgh, PA, USA) and TSS content (%) was
measured moisture content of the raw material was 117 g kg−1 . determined using an automatic refractometer (ATC-1, Atago Co.
More detailed sample preparation procedures can be found in our Ltd, Tokyo, Japan) with temperature-compensated mode. Clarity
recent publication.25 was determined using a Genesys 10Bio UV–visible spectrometer

Table 1. Industrial, color, spectral and antioxidant characteristics of liquid extracts from pomegranate marc peel before and after UHT sterilization

UHT sterilization

Quality category Quality indicator Before After

Industrial characteristics pH value 3.5 3.5

TSS content (%) 1.2 1.4
Clarity 0.095a 0.075b
Color characteristics Redness, a* 2.6a 1.8b
Yellowness, b* 33.5a 33.7a
Lightness, L* 60.1a 59.9a
Chroma, C* 33.6a 33.8a
Hue angle, H* (◦ ) 85.6b 86.9a
Total color difference, E* — 0.9
Spectral characteristics Peak A λA (nm) 256a 258a
AA 1.195a 1.142a
Peak B λB (nm) 368a 367a
AB 0.183a 0.179a
Antioxidant characteristics Concentration of specific phenolic compounds (mg L−1 ) Gallic acid 32.6a 21.7b
Ellagic acid 40.1b 176.0a
Punicalagin A 43.2b 187.4a
Punicalagin B 98.1a 99.2a
Total soluble phenolic content (×103 mg L−1 ) 2.3a 2.7a
Scavenging activity (g g−1 ) DPPH scavenging activity 6.1a 6.2a

Different letters denote significant differences (P < 0.05) and same letters denote no significant differences (P > 0.05) between samples before and
1006

after UHT sterilization.

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(a) 2.1 (b)

Before UHT sterilization 2.1
1.8 0d
After UHT sterilization 1.8 1d
1.5 5d
A 1.5

Absorbance
A 10 d

Absorbance
1.2 30 d
1.2
60 d
0.9 0.9 90 d
180 d
0.6 0.6
B B
0.3 0.3
0.0 0
200 300 400 500 200 300 400 500
Wavelength (nm) Wavelength (nm)

(d)
(c) 2.1
2.1
0d
1.8 1d 0d
1.8
5d 1d
1.5 A 1.5 A
10 d 5d
Absorbance

Absorbance
1.2 30 d 10 d
1.2
60 d 30 d
0.9 90 d 0.9 60 d
180 d 90 d
0.6 B 0.6
180 d
B
0.3 0.3

0 0
200 300 400 500 200 300 400 500
Wavelength (nm) Wavelength (nm)

Figure 1. UV-visible spectra of liquid extracts from pomegranate marc peel before and after UHT sterilization (a) and under different storage temperatures
(4 ◦ C (b), 25 ◦ C (c), and 37 ◦ C (d)) for up to 180 days.

(Thermo Fisher Scientific Inc., Waltham, MA, USA) and expressed
as absorbance at a wavelength of 660 nm, according to reported
methods.20 A higher absorbance indicates a lower clarity or higher
turbidity.
Determination of color characteristics
The reported method was applied to the determination of color
characteristics.27 Color was measured on each 1 mL liquid extract
sample in a transparent glass vial (diameter 18.5 mm and height
9.6 mm) against a white tile (a* = −0.5, b* = 2.3, L* = 97.7) using
a chroma meter (CR-200, Minolta Camera Co. Ltd, Osaka, Japan).
Measurements were made with a 0◦ viewing angle and illuminant
D65 diffused illumination. The test parameters included a* for
redness, b* for yellowness and L* for lightness. An additional three
parameters were calculated by the transformation of a* and b*
according to Eqns (1)–(3). They were chroma (C*), the correlate of
saturation or intensity; hue angle (H*, ◦ ), the correlate of chromatic
tonality; and total color difference (E*), the magnitude of color
change after treatment:

The total soluble phenolic (TSP) content in each sample was
C* = a*2 + b*2 (1)
H* = tan−1 (b*/a*)
 2
E* = a* − a0 + (b* − b0 )2 + (L* − L0 )2
where a0, b0 and L0 are the values of unsterilized liquid extract
sterilization effect study; however, for the storage effect study, a0 ,
b0 and L0 are the values for sterilized liquid extract samples stored
at 4 ◦ C and zero day (a0 = 1.8, b0 = 33.7, and L0 = 59.9).
Determination of spectral characteristics
After centrifugation, extract samples were diluted eightfold
using deionized water and placed in quartz glass UV cuvettes
(Fisher Scientific Inc., Pittsburgh, PA, USA). UV–visible spectra
were immediately measured in the scan range 200–500 nm
in 1 nm increments according to the reported method.19 Data
were recorded using a Lambda 750 UV–visible spectrometer
(PerkinElmer Inc, Shelton, CT, USA), after extract diluents were
prepared. The spectral data were used to describe the changes
in absorption wavelength and the corresponding absorbance.
Changes in spectral characteristics represented the change in
color intensity.
Determination of antioxidant characteristics
determined according to our modified Folin–Ciocalteu method
and reported in tannic acid equivalents.10 In brief, samples were
diluted fourfold using deionized water prior to the colorimetric
(2) analysis. The resulting diluted samples (60 µL) were combined with
2 mL Na2 CO3 (7.5%) and 2.5 mL Folin–Ciocalteu reagent (10-fold
diluted), and thoroughly mixed using a vortex mixer. The mixed
(3) solution was held in a water bath at 25 ◦ C for 30 min and then
its absorbance was measured at 760 nm using a Genesys 10Bio
spectrometer. The blanks were prepared by combining 60 µL
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samples (i.e. control with a0 = 2.6, b0 = 33.5 and L0 = 60.1) for the deionized water with 2 mL Na2 CO3 and 2.5 mL Folin–Ciocalteu

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concentration in blank (mg L−1 ), Ct is the TSP concentration in the

Table 2. Industrial characteristics of liquid extracts from
pomegranate marc peel under different storage temperatures and extracts (mg L−1 ), Vt is the total volume of extracts (L), and n is the
times dilution factor of extracts, which is 20 in this study.

Temperature Time TSS Absorbance

(◦ C) (day) pH value content (%) at 660 nm Statistical analysis
All measurements were made in triplicate for each sample in three
4 0 3.5 1.4 0.075 replicates. Data were presented as means (n = 3) in tables. Tukey’s
1 3.5 1.2 0.095 studentized range (HSD) test, using SAS software (Version 9.2., SAS
5 3.5 1.2 0.107 Institute Inc., Cary, NC, USA), was performed to determine whether
10 3.5 1.2 0.088 there were significant differences in the clarity, color, characteristic
30 3.4 1.2 0.062 absorption wavelength, absorbance values and antioxidant
60 3.4 1.4 0.064 characteristics of samples before and after UHT sterilization and
90 3.5 1.4 0.051 at different storage temperatures. The significance of mean values
180 3.3 1.1 0.070 was determined using least significant difference (LSD) (α = 0.05).
25 0 3.5 1.4 0.075
1 3.4 1.2 0.116
5 3.5 1.2 0.094
10 3.4 1.2 0.094
RESULTS AND DISCUSSION
Effect of thermal sterilization on quality stability
30 3.4 1.2 0.087
Table 1 shows the industrial, color, spectral and antioxidant char-
60 3.4 1.2 0.087
acteristics measured in liquid pomegranate peel extracts before
90 3.4 1.2 0.117
and after UHT sterilization. Statistical analysis results revealed
180 3.3 1.2 0.131
that thermal sterilization treatment had a significant effect on
37 0 3.5 1.4 0.075
the clarity in industrial characteristics a* and H*, and component
1 3.5 1.2 0.103
concentration of antioxidants except for punicalagin B (P < 0.05).
5 3.5 1.2 0.100
For the industrial characteristics, we observed that the sterilization
10 3.4 1.2 0.094
treatment had no effect on pH value (3.5 for samples both
30 3.4 1.2 0.112
before and after sterilization) and minimal influence on the TSS
60 3.3 1.2 0.147
content (1.2% and 1.4% for samples before and after sterilization)
90 3.3 1.2 0.134
but significantly increased the clarity (absorbance at 660 nm
180 3.2 1.0 0.172
decreased from 0.095 of the original to 0.075 after sterilization) of
Overall — — 4 ◦C c
liquid extracts. The increase in clarity caused by the sterilization
25 ◦ C b
37 ◦ C a
was desirable for liquid products and might be due to the polymer-
ization settlement of pectin and protein macromolecules present
Different letters denote significant differences (P < 0.05) and same in the extracts. Fang et al.26 reported a similar trend in bayberry
letters denote no significant differences (P > 0.05) between samples juice after UHT processing. The present results indicated that the
under different storage temperatures.
industrial characteristics of the extracts were resistant to UHT.
When evaluating the color characteristics of liquid extracts, b*,
L* and C* values were not influenced by thermal sterilization,
reagent using the above procedure. TSP content (mg L-1 ) was
but H* value was significantly increased based on the statistical
calculated using a standard curve of tannic acid (0–14.0 mg L−1 ).
analysis. H* values are stepped from 0◦ to 360◦ (magenta red)
The concentrations of specific phenolic compounds, including
across a continuously fading hue circle, indicating yellow (90◦ ),
gallic acid, punicalagin A, punicalagin B and ellagic acid were
bluish green (180◦ ) and blue (270◦ ). Therefore the liquid extracts
determined using our established HPLC method.28 Quantification
after thermal sterilization treatment became brighter yellow in
was performed by external calibration and results were reported
color than before sterilization, which was also reflected by a
as milligrams per liter.
significantly decreased a* value. In addition, the E* value was as
The DPPH scavenging activity in each sample was determined
low as 0.9, which meant that the total color differences between
using our adapted colorimetric procedure.10 Extract samples were
sterilized and unsterilized extract samples were relatively small.
diluted 20-fold using deionized water prior to the colorimetric
In general, the pomegranate peel extracts maintained good color
analysis. The diluted sample (60 µL) was reacted with 3 mL
characteristics with a* of 1.8, b* of 33.7, L* of 59.9, C* of 33.8 and
DPPH solution in methanol (50 mg L−1 ). The solution was mixed
H* of 86.9◦ after thermal sterilization. Lee and Coates12 reported
thoroughly using a vortex mixer and held in a water bath at
that the color characteristics of orange juice were significantly
25 ◦ C for 20 min. The absorbance of the solution was measured at
affected by the thermal sterilization process, which led to juice
517 nm using a Genesys 10Bio spectrometer. Deionized water was
color becoming lighter and more saturated. In another study it was
used as the control and blanks were prepared by combining 60 µL
also found that thermal sterilization changed the color parameters
extract diluent with 3 mL methanol. The DPPH scavenging activity
of grapefruit juice, causing a slight color shift towards lightness and
(g g−1 ) was calculated using Eqn (4):
brightness,11 which were similar to our results on the pomegranate
nVt [Cc − (Ce − Cb )] peel extract products.
DPPH scavenging activity = (4) Figure 1(a) shows the UV–visible spectra of liquid extract
C t Vt
samples before and after UHT sterilization. Two characteristic
where Cc is the DPPH concentration in control (mg L−1 ), Ce is the peaks appeared and were recorded as peaks A and B, and then
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DPPH concentration in diluted sample (mg L−1 ), Cb is the DPPH their absorption wavelengths (λA and λB ) and absorbance values

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Table 3. Color characteristics of liquid extracts from pomegranate marc peel under different storage temperatures and times

Temperature (◦ C) Time (day) Redness, a* Yellowness, b* Lightness, L* Chroma, C* Hue angle, H* (◦ ) Total color difference, E*

4 0 1.8 33.7 59.9 33.8 86.9 —

1 1.7 33.6 58.5 33.7 87.1 1.4
5 1.5 33.7 60.2 33.8 87.4 0.4
10 1.8 34.3 59.9 34.3 87.1 0.6
30 1.4 34.0 60.3 34.0 87.7 0.7
60 1.6 34.1 60.1 34.1 87.3 0.5
90 1.2 32.9 60.8 32.9 88.0 1.3
180 2.5 33.7 59.2 33.8 85.8 1.0
25 0 1.8 33.7 59.9 33.8 86.9 —
1 1.4 34.0 59.9 34.1 87.6 0.5
5 1.4 34.3 60.5 34.3 87.6 0.9
10 1.7 34.4 59.3 34.5 87.2 1.0
30 1.9 33.9 59.6 33.9 86.8 0.4
60 2.4 33.4 58.9 33.5 85.9 1.3
90 3.3 33.3 58.4 33.5 84.3 2.2
180 3.8 33.9 57.6 34.1 83.7 3.0
37 0 1.8 33.7 59.9 33.8 86.9 —
1 1.9 34.4 59.8 34.5 86.9 0.7
5 1.7 34.2 60.2 34.3 87.2 0.6
10 2.2 34.2 59.2 34.3 86.3 0.9
30 3.6 33.8 57.8 34.0 83.9 2.8
60 3.7 32.9 57.8 33.1 83.6 2.9
90 4.9 34.3 56.1 34.7 82.0 4.9
180 5.5 34.8 54.9 35.3 81.0 6.3
Overall 4 ◦C b 4 ◦C a 4 ◦C a 4 ◦C c 4 ◦C a —
25 ◦ C b 25 ◦ C a 25 ◦ C a 25 ◦ C b 25 ◦ C a
37 ◦ C a 37 ◦ C a 37 ◦ C b 37 ◦ C a 37 ◦ C b

Different letters denote significant differences (P < 0.05) and same letters denote no significant differences (P > 0.05) between samples under
different storage temperatures.

(AA and AB ) are reported in Table 1. The UV–visible spectra
of extract samples were not significantly influenced by thermal
sterilization treatment. Moreover, the λA (256 and 258 nm), λB
(368 and 367 nm), AA (1.195 and 1.142) and AB (0.183 and 0.179)
of the samples were not significantly different before and after
thermal sterilization. Dissimilarly, Lee and Coates11 reported that
the reflectance spectrum in the visible region (400–700 nm) clearly
showed changes in the spectral distribution of light reflected from
grapefruit juice after thermal sterilization (91 ◦ C for 30 s). This could
be due to different sterilization conditions and material properties
used in different studies. In general, the results indicated that
pomegranate peel extracts still had good stability in spectral
characteristics after thermal sterilization treatment.
Table 1 shows the TSP content, scavenging activity and concen-
tration of specific phenolic compounds measured in liquid extracts
before and after UHT sterilization. In our recent publication, HPLC
data showed that the major phenolic compounds of these extracts
were punicalagins A and B, and ellagic and gallic acids.28 Therefore
the concentrations of specific phenolic compounds, including
gallic acid, punicalagin A, punicalagin B and ellagic acid, were
determined in this study. The concentrations of specific phenolic
compounds except for punicalagin B were significantly influenced
by the thermal sterilization treatment, in which the concentrations
of punicalagin A and ellagic acid were increased and gallic acid
concentration was decreased. The increase in punicalagin might
be from the increased combination of gallic acid or gallagic
temperature; therefore the gallic acid content showed part of
the decrease.29 Moreover, the increase in ellagic acid at a higher
temperature might be due to the degradation of ellagitannins
or ellagic acid–glycosides.29,30 However, the TSP content and
scavenging activity were not significantly changed by the thermal
sterilization treatment. Therefore pomegranate peel extracts after
thermal sterilization still had high TSP content (2.7 × 103 mg
L−1 ) and scavenging activity (6.2 g g−1 ), composed of gallic acid
(21.7 mg L−1 ), ellagic acid (99.2 mg L−1 ) and punicalagins A and
B (176.0 and 187.4 mg L−1 ) even though their concentrations
showed some changes.
In general, the liquid extracts after thermal sterilization
maintained good industrial, color, spectral and antioxidant
characteristics. Therefore the tested sterilization method can be
used in industry to sterilize the liquid pomegranate peel extracts.
Effect of storage on industrial characteristics stability
Table 2 shows the industrial characteristics of liquid extract
samples at different storage temperatures and periods. The
overall pH value (3.2–3.5) and TSS content (1.0–1.4%) of extract
samples showed no significant differences at different storage
temperatures (P > 0.05) during the entire storage period. The
storage time also did not have a great effect on the pH value
and TSS content. A related report showed that the pH value of
pomegranate juice did not significantly change with time during
1009

acid, punicalin, and glucose or gluconic acid with the increasing storage at 4 ◦ C, but the TSS content decreased after 15 h of

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storage.21 In another study it was also found that the pH value

Table 4. Spectral characteristics of liquid extracts from pomegranate
of grape juice did not significantly change before 3 months of marc peel under different storage temperatures and times
storage time, but the soluble solids decreased after 3 months.31
The statistical analysis results indicated that the overall clarity of Peak A Peak B
Temperature Time
liquid extracts at various storage periods significantly decreased (◦ C) (day) λA (nm) AA λB (nm) AB
with increased storage temperatures (P < 0.05). The low clarity
at high temperature might be due to increased cloudiness and 4 0 258 1.142 367 0.179
turbidity caused by pectin, protein and other macromolecule 1 257 1.161 367 0.179
polymerization.20,22 The clarity of samples at various storage 5 257 1.133 364 0.198
temperatures showed different trends during the entire storage 10 257 1.155 368 0.181
period. The different clarity trends with time might be due to 30 257 1.134 367 0.175
different time segments of cloud formation and precipitation. 60 256 1.214 368 0.183
Under the high storage temperature (37 ◦ C), the absorbance values 90 257 1.092 369 0.173
of samples at 660 nm increased from 0.103 to 0.172 within 1 to 180 259 1.173 360 0.238
180 days of storage and showed a significantly decreased trend 25 0 258 1.142 367 0.179
in the clarity with time. Moreover, the overall clarities of samples 1 257 1.111 368 0.171
were higher than that of the original (0.075) and therefore clarity 5 258 1.153 361 0.213
was significantly influenced by the storage time. Similarly, the 10 256 1.180 367 0.183
overall clarity of samples stored at a moderate temperature (25 ◦ C) 30 256 1.186 365 0.206
was significantly decreased by storage time. The overall clarity of 60 256 1.204 367 0.189
samples stored at a low temperature (4 ◦ C) was relatively stable 90 255 1.225 367 0.197
during 180 days of storage, which indicated that the low storage 180 260 1.091 360 0.237
temperature and even long time were still beneficial to maintain 37 0 258 1.142 367 0.179
the high stability in clarity compared to the high temperature and 1 258 0.991 365 0.161
short time. 5 258 1.124 360 0.218
The present results indicated that high storage temperature 10 256 1.162 365 0.199
significantly decreased the clarity of extracts, even though it had no 30 256 1.226 367 0.193
significant effect on pH and TSS (P > 0.05). Therefore, to maintain 60 257 1.054 367 0.173
the high stability in industrial characteristics, it is recommended 90 257 1.136 367 0.188
that the pomegranate peel extracts should be stored at a low 180 258 1.035 363 0.224
temperature, such as 4 ◦ C for any time during 180 days of storage. Overall 4 ◦C a 4 ◦C a 4 ◦C a 4 ◦C a
25 ◦ C a 25 ◦ C a 25 ◦ C a 25 ◦ C a
37 ◦ C a 37 ◦ C a 37 ◦ C a 37 ◦ C a
Effect of storage on color characteristics stability
Table 3 shows the color characteristics of liquid pomegranate peel Different letters denote significant differences (P < 0.05) and same
extracts at different storage temperatures and periods. All color letters denote no significant differences (P > 0.05) between samples
under different storage temperatures.
characteristics of samples except for b* at various storage times
revealed significant changes with increased storage temperatures
(P < 0.05). The significantly low L* and H* values but high C* value
at high temperature indicated that the liquid extracts became Fig. 1. There were two characteristic peaks, recorded as peaks
more opaque and darker red, compared with the clarified and A and B. The corresponding absorption wavelengths (λA and
attractive yellow color of samples stored at low temperature, λB ) and absorbance values (AA and AB ) are listed in Table 4. All
which was also reflected by a significantly higher a* value. In UV–visible spectra and absorption properties of samples stored at
addition, compared to the original, the E* value of samples various times did not significantly change with increased storage
stored at high temperature was much higher than that at low temperatures (P > 0.05). All λA and λB of characteristic peaks
temperature. This indicated that the high temperature led to gathered around 255–260 nm and 360–369 nm, which were very
significant total color difference between the samples stored for a close to the absorption wavelengths of polyphenol components
period of time and the original. A similar report also showed that (254, 270 and 378 nm). Based on our HPLC data results of these
color degradation depended on storage temperature.32 Moreover, extracts, the two major peaks were attributed to the phenolic
the storage time also had a great effect on the color characteristics. compounds of punicalagins, and gallic and ellagic acids.28 Thus
Based on the available data, the recommended storage times were we predicted that storage temperature had no significant changes
≤180 days for samples stored at 4 ◦ C, ≤60 days for samples at 25 ◦ C in the total polyphenol amount in the extracts. Moreover, this
and ≤10 days for samples at 37 ◦ C. The present results indicated indicated that stable spectral characteristics of pomegranate peel
that storage temperature was a significant factor influencing extracts were obtained under all tested temperatures.
color characteristics. By considering the negative effect of a high
temperature on color characteristics, pomegranate peel extracts Effect of storage on stability of antioxidant characteristics
should be stored at a low temperature for the recommended time
Table 5 summarizes the TSP concentration, scavenging activity
to maintain the vivid color characteristics.
and concentrations of specific phenolic compounds measured in
liquid extracts at different storage temperatures and periods. It
Effect of storage on stability of spectral characteristics can be seen that TSP concentration and scavenging activity had
The UV-visible spectra of liquid pomegranate peel extracts no significant differences at different storage temperatures, but
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at different storage temperatures and periods are shown in storage temperature had a significant effect on the concentrations

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Sterilization and storage stability of pomegranate peel extract product www.soci.org

Table 5. Antioxidant characteristics of liquid extracts from pomegranate marc peel under different storage temperatures and times

Concentration of specific phenolic compounds (mg L−1 )

Temperature Time Total soluble phenolic DPPH scavenging
(◦ C) (day) Gallic acid Punicalagin A Punicalagin B Ellagic acid content (×103 mg L−1 ) activity (g g−1 )

4 0 21.7 176.0 187.4 99.2 2.7 6.2

1 20.8 154.0 167.7 92.6 2.5 (7%) 6.0 (3%)
5 19.4 155.6 166.1 80.9 2.2 (19%) 5.6 (10%)
10 20.0 154.2 171.7 84.2 2.0 (26%) 5.4 (13%)
30 20.8 160.8 181.8 85.2 2.2 (19%) 5.1 (18%)
60 17.6 178.0 200.4 75.7 2.2 (19%) 4.8 (23%)
90 16.9 177.8 198.5 68.1 2.1 (22%) 4.8 (23%)
180 19.3 197.2 221.1 92.4 1.8 (33%) 3.6 (42%)
25 0 21.7 176.0 187.4 99.2 2.7 6.2
1 20.0 144.8 155.6 87.6 2.4 (11%) 6.1 (2%)
5 20.8 156.4 164.4 91.2 2.2 (19%) 5.3 (15%)
10 20.0 151.6 172.0 92.0 2.0 (26%) 5.3 (15%)
30 21.2 154.4 174.8 96.4 2.2 (19%) 5.3 (15%)
60 22.4 157.6 180.0 107.2 2.0 (26%) 4.5 (27%)
90 24.0 160.0 181.6 135.2 2.026%) 4.3 (31%)
180 24.4 143.2 162.8 57.2 1.7 (37%) 3.7 (40%)
37 0 21.7 176.0 187.4 99.2 2.7 6.2
1 20.4 156.0 165.2 92.8 2.5 (7%) 5.4 (13%)
5 21.2 154.8 164.8 107.2 2.4 (11%) 5.3 (15%)
10 21.2 149.2 169.2 102.4 2.0 (26%) 5.3 (15%)
30 21.2 135.6 157.6 100.8 2.1 (22%) 5.2 (16%)
60 26.8 142.0 164.0 110.0 2.0 (26%) 4.1 (18%)
90 29.6 134.8 157.6 88.0 1.9 (30%) 4.3 (31%)
180 36.0 102.4 124.8 106.0 1.5 (44%) 3.7 (40%)
Overall 4 ◦C b 4 ◦C a 4 ◦C a 4 ◦C b 4 ◦C a 4 ◦C a
25 ◦ C b 25 ◦ C b 25 ◦ C b 25 ◦ C a 25 ◦ C a 25 ◦ C a
37 ◦ C a 37 ◦ C b 37 ◦ C b 37 ◦ C a 37 ◦ C a 37 ◦ C a

Different letters denote significant differences (P < 0.05) and same letters denote no significant differences (P > 0.05) between samples under
different storage temperatures. Values in parentheses are the reducing rate compared to the original extract (0 days).

of specific phenolic compounds (P < 0.05). The stable TSP con-
centration in extracts at different storage temperatures had been
predicted by the spectral characteristics of the extracts. According
to reports, scavenging activity was related to the amount of
polyphenols.2 Therefore, scavenging activity was also stable.
Storage temperature significantly increased the concentrations
of gallic and ellagic acids but decreased punicalagin (A + B) con-
centrations. If pomegranate marc peel is used for the isolation of
organic acids (gallic and ellagic acids), we would recommend that
a high storage temperature of 37 ◦ C be used. If punicalagin is the
desired product, the preferred storage temperature should be low.
During the entire storage period, both TSP concentration and
scavenging activity decreased over time, irrespective of temper-
ature. For example, under high storage temperature (37 ◦ C), TSP
concentrations decreased by 7%, 11%, 26%, 22%, 26%, 30% and
44% when samples were stored for 1, 5, 10, 30, 60, 90 and 180 days,
respectively. The scavenging activities accordingly decreased by
13%, 15%, 15%, 16%, 18%, 31% and 40%, respectively. There were
similar trends for samples stored at low temperatures (4 and 25 ◦ C).
This indicated that the long storage time caused much more loss
of TSP concentration and scavenging activity in extracts compared
to the short time. However, compared to the original extract, liquid
extracts stored at three temperatures can still maintain >50%
of TSP concentration, scavenging activity and concentrations of
The above results indicated that under all tested temperatures
the pomegranate peel extracts still maintained relatively
acceptable antioxidant characteristics.
CONCLUSIONS
The quality stability of liquid antioxidative extracts prepared from
pomegranate peel subjected to UHT sterilization (121 ◦ C, 10 s) and
three storage temperatures (4, 25 and 37 ◦ C) for up to 180 days was
evaluated. Thermal sterilization treatment had no negative effects
on the industrial, color, spectral and antioxidant characteristics of
the liquid extracts. During the entire storage period, the storage
temperature caused negative effects on the industrial, color and
antioxidant (individual phenolics) characteristics of the extracts.
Therefore it was recommended that the liquid extracts should
be stored at a low temperature to maintain acceptable quality
stability. Such a product stored at 4 ◦ C for as long as 180 days
can retain 67% of the initial total soluble phenolic content and
58% of the original scavenging activity. The major antioxidant
components in the product were gallic acid, punicalagin A,
punicalagin B and ellagic acid, having concentrations of 19.3,
197.2, 221.1 and 92.4 mg L−1 , respectively. It was concluded that a
functional antioxidant product, with a reasonable shelf life and an
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specific phenolic compounds when stored for as long as 180 days. acceptable quality, can be produced from pomegranate peel.

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 www.soci.org
ACKNOWLEDGEMENTS
The authors wish to thank POM Wonderful LLC for providing the
pomegranate marc materials, and Don Olson for preparing the
pomegranate samples. The authors also wish to express their
sincere thanks to the Youth Natural Science Fund of Jiangsu
Province (No. BK2012287), Postdoctoral Funds of Jiangsu Province
(No. 1101039C), Senior Professional Research Start-up Fund of
Jiangsu University (No. 10JDG121), Priority Academic Program
Development (PAPD) of Jiangsu Higher Education Institutions,
and College Students’ Scientific Research Project (No. 12A023),
People’s Republic of China, for their support.
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