Industrial Crops and Products 40 (2012) 81–89

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Industrial Crops and Products

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Antioxidant activity and physico-chemical properties of Tunisian grown

pomegranate (Punica granatum L.) cultivars
Faten Zaouay a,∗ , Pedro Mena b , Cristina Garcia-Viguera b , Messaoud Mars a
a
Research Unit on Agrobiodiversity, Higher Agronomic Institute, Chott-Mariem, IRESA-University of Sousse, Tunisia
b
Department of Food Science and Technology, CEBAS-CSIC, P.O. Box 164, E-30100, Espinardo, Murcia, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The objective of this study was to compare the physico-chemical characteristics and antioxidant activity
Received 4 January 2012 of 13 pomegranate cultivars grown in southern Tunisia. Fruit size, skin color, skin thickness, juice yield,
Accepted 29 February 2012 juice color, total soluble solids, pH, titratable acidity, total phenolics, total and individual anthocyanin
and antioxidant activity were measured in the pomegranate cultivars. The results showed great differ-
Keywords: ences in the characteristics and the composition of the pomegranate cultivars. The considerable variation
Pomegranate
was consistent with ANOVA and PCA results. Three cultivars were found isolated with particular char-
Cultivar
acteristics; CH8-2 distinguished with large fruit size, MZ2 with red-colored peel, sour sweet juice and
Fruit characteristics
Juice color
high antioxidant activity and GS1 with the smallest fruit, the lowest juice content, highest juice acidity
Anthocyanins and with its considerable antioxidant activity. In addition, the cultivar RF1was also detached from other
Antioxidant activity cultivars with considerable amounts of phenolics and anthocyanins. Results showed also significant cor-
relations between total phenolics content and antioxidant activity indicating the contribution of phenolic
compounds in juice antioxidant capacity. Moreover, significant correlations were found between the red
color intensity a* and antioxidant capacity, or total phenolic content and some anthocyanins pigments
suggesting the richness of red juices in antioxidants, phenolic compounds and anthocyanin pigments.
The correlation observed between the juice visual color and color indices of CIE L*a*b* emphasize the
efficiency of the grading color scale to determine the color of pomegranate juice in the absence of the CIE
system. These results would be a guide in the selection of potential cultivars which are used in food and
chemical industry and fresh market.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction C, and certain phenolic compounds as punicalagin, ellagic acid, gal-

New life styles have driven consumers away from healthy
dietary habits. As a matter of fact, their increasing concern about
their health has prompted the industry to become involved in
the need for food products which contribute to the prevention of
illness. Fruit juices with a high content of bioactive compounds
may represent specific products. Pomegranate (Punica granatum
L.) fruit sets a best example for such products. The consumption of
pomegranate fruit has been reported to have many positive health
benefits (Balasundram et al., 2006; Mertens-Talcott et al., 2006;
Seeram et al., 2007; Basu and Penugonda, 2009). Pomegranate fruits
are widely consumed fresh and in other processed forms as juice,
jam, jelly, vinegar, wine, oil and in extract supplements (Gil et al.,
2000; Maestre et al., 2000; Vardin and Fenercioglu, 2003). In fact,
pomegranate fruits are a source of carbohydrates, minerals, crude
fibres, and various biologically active compounds, such as vitamin
∗ Corresponding author. Tel.: +216 73 327 544; fax: +216 73 327 591.
E-mail address: fatenzaouay@hotmail.com (F. Zaouay).
lotannins, anthocyanins (Kaplan et al., 2001; Noda et al., 2002;
Cerda et al., 2003), which are known to act as natural antioxidants.
The natural antioxidants found in pomegranate fruit can protect
humans against the oxidative stress and reduce consequently the
risk of chronic diseases and prevent disease progression (Lansky
and Newman, 2007; Viuda-Martos et al., 2010).
It is known that varietal, geographical, seasonal, and maturity
differences, as well as processing conditions, greatly affect the
composition of both fruits and juices (Gil et al., 1995; Al-Maiman
and Ahmad, 2002; Kulkarni and Aradhya, 2005; Mirdehghan and
Rahemi, 2007; Borochov-Neori et al., 2009). Some properties of
pomegranate fruits, particularly the total antioxidant capacity, total
phenol content, the ellagitannins and anthocyanins content, are
useful to monitor their quality during ripening. These compounds
also contribute to the nutritional quality of both fresh fruits and
juices. As well, they are useful markers for evaluating the condi-
tions of fruit processing and storage and the authenticity of fruit
juice (Ozgen et al., 2008; Çam et al., 2009; Mena et al., 2011).
However, the increasing interest in fruit quality makes it possi-
ble to enhance the genotypes characterization of the pomegranate

0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.indcrop.2012.02.045
82 F. Zaouay et al. / Industrial Crops and Products 40 (2012) 81–89

Table 1 1,2-phenylenediamine dihydrochloride (OPDA), from Fluka

Cultivars used in pomegranate assessment.
Chemika (Neu-Ulm, Switzerland); cyanidin–glucoside polyphe-
No. Cultivar Code Collection site nols, Sandnes, Norway); potassium dihydrogen phosphate
1 Chelfi8-2 CH8-2 Djerba and sodium carbonate anhydrous from Panreac Química S.A.
2 Jerbi1 JR1 Djerba (Barcelona, Spain) were used as standards.
3 Zehri11 ZH11 Djerba Milli-Q water used was produced using an Elix3 Millipore water
4 Zehri4 ZH4 Sidi Bou Ali purification System (Molsheim, France).
5 Tounsi9-2 TN9-2 Djerba
6 Gabsi11 GB11 Djerba
7 Gabsi1 GB1 Zerkine 2.3. Titratable acidity, pH and total soluble solids
8 Rafrafi1 RF1 Zerkine
9 Mezzi2 MZ2 Tozeur Titratable acidity (TA), pH and total soluble solids (TSS) were
10 Garsi1 GS1 Tozeur
evaluated as juice quality indexes. The pH values were measured
11 Jebali8 JB8 Sbikha
12 Nebli1 NB1 Esslouguia using a Jenway pH-meter. The TA was determined by titrating 10 ml
13 Zaghouani1 ZG1 Zaghouan of with 0.1 N NaOH (pH 8.1). Results were expressed as g malic acid
per 100 ml of sample, in accordance with AOAC (1984). The TSS
contents were recorded in an Atago refractometer at 20 ◦ C with val-
germplasm and creating high antioxidants lines with potential ues being expressed as ◦ Brix. The ratio, also known as the maturity
health promoting effects for human diet. In this context, multivari- index, was calculated as the relation between TSS and TA.
ate analysis has been usefully employed for fruit characterization
and germplasm evaluation of different fruit species like cherimoya 2.4. Color quantification
(Andrés-Agustín et al., 2006) peach and nectarine (Cantín et al.,
2010), apricot (Leccese et al., 2010) and pomegranate (Zaouay and A quartz cell of 2 mm path length (CT-A22) was filled with
Mars, 2011) in order to study relationships among cultivars and homogenized juice. Color measurements were performed in a
correlations among fruit traits. Minolta CM-508i® tristimulus spectrophotometer colorimeter
Pomegranate is cultivated all over the world and Tunisia is con- (Osaka, Japan) coupled with a CM-A760 transmittance adaptor,
sidered as a micro-genecenter of this species with more than 60 illuminant D65 and 10◦ viewing angle according to the CIELAB 76
local ecotypes already collected (Mars, 2001). Nevertheless, some convention. CIE color data (L*, a*, b*) were recorded and processed
cultivars are threatened by extinction despite their high poten- using the Minolta Software Chroma control S, PC-based colorimet-
tial of valorization. Hence, the knowledge of fruit characteristics ric data system. Hue angle (H*) was calculated from tan−1 (b*/a*)
is compulsory for the preservation of these cultivars. It is also ben- and Chroma (C*) from (a*2 + b*2 )1/2 . Data were obtained in tripli-
eficial for fresh market and food industry development as well as cate.
for breeding research. Thus, the current study focused on Tunisian
pomegranate cultivars aims to evaluate the antioxidant potential, 2.5. Determination of total phenols by Folin–Ciocalteau’s reagent
some morphological and chemical fruit characteristics which are
routinely monitored through every quality control program in the Total phenol content TPC was determined with the
industry and fresh market. Folin–Ciocalteu method (Arnous et al., 2001). In a 1.5-ml Eppendorf
microtube, 790 ␮l of Milli-Q water, 10 ␮l of sample appropriately
2. Materials and methods diluted with MeOH, and 50 ␮l of Folin–Ciocalteau reagent were
added and vortexed. After 1 min, 150 ␮l of aqueous 20% sodium
2.1. Plant material and sample preparation carbonate were added, vortexed again and allowed to stand at
room temperature in the dark, for 2 h. The final volume of the
Thirteen cultivars of pomegranate were collected from differ- assay was 1 ml. Reaction was followed with a spectrophotometer
ent localities (Table 1). These cultivars were maintained in the (UV-1603 Shimadzu, Tokyo, Japan). Results were expressed as mg
same ex situ collection in Gabès in Southern Tunisia (Mars and of Gallic acid/100 ml of juice.
Marrakchi, 1998). Ten pomegranate fruits of each cultivar were
randomly selected and many fruit attributes were recorded using 2.6. HPLC analysis, identification and quantification of
methods developed by Mars and Marrakchi (1999): fruit weight anthocyanins
FW (g), fruit height FH (mm), fruit diameter FD (mm), skin thick-
ness at the equatorial area STH (mm) measured by a digital caliper All samples were centrifuged during 5 min at 10,480 g (model
with 0.001 mm accuracy, skin color SC assessed according to grad- EBA 21; Hettich Zentrifugen) at room temperature.10. The super-
ing scale (2: yellow greenish to 18: dark red purple) and aril yield natant (soluble fraction) was filtered through a 0.45 ␮m nylon
(AY) as a percentage of edible part. membrane (Waters Corporation) before injection into the HPLC. For
The juice yield (%) JY was obtained by pressing 100 g of fleshy identification and quantification of anthocyanins the method pre-
arils in a domestic squeezer and filtered through a cloth tissue. Juice viously reported by Pérez-Vicente et al. (2004) was followed. Each
color JC was assessed visually according to grading scale of color sample was analyzed on a Merck-Hitachi L6200 liquid chromato-
intensity ranging 2 (light pink: the lightest color) to 18 (reddish graph (Tokyo, Japan), equipped with a Diode Array Detector UV–vis
purple: the darkest color). Shimadzu SPD-M6A (Kyoto, Japan) and an autoinjector (Gilson
International, model 234, Barcelona, Spain). Chromatograms were
2.2. Reagents and standards recorded and processed on a LC Work station Class M10 A Shi-
madzu PC-based chromatography data system. A 20 ␮L sample was
2,2-Diphenyl-1-picrylhydrazyl (DPPH• ), cetrimide analyzed on a Luna C18 column (25 cm × 0.46 cm, 5 ␮m particle
(cetyltrimethylammonium bromide), 2,2-azino-bis (3-ethyl- size; Phenomenex, Macclesfield, UK) with a security guard C18-
benzothiazoline-6-sulfonic acid) diammonium salt (ABTS), ODS (4.0 mm × 3.0 mm) cartridge system (Phenomenex), using a
Folin Ciocalteu reagent, formic acid and methanol, all of mobile phase of water/formic acid (95:5, v/v) (solvent A) and HPLC
analytical grade, from Scharlau (Sentmenant, Spain); 6-hydroxy- grade methanol (solvent B). Elution was performed at a flow rate of
2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and 1 mL min−1 . The linear gradient started with 1% B, keeping isocratic
 F. Zaouay et al. / Industrial Crops and Products 40 (2012) 81–89
conditions during 5 min, reaching 20% B at 20 min, 40% B at 30 min,
95% B at 35 min and 1% B after 41 min. UV chromatograms were
recorded at 280, 360 and 520 nm. The anthocyanins were charac-
terized by chromatographic comparison with analytical standards
and quantified as Cyanidin-3-glucoside (detected at 520 nm).
2.7. DPPH, ABTS and FRAP assays of antioxidant capacity
All samples were centrifuged at 10,480 g (model EBA 21; Hettich
Zentrifugen) for 5 min at room temperature.
2.7.1. DPPH• radical scavenging activity assay
The reaction mixture consisted of 2 ␮l of the diluted sample and
an aqueous methanolic solution (250 ␮l) containing the free radi-
cal (DPPH• ). The reduction of the DPPH radical was determined by
measuring the absorption at 515 nm after 50 min of reaction (Espín
et al., 2000; Llorach et al., 2004).
2.7.2. ABTS+ assay
The reaction started by adding 2 ␮l of the diluted sample to
the stock solution containing 250 ␮l of the free radical (ABTS+ )
water solution. The radical was chemically generated with MnO2
as described by Espín et al. (2000). The experiments were always
performed on freshly made up solutions. The disappearance of
ABTS+ was determined by measuring the decrease in absorbance
at 414 nm for 1 h at 25 ◦ C.
2.7.3. FRAP assay
Ferric reducing antioxidant power (FRAP) assay was performed
according to Benzie and Strain (1996) method with some modifi-
cations (Llorach et al., 2004). The freshly made up FRAP solution
contained 25 ml of 0.3 M acetate buffer (pH 3.6) plus 2.5 ml of
10 mM TPTZ solution in 40 mM HCl (previously prepared) and
2.5 ml of 20 mM ferric chloride (FeCl3 ·6H2 O). This solution was
used as blank. FRAP solution (250 ␮l of warmed solution, 37 ◦ C)
was mixed with 2 ␮l of the corresponding diluted sample. The ferric
reducing ability of pomegranate juice was measured by monitoring
the increase of absorbance at 593 nm for 40 min.
All the antioxidant assays were performed using 96-well micro-
plates (Nunc, Roskilde, Denmark) and Infinite® M200 micro-plate
reader (Tecan, Grödig, Austria). The results were expressed as
mmol L−1 of Trolox.
2.8. Statistical analysis
Statistical analyses were performed using a one-way analy-
sis of variance ANOVA, and the significant difference between
means was determined by Duncan’s multiple range test. Dif-
ferences at P < 0.05 were considered statistically significant. The
results were presented as mean values ± SD (standard deviations).
Data were analyzed also to determine whether there is any cor-
relation between fruit attributes using Pearson correlation. Cluster
analysis was applied to the standardized data to obtain hierarchical
associations employing Squared Euclidean distance method as dis-
similarity measure. Principal component analysis (PCA) was carried
out using the SPSS 13.0 software.
3. Results and discussions
3.1. Fruit external characteristics
Fruit size is a varietal characteristic that may fluctuate depend-
ing on climatic and agricultural conditions (Shulman et al., 1984).
Based on weight we distinguish small (101 g), medium-sized
(200–400 g) and big sized fruits (>400 g). On the basis of size
(height + diameter/2), we divide pomegranate fruits into small
83
(up to 54.19 mm), medium-sized (70–80 mm) and large fruits
(>80 mm). Among analyzed cultivars, the smallest fruits were found
in cultivar GS1 (101.3 g and 51.5 mm) (Table 1). The biggest fruits
were reported for cv CH8-2, with 549.7 g and large size with an
average of 95.1 mm. However, most of cultivars were of medium-
sized fruits and that is relevant in the design of appropriate
packaging for fruit handling and storage (Valero and Ruiz-Altisent,
2000). Previously, average fruit weight of Tunisian pomegranates
was reported between 87 and 590 g (Zaouay and Mars, 2011), while
fruit weight of Iranian cultivars did not exceed 347 g (Tehranifar
et al., 2010; Zarei et al., 2010). According to Al-Said et al. (2009),
commonly grown cultivars in Sultanate Oman were of 90 mm fruit
diameter and this is in accordance with our results.
Skin color directly affects the consumer acceptability of fruit.
It varied largely among the studied cultivars (Table 2). ZH4 and
MZ2 fruits had red purple coloration, while GS1 fruits exhibited a
green yellowish color. Aril yield, which is a highly desirable prop-
erty in the food processing industry, ranged from 37.8 to 61.7%.
GS1 had the lowest percentage followed by TN9-2. CH8-2 with
big fruits showed high aril yield, while ZH4 and ZH11 which did
not exhibit higher fruit size, yielded more arils per fruit. Similar
results were found with Tunisian and Iranian cultivars (Tehranifar
et al., 2010; Zaouay and Mars, 2011). There were significant dif-
ferences in fruit skin thickness among the cultivars, with NB1
having the thickest skin (6.4 mm) and ZH11 having the thinnest one
(3.6 mm).
Results revealed that CH8-2 is a promising cultivar combining
fruit size and aril yield. ZH11 also is considered promis-
ing cultivar with red fruit color and high aril percentage.
These properties are highly desirable in food processing and
marketing.
3.2. Chemical properties
One of the most important attributes from an industrial point
of view is high juice yield in pomegranate arils (Maestre et al.,
2000). The juice content of the studied cultivars varied from
58.7 ml/100 g of arils for GS1 to 88.9 ml/100 g for ZH11. In general,
most of cultivars showed an important juice content exceeding
70%. This percentage is higher compared to those reported for
pomegranate fruits of different cultivars grown in Turkey, Spain
and Iran (Martinez et al., 2006; Zarei et al., 2010; Poyrazoglu et al.,
2002).
In terms of juice pH, mean values ranged from 2.72 (GS1) to 4.24
(CH8-2). Moderately significant differences were found between
cultivars in term of total soluble solids content (TSS). It ranged from
14.33 to 16.30 ◦ Brix. MZ2 and GB1 cultivars had significantly higher
soluble solids contents (>16 ◦ Brix) than other cultivars (Table 3). TSS
content for all cultivars ranged higher than the minimum thresh-
old generally required for commercial use (12%) and felled into
the range of others cited pomegranate genotypes grown under
Spanish (Martinez et al., 2006), Italian (Barone et al., 2001) and Ira-
nian (Tehranifar et al., 2010) conditions. However, Poyrazoglu et al.
(2002) and Zarei et al. (2010) reported higher levels of soluble solids
for up to 19 ◦ Brix. Titratable acidity expressed as % of malic acid var-
ied from 0.14 to 2.43%. GS1 gave the highest acidity level followed
by MZ2 and GB1, while the rest of the cultivars had considerably
lower acidity values. The maturity index (MI = TSS/TA), influencing
the taste and flavor of pomegranate, is one of the important factors
and used to classify cultivars (Martinez et al., 2006; Melgarejo et al.,
2000). MI varied considerably from 6.03 in GS1 to 104.94 in GB11.
According to the classification established for Spanish cultivars, GS1
is classified as sour cultivar, MZ2 and GB1 as sour–sweet and the
rest of the cultivars are considered sweet ones and appropriate for
fresh consumption.
84 F. Zaouay et al. / Industrial Crops and Products 40 (2012) 81–89

Table 2
Fruit characteristics of pomegranate cultivars.

VAR FW (g) FH (mm) FD (mm) HF + DF/2 SC AY (%) STH (mm)

ZH11 253.4 ± 73.3e 67.38 ± 7.7d 77.49 ± 8.0f 72.44 15.75 ± 1.7ab 61.7 ± 0.5a 3.6 ± 0.9c
red purple
ZH4 355.3 ± 58.2cd 73.16 ± 5.2cd 89.23 ± 5.1cde 81.19 17.60 ± 0.8a 60.0 ± 0.6ab 4.1 ± 0.8bc
dark red purple
RF1 362.3 ± 72.7cd 76.22 ± 8.8abc 90.59 ± 6.7abcde 83.40 14.40 ± 3.4b 56.8 ± 0.5abc 5.1 ± 0.6b
red pink
CH8-2 549.7 ± 119.8a 88.85 ± 8.7a 101.33 ± 6.7a 95.09 8.00 ± 1.3c 58.4 ± 0.4abc 4.8 ± 0.5bc
pink yellowish
JB8 306.3 ± 88.5d 74.91 ± 7.0bcd 91.19 ± 8.5abcd 83.05 14.00 ± 2.1b 41.2 ± 0.4e 4.9 ± 0.8b
red pink
JR1 372.3 ± 98.3d 80.11 ± 6.4abc 90.09 ± 8.3bcde 85.10 10.67 ± 2.0c 55.0 ± 0.6abc 5.0 ± 0.6bc
pink
ZG1 419.3 ± 84.5ab 78.38 ± 5.1abc 94.81 ± 6.2ab 86.60 12.40 ± 3.4b 51.0 ± 0.4cd 5.1 ± 0.9b
pink reddish
NB1 294.6 ± 39.8de 78.08 ± 9.4bcd 85.12 ± 3.5def 81.60 14.33 ± 1.5b 45.7 ± 0.3de 6.4 ± 1.3a
red pink
TN9-2 404.3 ± 76.3abc 81.12 ± 6.4ab 91.78 ± 7.0abc 86.45 11.64 ± 2.0c 37.9 ± 0.1e 5.0 ± 0.7bc
pink reddish
GB11 309.7 ± 51.9de 73.62 ± 6.3cd 85.33 ± 6.0ef 79.47 13.40 ± 1.6b 56.7 ± 0.5abc 4.9 ± 1.0b
red pink
GB1 330.8 ± 90.5d 71.36 ± 6.7cd 88.64 ± 8.2abcde 80.00 9.60 ± 1.3c 57.0 ± 0.6abc 4.9 ± 0.9bc
pink
MZ2 345.1 ± 60.3bcd 73.21 ± 5.1abc 89.89 ± 5.0abcd 81.55 17.87 ± 0.5a 51.8 ± 0.4bcd 4.9 ± 1.0b
dark red purple
GS1 101.3 ± 32.6f 51.54 ± 5.1e 56.83 ± 6.2g 54.19 6.00 ± 0.0d 37.8 ± 0.4e 4.0 ± 0.5c
green yellowish
** ** ** ** ** ** **
Sig. degree

FW: fruit weight; FH: fruit height; FD: fruit diameter; SC: skin color; AY: aril yield; STH: skin thickness.
**
Means with different letters, in the same column, indicate significant differences at P < 0.01.

3.3. Juice color
The color of food has always been an important quality attribute.
The attractive red color of pomegranate juice is one of the param-
eters that are evaluated for the commercial classification of the
product in relation to its quality, which influences consumer behav-
ior. The juice color was measured using a colorimeter. Also, it is
evaluated visually using a grading scale. Visually, it was found that
juice from ZG1 cultivar was the most colored followed by JR1, RF1
and NB1 juices while CH8-2 and GB11 showed a light colored juices.
The sour (GS1) and the sour–sweet cultivars (MZ2 and GB1) had
red-colored juices.
When evaluated in the CIE L*a*b* system, juice color indices
varied significantly among the studied pomegranate cultivars
(Table 4). On the basis of lightness (L* = 0 denotes black and L* = 100
indicates diffuse white), the results indicate that juices from most
of cultivars have a light color and there were consistent similarities
in color indices. However, JR1 had the less light juice and juice
from ZH4 cultivar was the lightest one among studied cultivars.
The (a*) and (b*) indices which indicates respectively redness and
yellowness showed significant differences (P ≤ 0.001). RF1 had the
highest redness color value while ZH4 and CH8-2 showed the low-
est values. For yellowness color (b*), JR1 and ZG1 showed the
highest values whereas ZH4, TN9-2 as well as MZ2 and GS1 pre-
sented the lowest values (Table 4). Regarding the Chroma value
(C) which represents the “purity” or intensity of a color, the culti-
var ZH4 showed the less intensity of color whereas RF1 and ZG1
with the highest C value being more pure (C > 30). Results showed
also similar color intensities among sour and some sweet cultivars.
Large significant differences among the cultivars existed in the hue
angle (H) of the juices, ranging from 17.1◦ (MZ2) to 68.8◦ (CH8-2).
Nevertheless, the tested cultivars did not exhibited dark red color
compared to Wonderful variety or Spanish cultivars (Mena et al.,
2011).

Table 3
Juice yield, pH, total soluble solids, titratable acidity and maturity index of Tunisian pomegranate cultivars.

Cultivar JY (ml/100 g) PH TSS (◦ Brix) TA% MI

a cd
ZH11 81.9 ± 3.00 3.83 ± 0.08 15.6 ± 0.57abc 0.3 ± 0.03cd 49.9 ± 17.40e
ZH4 79.7 ± 2.28ab 3.98 ± 0.09bc 15.3 ± 0.79bcd 0.2 ± 0.02d 75.1 ± 48.64b
RF1 76.9 ± 4.07bc 3.99 ± 0.14bc 15.2 ± 0.46cde 0.2 ± 0.02d 78.9 ± 30.53b
CH8-2 73.2 ± 7.60c 4.24 ± 0.04a 14.8 ± 0.46cdef 0.3 ± 0.06cd 48.3 ± 7.64e
JB8 76.6 ± 4.35bc 4.01 ± 0.05bc 15.5 ± 0.50abc 0.2 ± 0.02cd 67.7 ± 20.85c
JR1 75.2 ± 3.38abc 4.06 ± 0.16ab 14.3 ± 0.37f 0.2 ± 0.02cd 68.56 ± 18.58c
ZG1 75.2 ± 4.18c 3.79 ± 0.12d 15.0 ± 0.70cdef 0.4 ± 0.05c 38.6 ± 14.88f
NB1 76.5 ± 3.39abc 4.08 ± 0.04ab 14.6 ± 0.58ef 0.3 ± 0.01cd 57.5 ± 40.73d
TN9-2 78.0 ± 3.03bc 3.92 ± 0.07bc 14.6 ± 0.55def 0.3 ± 0.01cd 58.2 ± 47.06d
GB11 73.0 ± 4.14c 4.08 ± 0.07ab 14.9 ± 0.78cdef 0.1 ± 0.08d 104.9 ± 10.38a
GB1 73.6 ± 2.17c 3.11 ± 0.07e 16.1 ± 0.36ab 1.4 ± 0.23b 11.5 ± 1.58g
MZ2 75.3 ± 3.29c 3.02 ± 0.31e 16.3 ± 0.53a 1.6 ± 0.29b 10.2 ± 1.81gh
GS1 58.7 ± 2.06d 2.72 ± 0.06f 14.6 ± 0.75cdef 2.4 ± 0.29a 6.0 ± 2.57h
** ** ** ** **
Sig. degree

JY: juice yield; PH: juice pH; TSS: total soluble solids content; TA: titratable acidity; MI: maturity index.
**
Means with different letters, in the same column, indicate significant differences at P < 0.01.
 F. Zaouay et al. / Industrial Crops and Products 40 (2012) 81–89 85

Table 4
Juice color and color parameters (CIE Lab) of the investigated pomegranate cultivars.

Cultivar JC Color parameters

L* a* b* C H

JR1 15.5 ± 1.93ab 51.7 ± 19.2e 16.7 ± 4.6cd 23.0 ± 15.2a 28.9 ± 14.8ab 49.6 ± 11.9bc
RF1 15.6 ± 3.10abc 61.6 ± 8.4bcde 29.7 ± 8.1a 12.4 ± 3.5bc 32.3 ± 8.2a 23.1 ± 5.9fg
ZG1 17.2 ± 1.97a 56.1 ± 14.3de 24.4 ± 12.1abc 23.7 ± 10.6a 34.1 ± 16.1a 44.9 ± 2.3bcd
NB1 15.7 ± 0.82abc 70.2 ± 5.6ab 16.2 ± 4.3cd 11.4 ± 2.0bc 19.9 ± 4.5bc 35.7 ± 3.6de
ZH11 12.5 ± 4.63bcd 71.1 ± 12.8ab 11.5 ± 6.3de 14.6 ± 5.7bc 18.9 ± 7.7bc 54.2 ± 12.8b
ZH4 11.7 ± 4.83de 83.9 ± 0.4a 6.2 ± 1.1e 9.2 ± 0.6c 11.2 ± 0.3c 56.3 ± 6.1b
JB8 12.8 ± 3.43cd 62.1 ± 2.9bcde 22.2 ± 7.3abc 12.3 ± 2.7bc 26.0 ± 5.0ab 31.1 ± 14.5ef
CH8-2 7.2 ± 1.79e 70.9 ± 2.9ab 7.0 ± 1.4e 18.3 ± 1.9ab 19.7 ± 1.6bc 68.8 ± 4.9a
TN9-2 14.6 ± 1.81abcd 73.2 ± 1.5ab 19.1 ± 2.6bcd 9.1 ± 1.4c 21.3 ± 2.3bc 25.7 ± 4.8efg
GB11 6.3 ± 1.98e 66.6 ± 5.8bcd 15.6 ± 5.5cd 14.1 ± 2.4bc 21.1 ± 5.5bc 43.5 ± 7.4cd
GB1 14.6 ± 1.90abcd 56.9 ± 1.7cde 18.4 ± 1.0bcd 17.5 ± 1.1ab 25.5 ± 1.5ab 43.5 ± 0.8cd
GS1 14.6 ± 3.60abcd 69.8 ± 6.6bc 22.2 ± 9.0abc 7.1 ± 0.8c 24.5 ± 7.6ab 20.0 ± 8.5g
MZ2 14.6 ± 2.53abcd 62.9 ± 13.4bcde 26.0 ± 1.5ab 8.3 ± 5.2c 27.6 ± 2.6ab 17.1 ± 9.6g
** ** ** ** ** **
Sig. degree

JC: juice color; L*: color lightness; a*: color redness; b*: color yellowness; C: Chroma; H: hue angle.
**
Means with different letters, in the same column, indicate significant differences at P < 0.01.

3.4. Total phenolic content
The content of total phenolics is one of the most important
parameters for appraising the characterization of pomegranate cul-
tivars, with respect to their nutraceutical value and potential use for
different purposes. As one of the most important antioxidant plant
components, phenolic compounds have been widely investigated
in many fruits (Djeridane et al., 2006). Their activity is believed
to be mainly because of the irredox properties (Zheng and Wang,
2001), which play an important role in adsorbing and neutralizing
free radicals (Laranjinha et al., 1995). The amount of total pheno-
lics varied widely in pomegranate juices and ranged from 133.93
to 350.06 g/100 ml of juice (Fig. 1). The concentration of phenolics
was dependent on cultivar. Among sweet cultivars, low levels were
found in NB1 (145.28 g/100 ml), ZH11 (145.4 g/100 ml) and CH8-2
juices (148.39 g/100 ml), whereas RF1 contained high amounts of
phenolics (350.06 g/100 ml). Sour cultivars excluding GB1, had rel-
atively high contents of phenolic substances (174.55 g GAE/100 ml
with GS1 and 181.84 g GAE/100 ml with MZ2). Considerable varying
amounts of total phenolic contents have been reported by various
authors (Gil et al., 2000; Poyrazoglu et al., 2002; Mousavinejad et al.,
2009; Tezcan et al., 2009; Çam et al., 2009; Tehranifar et al., 2010;
Fawole et al., 2011).
3.5. Anthocyanin determination
The anthocyanin composition is an important quality parameter
of pomegranate fruit, due to the importance of these compounds
in the color of the respective juices. The anthocyanins in the
pomegranate vary greatly with the cultivars, maturity, produc-
tion area and seasonal conditions (Gil et al., 1995; Borochov-Neori
et al., 2009). As pigments, they are almost exclusively responsible
for the red, blue and purple colors in fruits. Total and individual
anthocyanin contents of the cultivars are presented in Table 5.
The total content of anthocyanin varied from 50.5 mg L−1 (CH8-2)
to 490.4 mg L−1 (JR1). Sour cultivars did not reveal high antho-
cyanin amounts compared to sweet ones. The levels of anthocyanin
found with our juices were lower than those reported for Ira-
nian and Chilean genotypes (Alighourchi and Barzegar, 2008;
Sepúlveda et al., 2010). Regarding individual anthocyanins con-
centration, a significant variation was found between cultivars.
Hence, Delphinidin-3,5-diglucoside was the predominant pigment
in all cultivars except TN9-2 which had Delphinidin-3-glucoside as
the main pigment (Table 5). The highest amounts of Delphinidin-
3,5-diglucoside were detected in JR1 and RF1 cultivars with
respectively 406.7 and 349.5 mg L−1 , whereas, GS1 showed the low-
est concentration (33.2 mg L−1 ). Delphinidin-3-glucoside was also
a characteristic pigment of Tunisian pomegranate juices especially
for TN9-2 with high amounts of 84.7 mg L−1 . Cyanidin-3-glucoside
was also present in all juices with a range of 1.2–26.8 mg L−1 , while
Cyanidin-3,5-diglucoside was particularly present in some sweet
juices of RF1, TN9-2, NB1, JR1 and JB8 (Table 5). Our results are
consistent with those found by Mousavinejad et al. (2009) and
Noda et al. (2002) who reported that Delphinidin-3,5-diglucoside
was a major anthocyanin in pomegranate juices. However, these
findings are in contrast with other findings reporting that

Fig. 1. Total phenolics content of pomegranate cultivars. Means with different letters indicate significant differences at P ≤ 0.01 according to Duncan range test.
86 F. Zaouay et al. / Industrial Crops and Products 40 (2012) 81–89

Table 5
Total and individual anthocyanin composition (mg L−1 ) of pomegranate juices.

Cultivar A1 A2 A3 A4 A5 A6 TAC

JR1 406.72 ± 1.0 8.33 ± 5.0ab 0.86 ± 0.04a 62.12 ± 6.3bc 11.18 ± 0.1bc 1.21 ± 0.9a 490.42 ± 5.0a
ZH11 45.05 ± 2.0 5.09 ± 2.8cd 0.24 ± 0.3f 20.46 ± 10.5ef 6.20 ± 0.1defg 0.60 ± 0.2cd 77.65 ± 2.9b
RF1 349.51 ± 3.0 6.06 ± 3.9bc 0.28 ± 0.07ef 62.71 ± 9.3bc 8.91 ± 0.2cde 1.23 ± 0.2a 428.70 ± 3.9bc
GB11 50.30 ± 4.0 2.98 ± 0.6de 0.42 ± 0.06def 25.57 ± 3.4ef 4.28 ± 0.0fgh 0.83 ± 0.1bc 84.37 ± 10.3b
ZG1 61.10 ± 5.0 4.65 ± 1.6cd 0.43 ± 0.06de 73.41 ± 5.7ab 14.22 ± 0.1b 1.31 ± 0.1a 155.13 ± bc

MZ2 49.73 ± 6.0 2.99 ± 0.9de 0.53 ± 0.05bcd 29.50 ± 6.6de 5.08 ± 0.1efgh 0.93 ± 0.1b 88.76 ± 7.5b
GB1 52.84 ± 7.0 4.95 ± 1.8cd 0.62 ± 0.03bc 18.46 ± 1.9ef 4.65 ± 0.1fgh 0.62 ± 0.1cd 82.14 ± 2.9b
GS1 33.16 ± 8.0 3.10 ± 1.5de 0.28 ± 0.04ef 14.94 ± 6.9ef 2.33 ± 0.1gh 0.22 ± 0.03e 54.02 ± 1.6b
JB8 77.20 ± 9.0 7.81 ± 4.4ab 0.70 ± 0.07ab 40.97 ± 2.0d 10.18 ± 0.1bcd 0.57 ± 0.1d 137.43 ± 29.5bc
NB1 93.92 ± 10.0 8.61 ± 1.7a 0.67 ± 0.02b 55.73 ± 2.1c 11.01 ± 0.1bc 0.54 ± 0.01d 170.48 ± 11.3bc
TN9-2 77.73 ± 11.0 9.32 ± 1.4a 0.60 ± 0.01bcd 84.69 ± 6.0a 26.76 ± 0.1a 1.27 ± 0.2a 200.37 ± 21.9bc
ZH4 58.39 ± 12.0 9.80 ± 3.8a 0.68 ± 0.01b 16.91 ± 2.9ef 7.96 ± 0.2cdef 0.21 ± 0.01e 93.95 ± 12.8b
CH8-2 35.83 ± 13.0 2.02 ± 0.4e 0.47 ± 0.04cd 10.85 ± 2.0f 1.16 ± 0.1h 0.19 ± 0.01e 50.52 ± 8.4b
** ** ** ** ** **
Sig. degree NS

A1: Delphinidin-3,5-diglucoside; A2: Cyanidin-3,5-diglucoside; A3: Pelargonidin-3,5-diglucoside; A4: Delphinidin-3-glucoside; A5: Cyanidin-3-glucoside; A6: Pelargonidin-
3-glucoside; TAC: total anthocyanins content. NS: non-significant (P > 0.05).
**
Means with different letters, in the same column, indicate significant differences at P < 0.01.

Table 6
Antioxidant capacity tested by three methods for pomegranate juices.

Cultivar ABTS (mmol L−1 Trolox) FRAP (mmol L−1 Trolox) DPPH (mmol L−1 Trolox)
bc de
JR1 17.34 ± 1.41 10.86 ± 1.00 18.42 ± 2.63bc
GB11 15.71 ± 1.15cde 11.02 ± 1.82de 18.30 ± 2.59bc
RF1 18.22 ± 1.81b 13.74 ± 2.89b 20.30 ± 3.36ab
ZH11 15.73 ± 1.84cde 9.57 ± 1.09e 16.05 ± 2.44cd
ZG1 15.86 ± 2.86bcd 12.54 ± 3.27bcd 17.58 ± 3.25bc
MZ2 21.85 ± 3.20a 21.07 ± 4.74a 22.50 ± 3.27a
GB1 15.16 ± 3.33defg 12.79 ± 2.37bcd 18.96 ± 2.58ab
TN9-2 13.68 ± 1.67efg 14.15 ± 3.92bcd 16.01 ± 3.76cd
GS1 15.93 ± 1.75cde 19.91 ± 2.93a 17.59 ± 2.39bc
JB8 14.45 ± 1.87cdef 13.65 ± 1.80bc 13.46 ± 0.91de
ZH4 12.38 ± 1.31gh 11.14 ± 0.90de 11.91 ± 1.31e
CH8-2 11.24 ± 1.70h 10.52 ± 1.49de 12.64 ± 1.58e
NB1 12.75 ± 2.58fgh 11.13 ± 1.32cde 12.27 ± 3.76e
** ** **
Sig. degree
**
Means with different letters, in the same column, indicate significant differences at P < 0.01.

Cyanidin-3,5-diglucoside were the major pigments in Tunisian 3.7. Correlation analysis

pomegranate juices (Hasnaoui et al., 2011) while Cyanidin-3-
glucoside was characteristic of ‘Mollar de Elche’ varieties (Mena Correlation analysis was performed for correlations among TPC,
et al., 2011). FRAP, DPPH, total anthocyanins content, and L*, a* and b* param-
eters of pomegranate cultivars. The results of TPC in the studied
samples correlated significantly and positively with their antiox-
3.6. Antioxidant activity idant capacity determined by ABTS, FRAP and DPPH methods
(Table 7). Significant linear correlations between total phenolics
Three in vitro assays (DPPH, ABTS and FRAP) were used as content and antioxidant capacities of 15 Spanish varieties analyzed
complementary methods to evaluate the potential antioxidant by ABTS, DPPH and FRAP methods were demonstrated by Mena
activity. Significant differences were observed between the dif- et al. (2011). Therefore, the total phenolics assay could be a suitable
ferent varieties in the three assays. The DPPH scavenging activity candidate for measuring the antioxidant capacity of pomegranate
of pomegranate juices was significantly different with a range fruit juice. Nevertheless, we did not find a significant correlation
from 11.91 to 22.50 mg TEAC. MZ2 showed the highest antioxi-
dant activity value (22.50 mmol L−1 Trolox) while JB8, ZH4, CH8-2
and NB1 showed the lowest values (Table 6). The ABTS scaveng- Table 7
ing activity was also significantly depending on cultivar showing Correlation coefficients of antioxidant capacity, total phenolic content, total antho-
a range between 11.24 and 21.85 mmol L−1 Trolox. In this assay, cyanin content and color parameters.
once again, MZ2 showed the highest antioxidant activity, CH8-2 ABTS DPPH FRAP TPC
showed the lowest value (Table 6). The ferric reducing antioxidant
DPPH 0.60* 1.00 0.55 0.47**
power (FRAP) showed the same trend observed in the previous FRAP 0.91** 0.55 1.00 0.32*
DPPH and ABTS assays. In this assay, once more, the sour cultivar TPC 0.32* 0.47** 0.32* 1.00
MZ2 showed the highest antioxidant activity value (21.07 mmol L−1 TAC 0.07 0.07 0.29 0.32*
Trolox), while ZH11 showed minor value (9.57 mmol L−1 Trolox) CIE L* −0.37* −0.15 −0.46** −0.34*
CIE a* 0.57** 0.57** 0.52** 0.75**
(Table 6). Thus, sour cultivars showed significantly higher activities CIE b* −0.05 −0.31* 0.1 −0.06
than sweet ones. These results agree with previous works reporting Chroma 0.47** 0.39** 0.50** 0.64**
high antioxidant power in pomegranate (Ozgen et al., 2008; Çam H −0.57** −0.68** −0.37* −0.53**
et al., 2009; Mousavinejad et al., 2009; Fawole et al., 2011; Mena *
Correlations are significant at P ≤ 0.05 according to Pearson correlation.
et al., 2011). **
Correlations are significant at P ≤ 0.01 according to Pearson correlation.
 F. Zaouay et al. / Industrial Crops and Products 40 (2012) 81–89 87

Table 8
Correlation coefficients of juice color, color parameters, total and individual anthocyanins.

JC

JC 1
CIE L* −0.42**
CIE a* 0.52**
CIE b* 0.11
Chroma 0.46**
H −0.44**

CJ CIE L* CIE a* CIE b* Chroma

Dp-3,5-diglc 0.24 −0.26 0.28 0.13 0.28

Cy-3,5-diglc 0.12 −0.04 0.06 0.03 0.04
Pg-3,5-diglc −0.05 −0.254 −0.04 0.22 0.05
Dp-3-glc 0.42** −0.43** 0.46** 0.29 0.48**
Cy-3-glc 0.24 −0.10 0.15 0.09 0.12
Pg-3-glc 0.41** −0.48** 0.45** 0.31* 0.48**
Total anthocyanins 0.30 −0.31* 0.34* 0.163 0.33*
*
Correlations are significant at P ≤ 0.05 according to Pearson correlation.
**
Correlations are significant at P ≤ 0.01 according to Pearson correlation.

between total anthocyanins and antioxidant activity assays which by fruit weight, fruit height, fruit diameter, juice yield, juice pH,
indicates that anthocyanins compounds does not contribute to the maturity index and hue angle and negatively by titratable acidity
antioxidant activity. This result corroborate with the findings of and DPPH. PC2 accounted for 24.84% of the total variation. It cor-
Fawole et al. (2011) but it disagree with results reported by Mena related positively with juice colour, ABTS, FRAP, Total phenolics
et al. (2011) who found that total anthocyanins is well correlated content, CIE a*, Chroma, Delphinidin-3,5-diglucoside, Delphinidin-
to antioxidant capacity assays. Besides, significant correlation was 3-glucoside, Pelargonidin-3-glucoside and total anthocyanins
found between analytical methods ABTS, FRAP and DPPH used to content and inversely with CIE L* (Fig. 2a).
determine the antioxidant potential of pomegranate cultivars but The biplot showed that some cultivars as ZG1, JB8, TN9-2 and
no significant correlation was found between DPPH and FRAP. JR1 were laid relatively close to each other along the X-axis (PC1).
Correlation analysis of the data indicated also that the antioxi- Sour cultivars GS1, MZ2 as well as GB1 had large negative scores
dant capacity of the studied pomegranate cultivars determined by
ABTS and DPPH assays and total phenolics content, significantly
negatively correlated with their luminosity, L* (Table 8). This fact
suggests that darker juices contain higher levels of antioxidants and
total phenolics. On the contrary, significant positive correlations
were calculated between ABTS, FRAP, DPPH or total phenolics con-
tent values and the red color intensity (a*) indicating the richness of
red juices in antioxidants and phenolic compounds (Table 8). Sim-
ilar correlation was found by Borochov-Neori et al. (2009) for the
antioxidant capacity of diverse pomegranate cultivars as a function
of the internal red color intensity, whereas a* of the arils did not
correlate with the total phenolics concentration. Further studies on
rapeseed cultivars confirm the correlation between red color index
and antioxidant capacity or total phenolics content (Szydłowsk-
Czerniak et al., 2011). However, the results of ABTS and DPPH or TPC
in the studied juice samples and their yellow color intensity (b*) are
not significantly correlated. The correlation observed between the
juice color measured by visual grading scale and different indices
of CIE L*a*b* emphasizes the efficiency of the grading color scale
to determine the color of pomegranate juice in the absence of
the CIE system. Visual color assessment (JC) was also correlated
to Delphinidin- and Pelargonidin-3-glucosides anthocyanins that
provide the orange and purple colors of the juice. In addition, the
positive correlation between CIE a*, Chroma and total anthocyanins
indicates that red-colored juices are in general rich in anthocyanins
pigments including Delphinidin- and Pelargonidin-3-glcucosides.
Whereas, the lightness CIE L* of the juices was found to be nega-
tively correlated to total and individual anthocyanins.

3.8. Principal component analysis

Principal component analysis (PCA) was used, as an unsu-

pervised method, to examine the similarity among varietal
pomegranate fruits. The first three principal components took into
account 65.84% of the total variation. The first component, PC1 rep-
resenting 28.94% of total variance was highly positively contributed
Fig. 2. (a) Plot of contribution of the physico-chemical investigated traits into the
first and second principal components. (b) Plot of the first and second principal com-
ponents resulting from a PCA of the pomegranate cultivars using physico-chemical
characters.
88 F. Zaouay et al. / Industrial Crops and Products 40 (2012) 81–89
on the PC1 and they were separated from the other cultivars across
PC1 (Fig. 2b). They distinguished with high acidity, and high antiox-
idant capacity tested by DPPH. The cultivars GS1 and MZ2 were also
isolated from others on the second axis.
RF1 had large positive scores on the PC2 axis. It was opposed to
ZH4 and CH8-2, which had large negative scores in terms of juice
color, ABTS, FRAP, CIE a*, Delphinidin-3,5-diglucoside, Delphinidin-
3-glucoside, Pelargonidin-3-glucoside and total anthocyanin con-
tent.
Some attributes such as skin color, skin thickness and total
soluble solids content were revealed not efficient to discrimi-
nate pomegranate cultivars. Principle component analyses were
previously employed by several researchers to characterize
pomegranate germplasm and to highlight the most discriminat-
ing characters (Mars and Marrakchi, 1999; Drogoudi et al., 2005;
Durgaç et al., 2008; Hasnaoui et al., 2011; Mena et al., 2011; Zaouay
and Mars, 2011).
4. Conclusions
This investigation clearly shows the potential value of
pomegranate germplasm. The fruit quality of pomegranate fruit
was affected by cultivar to a large extent. The results of this study
might improve the diversification of cultivars and their fruit quality
control. Fruit size, aril yield, soluble solids content and acidity were
varied significantly among cultivars. These attributes are the most
important considerations in pomegranate marketing as well as in
breeding programs to obtain new promoting cultivars.
Interestingly sour cultivars with their red colored juices provide
high antioxidant capacity besides to high amounts of polyphe-
nols compounds. JR1 and RF1 revealed the richest cultivars in
total anthocyanins content. It is noteworthy that there are linear
and significant correlations between total phenolic content and
antioxidant capacity of pomegranate cultivars. Moreover, antiox-
idant capacity significantly correlated with their luminosity (L*),
red color intensity (a*). Thus, darker juices contain higher levels of
antioxidants. In addition, the correlation observed between visual
juice color assessment and different indices of CIE L*a*b* and indi-
vidual anthocyanins emphasizes the efficiency of the grading color
scale to determine the color of pomegranate juice. PCA as well
as cluster results allowed singling out three cultivars with par-
ticular distinctive characters (GS1, CH8-2 and MZ2). These data
will help in the selection of pomegranate genotypes to improve
quality and processing characteristics in processing industry. In
consideration of the total qualities demanded in pomegranate juice
(nutrition, taste, appearance, etc.), using different pomegranate
cultivars to produce mixed juice (sour and sweet juices) may be
a good alternative in food industry. The high antioxidant capacity,
total phenolic content and hence high protection against oxida-
tive stress would also encourage nutritional studies to assess the
importance of pomegranate juice in the maintenance of health and
disease prevention. Furthermore, pomegranate may be useful in
food technologies and cosmetic industries. However, a long-term
study is required to estimate the interaction between the environ-
mental variability and the cultivation conditions on the quality of
pomegranate fruits and juices and further studies on nutritional
quality would be relevant in order to use it as a criterion to choose
production of different cultivars.
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