journal of functional foods 8 (2014) 161168

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Short communications

Pomegranate juice functional constituents after

alcoholic and acetic acid fermentation
Stella A. Ordoudi *, Fani Mantzouridou, Eleni Daftsiou, Christine Malo,
Efimia Hatzidimitriou, Nikolaos Nenadis, Maria Z. Tsimidou
Laboratory of Food Chemistry and Technology, School of Chemistry, Aristotle University of Thessaloniki, 54124
Thessaloniki, Greece

A R T I C L E

I N F O

A B S T R A C T

Article history:

In this study, a two-stage process, alcoholic and acetic acid fermentation was applied for

Received 13 September 2013

the first time to fresh pomegranate juice to examine its effect on the phenolic content to-

Received in revised form 14 March

gether with the antioxidant activity of the fermented product(s). The total phenol content

2014

of the juice was not greatly affected during the process. On the contrary, the total antho-

Accepted 20 March 2014

cyanin content fluctuated; the alcoholic product was 10-fold poorer while the end product

Available online

was as rich as the fresh juice. Both fermented products had qualitatively similar HPLCDAD-MS profile of phenolic constituents with that of the starting material but lower content

Keywords:

(e.g. 49 and 32% of individual phenolic acid derivatives remained, respectively). The aceti-

Pomegranate vinegar

fication product retained 44% and 37% of the fresh juice activity to scavenge the DPPH and

Total phenol content

reduce cupric ions, respectively. These characteristics along with attractive red colour suggest

Total anthocyanin content

that the end product is a potential source of functional constituents.

DPPH

2014 Elsevier Ltd. All rights reserved.

CUPRAC
Colour

1.

Introduction

Pomegranate (Punica granatum L.) has gained great popularity

during the last decade due to the growing scientific evidence
for its high nutritional value and beneficial effect to human
health (Gil, Toms-Barbern, Hess-Pierce, Holcroft, & Kader,
2000). Bioactive phenolic compounds such as hydrolysable
tannins, monomeric anthocyanins, ellagic acid, gallic acid and
hydroxycinammic acids (Gil et al., 2000; Madrigal-Carballo,
Rodriguez, Krueger, Dreher, & Reed, 2009) that are present in
diverse parts of the fruit (Fischer, Carle, & Kammerer, 2011),
account for strong antioxidant and specific physiological func-

* Correspondence author. Tel.: +30 2310 997847; fax: +30 2310 997847.
E-mail address: steord@chem.auth.gr (S.A. Ordoudi).
http://dx.doi.org/10.1016/j.jff.2014.03.015
1756-4646/ 2014 Elsevier Ltd. All rights reserved.

tions (e.g. anti-tumour, anti-inflammatory) (Faria & Calhau, 2011;

Wu, Ma, & Tian, 2013). Pomegranate is traditionally consumed either as fresh arils or more conveniently, as processed juice. However, the processing including microfiltration,
clarification, enzymatic and thermal treatments may cause
severe losses of its functional constituents and may weaken
the nutritional value of the juice (e.g. Fischer, Carle, &
Kammerer, 2013; Mena et al., 2013).
Different fermentation technologies have been recently
applied to develop novel pomegranate products such as
probiotic juice (Filannino et al., 2013; Mousavi, Mousavi, Razavi,
Emam-Djomeh, & Kiani, 2011), wine (Mena, Girons-Vilaplana,
Mart, & Garca-Viguera, 2012b; Zhuang, Du, & Wang, 2011) and

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journal of functional foods 8 (2014) 161168

vinegar (Yae et al., 2007), as a good alternative to deal with functional characteristics of the raw material and fruit surplus. So
far, know-how in the alcoholic and acetic acid fermentations
of pomegranate juice is limited and has arisen mainly from
research on grape wine and cider-making technologies, as recently addressed in certain scientific papers (Mena et al., 2012b;
Yae et al., 2007; Zhuang et al., 2011) and patents (e.g. Chen et al.,
2012; Wang, Gao, Cheng, & Deng, 2012; Yao, 2012; Yu, Yu, & Yu,
2012; Zhou, 2012). Pomegranate varietal wines have been shown
to retain antioxidant properties due to their high content in
phenolic compounds and are considered promising as health
promoting beverages (Mena et al., 2012b; Zhuang et al., 2011).
To the best of our knowledge, no study exists about the functionality of the pomegranate product after a two-stage process,
which is alcoholic and acetic acid fermentation. In our study,
we examined the contents of total and individual polyphenols and anthocyanins as well as the antioxidant activity of
the end product in comparison with those of the fresh pomegranate juice. Colour characteristics were also evaluated in the
end product and were compared with those of commercial red
wine vinegars.

this study was selected on the basis of oenological properties

(short lag phase, rapid kinetics, polyphenol and colour stability). This strain was kindly donated by Tsantalis S.A. (Chalkidiki,
Greece). Yeast activation and inocula preparation took place
as described by Naziri, Mantzouridou, and Tsimidou (2011). Prior
to inoculation the yeast assimilable nitrogen (YAN) content of
the raw juice was ameliorated up to 300 mg/L by addition of
ammonium sulphate. Sodium metabisulphite (50 mg/L) was also
added for the inactivation of undesirable bacteria. To achieve
maximum ethanol production (75.4 0.6 g/L), the process temperature, the duration and the initial pH of the pomegranate
juice were adjusted to 25 C, 90 h, and 3.3, respectively, after
optimization experiments using the Response Surface Methodology (RSM) (unpublished data by Mantzouridou & Daftsiou,
2012). Cultivation of yeast cells was performed under semianaerobic conditions by allowing 300 mL of the juice to stand
in hydrophobic cotton-stopped Erlenmeyer flasks (500 mL). Fermentation experiments were carried out in triplicate. Fermentation efficiency (%) was calculated according to Grewal, Tewari,
and Kalra (1988).

2.4.

2.

Materials and methods

2.1.

Reagents and solvents

a-D-glucose monohydrate was from Duchefa Biochemie

(Haarlem, Netherlands), D-fructose was from Panreac Quimica
S.A. (Barcelona, Spain) ethanol and chlorogenic acid were from
Riedel-de Han AG (Seelze, Germany). Calcium disodium
ethylenediaminetetraacetic acid (CaNa2-EDTA) vanillic and
ferulic acids were from Sigma-Aldrich (Steinhem, Germany).
Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid)
was obtained from Aldrich Chemie (Steinheim, Germany). Gallic
acid and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were from Sigma
Chemical Co. (St. Louis, MO, USA). Copper chloride dihydrate
was from Merck (Darmstadt, Germany). Ammonium sulphate
was from Chem-Lab NV (Zedelgem, Belgium), sodium
metabisulphite and ellagic acid were from Alfa Aesar GmbH
& Co (Karlsruhe, Germany). Cyanidin-3-O-glucoside chloride was
from Extrasynthese (Genay, France).

2.2.

Fruit harvesting and raw juice preparation

Pomegranate fruits of a Northern Greek genotype (accession

11005) were sampled from the grove of the Pomology Institute (Imathia, Greece) in late October, when fully mature (26
October 2011). The fruits were placed into a ventilated chamber,
transferred within 2 h in the laboratory and stored at 4 C. Arils
(at about 100150 g lots) were hand-separated from the peels
and mesocarp and pressed using cotton filters to produce juice
that was then transferred to 0.5 L PET containers and stored
at 20 C. Prior to analysis and further processing, the juice was
centrifuged at 5862 g (4 C, 10 min).

2.3.

Alcoholic fermentation

Commercial Saccharomyces cerevisiae strain, Fermicru VR5 (DSM

Food Specialties B.V., Flemingham, The Netherlands) used in

Acetic acid fermentation

A gelatinous film of acetic acid bacteria used in acetification

of Xinomavro red wine was generously provided by the OIKOAGRO vinegar industry (Imathia, Greece). It was maintained in
a liquid medium (30:70, v/v; wine to vinegar mixture) at room
temperature (conditions suggested by the wine vinegar producer). A 10% (v/v) inoculum of acetic acid bacteria previously activated according to de Ory, Romero, and Cantero (1998)
was used. A semi-continuous submerged acetification process
took place in 250 mL Erlenmeyer flasks (working volume of
50 mL, 30 C, 250 rpm) in five cycles (total period of 31 days)
as follows: At the beginning of each cycle ethanol concentration was within the range of 3545 g/L. When it reached 510 g/L
in the substrate a certain volume was removed and replaced
by an equal volume of the alcoholic product (75.4 g ethanol/
L). During the first two acetification cycles the removed volume
was less than the typical 50% of the substrate in order to avoid
the risk of cell washout. All experiments were carried out in
duplicate and the results were reported as mean values. Process
yield (%) was calculated according to Rubio-Fernndez, Salvador,
and Fregapane (2004).

2.5.
Total soluble solids, total sugars, pH and total
titratable acidity
The total soluble solids (TSS) content (Brix) of fresh juice
samples was determined using an A. Krss Optronic GmbH
(Hamburg, Germany) refractometer (Association of Official
Analytical Chemists, 1998). Total sugars (TS), expressed as g
glucose/L juice, were determined using the phenolsulphuric
acid assay (Rubio-Fernndez et al., 2004). Measurement of pH
was achieved using a MP220 portable pH-meter (MettlerToledo, Switzerland). Total titratable acidity (TA) was determined according to recommended protocols for the raw juice,
the alcoholic product and the vinegar (Association of Official
Analytical Chemists, 1998).

journal of functional foods 8 (2014) 161168

2.6.

Glucose, fructose and ethanol by HPLC

Glucose, fructose and ethanol in the fresh and fermented juice

were separated on a Sugar Pak column (Waters, Milford, MA)
by isocratic elution with an aqueous solution of CaNa2-EDTA
(50 mg/L) at 80 C (Blanco-Gomis, Gutierrez-Alvarez,
Mangas-Alonso, & Noval-Vallina, 1988). The HPLC system was
composed of an LC-10Advp pump (Shimadzu, Kyoto, Japan),
and a refractive index detector (RID-6A, Shimadzu). The data
were processed with the aid of Clarity Software (DataApex,
Prague, Czech Republic). The flow rate was 0.4 mL/min and the
injection volume was 10 L. Quantification was made using
calibration curves for standard glucose, fructose and ethanol
(1055 g/L). Analysis of samples was carried out in
triplicate.

2.7.

Total phenols, total anthocyanins and HPLC analysis

Total phenol content (TPC) was determined using the Folin

Ciocalteau assay at 750 nm with gallic acid as reference
(Nenadis, Kyriakoudi, & Tsimidou, 2013). Total anthocyanin
content (TAC) was determined using the SO2 bleaching method
(Ribreau-Gayon, Glories, Maujean, & Dubourdieu, 2000). Results
were expressed as mean values of three measurements. The
RP-HPLC profile of phenolic compounds was examined on an
HPLC system that was consisted of a pump, model P4000
(Thermo Separation Products, San Jose, CA, USA), a Midas
autosampler (Spark, Emmen, The Netherlands), and a UV 6000
LP diode array detector (DAD; Thermo Separation Products).
Phenolic constituents were monitored at 260, 350, and 520 nm.
The data were processed with the aid of Chrom Quest software (version 3.0, Thermo Separation Products). Analysis was
carried out on a Nucleosil 100, C18 (250 mm, 4.6 mm, 5 m)
MZ-Analysentechnik GmbH (Mainz, Germany). The gradient
elution protocol was that proposed by Obn, Daz-Garca, and
Castellar (2011) using aqueous acetic acid (3.5% v/v CH3COOH)
(A) and acidified ACN (3.5% v/v CH3COOH) (B) as mobile phase
components. The flow rate was 0.9 mL/min and the injection
volume was 20 L. The identity of major peaks was examined using LC-MS/MS on a Hewlett-Packard 1100 series liquid
chromatograph, equipped with an AQUA reversed-phase C18
column (150 mm, 4.6 mm i.d., 5 m) Phenomenex (Torance, CA).
The capillary voltage was 3 V, and the capillary temperature
was 190 C. Spectra were recorded in positive and negative ionization mode between m/z 120 and 1500. The mass spectrometer was programmed to do a series of three scans: a full mass,
a zoom scan of the most intense ion in the first scan, and MS
MS of the most intense ion using relative collision energy of
30 and 60 (arbitrary units). The same elution protocol with a
flow rate of 0.5 mL/min was applied. Identification was based
on comparison of MS data with available ones from literature (Fischer et al., 2011; Mena et al., 2012a; Ye, Peng, Fan, &
Huang, 2007). Monomeric anthocyanins were quantified at
520 nm as cyanidin 3-glucoside; both pedunculagin isomers
and ellagic acid derivative were expressed as ellagic acid and
quantified at 360 and 280 nm, respectively (Mena et al., 2012b);
vanillic and ferulic acid hexosides as the corresponding acid
at 260 and 325 nm, respectively; caffeoyl-quinic acid as chlorogenic acid at 325 nm.

2.8.

163

Antioxidant activity evaluation

Antioxidant activity of juice and the fermented products were

assessed using two complementary assays; the DPPH radical
scavenging activity assay and the Cupric Ion Reducing Antioxidant Capacity (CUPRAC) one (Nenadis et al., 2013). All of the
samples were diluted with water (1:15, v/v) in duplicate prior
to analysis and each replicate was analyzed in triplicate. Results
were the mean value of six measurements and were expressed as mM of Trolox.

2.9.

Colour measurement

Colour measurements were carried out for the pomegranate

and selected commercial red wine vinegars (from Xinomavro
and Agiorgitiko grape cultivars) employing the protocol of
Garca-Parrilla et al. (1998). Measurements were made with a
1 cm path length cells at a Shimadzu UV1601 spectrophotometer. The rectangular coordinates L*, a*, b* and the cylindrical
coordinates C*, h* were calculated according to the CIEL*a*b*
colour system for a 10 standard observer and the illuminant
D65.

2.10.

Statistical analysis

Statistical comparisons of the mean values of all parameters

studied were performed by one-way ANOVA, followed by the
Tukeys test using the SPSS 14.0 software (SPSS Inc., Chicago,
IL, USA). A significance level p < 0.05 was adopted. Different
letters were used to label significantly different values.

3.

Results and discussion

3.1.
Alcoholic and acetic acid fermentation of the
pomegranate juice
The fresh juice from the selected pomegranate accession presented good yeast fermentation potential in terms of soluble
solids content (17.4 Brix), total sugar content (171.0 1.6 g/L,
as glucose), pH (3.3) and total titratable acidity values
(3.75 0.03 g/L, as citric acid). These values were in line with
those reported for other local genotypes (Drogoudi, Tsipouridis,
& Michailidis, 2005) and commercial pomegranate varieties
(Mena et al., 2012b). The content in total sugars was higher than
typical ones declared for fresh apple (ca. 100 g/L) (Joshi &
Sharma, 2009), mango (ca. 36 g/L) (Gonzalez & De Vuyst, 2009)
and strawberry juices (28 g/L) (Hidalgo, Mateo, Cerezo, Torija,
& Mas, 2010) that are also used for vinegar production. Glucose
and fructose were the only sugars detected in the fresh pomegranate juice with the latter being in slight excess (G/F = 0.84).
Under the applied operational conditions (process temperature 25 C, duration 90 h, initial pH of the pomegranate juice
of 3.3), which promoted the almost complete conversion of
sugars to ethanol (unpublished data by Mantzouridou &
Daftsiou, 2012), ethanol yield and volumetric productivity of
0.44 g/g of sugars and 0.83 g/Lh, respectively, were achieved.
Fermentation efficiency, equal to 86.5% of the theoretical value
(0.51 g/g), was of the same magnitude or even higher than that

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journal of functional foods 8 (2014) 161168

obtained by Mena et al. (2012b) (0.360.42 g/g) using two different pomegranate cultivars. The alcoholic product presented higher TA than that of the fresh juice by 1.00 g/L
(5.21 0.01 g/L, as citric acid). This increase in TA did not influence the pH value (pHalcoholic product = 3.4). It has been suggested that, alcoholic fermentation process causes marginal
changes in the concentration of individual organic acids present
in the fresh pomegranate juice (e.g. malic acid) or formed as
by-products (e.g. acetic acid) (Mena et al., 2012b; Yae et al., 2007).
During the five-cycle acetification process the acetic acid
content of the substrate significantly increased (see
supplementary Fig. S1 in the online version at doi:10.1016/
j.jff.2014.03.015) causing a concomitant decrease in the pH value
(2.93). In particular, from acetification cycle II to V the process
yield increased from 0.42 to 1.00 g/g, i.e. 32.0% to 76.7% of the
theoretical value of 1.304 g/g. Transformation equal to 0.74 and
0.93 (g/g) of ethanol to acetic acid has been also reported for
cashew and mango vinegar production processes, respectively (Ameyapoh et al., 2010; Silva, Torres Neto, Silva, Silva,
& Swarnakar, 2007). The fermentation was finished when the
TA reached a value of 45.3 g/L (as acetic acid) and the residual ethanol content was 6.0 g/L. The achieved values were
found to conform to basic requirements for fruit vinegar commercialization in Greece and elsewhere (Joshi & Sharma, 2009).

3.2.
Total phenol content, total anthocyanin content and
HPLC profiles
Data for the total phenol and total anthocyanin contents are
presented in Table 1. The juice content in total phenols (1387 mg
GAE/L) was regarded high in comparison with other phenolrich fruits (e.g. blueberries) that have been used as raw materials for vinegar production (Su & Chien, 2007). It was
interesting to find that the TPC value of the alcoholic product
was 1395 mg GAE/L nearly equal to that of the starting material. After acetification, the end product was also considered
rich in total phenols (1254 mg GAE/L). The magnitude of TPC
values is similar or even higher than those reported for vinegar
from other types of raw materials (e.g. Andlauer, Stumpf, &
Furst, 2000; Ubeda et al., 2012). This is the first time that data
about the TPC content of pomegranate vinegar are reported.
Considering the total anthocyanin content (TAC), a 10-fold
higher value was found in the fresh juice than in the alcoholic product (130.4 vs 12.8 mg/L) (Table 1). Several phenomena may account for this result (e.g. formation of colourless

products with SO2 or -dicarbonylated compounds, instability of anthocyanin aggregates due to ethanol formation, degradation of the least stable monoglycosidic members) (Mena
et al., 2012b; Ribreau-Gayon et al., 2000; Somers & Verette,
1988). The TAC value in the pomegranate vinegar was found
to be 126.0 mg/L (Table 1), similar to the corresponding value
of the fresh juice. The colour regeneration may partially be
related with pH-dependant dissociation of the colourless products with SO2 since the pH value of the vinegar was lower by
0.47. Consumption of ethanol during acetification may also
favour re-aggregation of anthocyanins and colour enhancement (Somers & Verette, 1988). The HPLC profiles of pomegranate juice and the two fermented products at 260, 350, and
520 nm were then investigated to gain more information about
changes in individual phenolic constituents (Fig. 1ac). It was
found that these profiles were qualitatively identical at any of
the three examined wavelengths (Fig 1). However, due to the
complexity of the profile at 260 nm (low resolution under the
adopted analytical conditions) bioactive constituents were selectively identified mainly at longer wavelengths (350, 520 nm).
Data given in Table 2 verified the presence of hydrolysable
tannins (ellagitannins), caffeic/ferulic acid derivatives and
anthocyanins. Gallic acid (t R = 5.0 min) and ellagic acid
(tR = 30.2 min) monomers were not detected in the fermented
products signifying that their complex derivatives had not been
extensively hydrolyzed during fermentation. Under the conditions of analysis, gradual quantitative losses of phenolic constituents were evidenced after each fermentation process (see
supplementary Table S1 in the online version at doi:10.1016/
j.jff.2014.03.015). In comparison with the fresh juice, the alcoholic product and the vinegar retained 40% and 27% of the
quantified phenolic constituents, respectively. This trend was
not in accordance with the one based on TPC values of the three
products. Alcoholic fermentation was found to heavily affect
monomeric anthocyanin levels (~91% loss). Still, the end product
was found richer than the alcoholic one since it retained ~25%
of the initial anthocyanin content. The semi-continuous acetification process along with colour regeneration phenomena
already discussed could account for this finding. Similar fluctuations were not observed in the contents of major phenolic constituents (see supplementary Table S1 in the online
version at doi:10.1016/j.jff.2014.03.015); thus, 4050% of these
constituents were still present in the alcoholic product while
33% of them were found in the vinegar.

3.3.

Table 1 Total phenol content (TPC) and total

anthocyanin content (TAC) of pomegranate juice,
alcoholic product and vinegar.

PJ
PA
PV

TPC
mg GAE/L

TAC
mg/L

1387 54
1395 48
1254 37

130.1 1.4
12.8 1.3
126.0 2.2

All values are shown as the mean standard deviation (n = 3).

PJ, pomegranate juice; PA, pomegranate alcoholic product; PV, pomegranate vinegar.

Antioxidant potential

As phenolic compounds contribute to the health promoting

properties of pomegranate juice mainly through their antioxidant properties, the abilities to scavenge the DPPH and to
reduce the cupric ions were examined. The results, expressed
as Trolox equivalents are presented in Table 3.
Concerning the antiradical activity, the value for the fresh
juice was within the range reported for juices from various
pomegranate cultivars (1022 mM of Trolox) (Gil et al., 2000).
After alcoholic fermentation, the DPPH scavenging activity was
8.3 mM of Trolox, that is, lower than that of the fresh juice by
almost 27%. Other authors who have also evidenced a drop by
16% to 39% in the antioxidant activity of pomegranate juice
after pomegranate winemaking (Mena et al., 2012b; Zhuang

journal of functional foods 8 (2014) 161168

Fig. 1 RP-HPLC-DAD chromatograms of pomegranate juice (PJ), alcoholic product (PA) and vinegar (PV) at (a) 260 nm, (b)
350 nm and (c) 520 nm.

165

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journal of functional foods 8 (2014) 161168

Table 2 Ellagitannins, hydroxycinnamic acid derivatives and anthocyanins detected in pomegranate juice, alcoholic
product and vinegar.
Peak no.

Rt (min)

[M]+ (m/z)

1
2
3
*
4
5
6
7
8
9
10
11
12
13
14
15

5.9
6.3
6.8
7.9
8.4
9.6
13.2
13.2
15.0
16.7
16.8
17.6
20.1
20.8
22.0
33.4

627

[M-H]- (m/z)
1083
783
329

611
785
465
465
325
785
449
355
433
551
551
475

MS2 (m/z)

Identification

465, 303
781, 601
481, 301
269, 209, 167
449, 287
633, 483, 301
303
303
nf
633, 483, 301
287
217, 193, 175
271
389
389
nf

Delphinidin-3,5-diglucoside
Punicalagin
Pedunculagin I
Vanillic acid hexoside
Cyanidin-3,5-diglucoside
Pedunculagin II
Delphinidin-3-hexoside
Delphinidin-3-hexoside
Caffeoyl-quinic acid
Pedunculagin II
Cyanidin-3-glucoside
Ferulic acid hexoside
Pelargonidin-3-glucoside
Ferulic acid derivative
Ferulic acid derivative
3,3-Di-O-methylellagic acid-4-O-L-rhamnopyranoside

Identification was based on previously reported MS data for pomegranate juice composition (Fischer et al., 2011; Mena et al., 2012a; Ye et al.,
2007).
Rt, retention time; [M]+ and [M-H], molecular mass under positive and negative ionization conditions, respectively; m/z, mass-to-charge ratio;
nf, no mass fraction.

Table 3 DPPH scavenging activity and Cupric Ion

Reducing Capacity of pomegranate juice, alcoholic
product and vinegar.

PJ
PA
PV

DPPH
mM Trolox

CUPRAC
mM Trolox

11.4 0.2
8.3 0.2
5.0 0.2

21.2 0.2
13.4 0.7
7.8 0.2

All values are shown as the mean standard deviation (n = 6).

PJ, pomegranate juice; PA, pomegranate alcoholic product; PV, pomegranate vinegar.

et al., 2011) suggested that it is associated with TPC

reduction (e.g. 1456%) (Mena et al., 2012b). After acetification, the end product presented an antiradical potential equivalent to 5.0 mmol/L of Trolox, almost 55% lower than that of the
fresh juice. The observed loss in activity implied that strong
radical scavengers were partially degraded during both fermentation processes. Quantitative data for the compounds 2,
3, 5, 7, 8 and 10 (Fig. 1; see supplementary Table S1 in the online
version at doi:10.1016/j.jff.2014.03.015) that are expected to con-

tribute significantly to antioxidant activity (e.g. Fernandes, Faria,

Calhau, de Freitas, & Mateus, 2013; Ivanov, Nomura, Malfanov,
& Ptitsyn, 2012, Mena et al., 2012a) justified this suggestion.
Despite the losses, the produced pomegranate vinegar retained a substantial ability to inhibit DPPH reduction (35.5%)
in comparison with respective values reported for other fruit
vinegars (e.g. 11% for apple, 52% for persimmon vinegars)
(Sakanaka & Ishihara, 2008). The aforementioned findings were
corroborated by those obtained using the CUPRAC assay, as
shown in Table 3.

3.4.

Colour evaluation

The colour of the pomegranate vinegar, associated with the

presence of anthocyanins was evaluated using the CIEL*a*b*
system of chromatic coordinates, an objective tool for colour
perception (Pathare, Opara, & Al-Said, 2013). Two red wine vinegars from the Greek market were used for comparison. The
results are presented in Table 4. The analytical protocol was
adopted from studies on the colour of wines (Garca-Parrilla
et al., 1998) since literature about objective measurement of
colour in vinegars is very limited. As shown in the table, pomegranate vinegar was considerably bright, almost as much as

Table 4 CIEL*a*b* chromatic coordinate values of commercial Greek red wine vinegars with regard to those of
pomegranate vinegar produced in this study.
Sample

Chromatic coordinates
L*

RWV1
RWV2
PV

a*

94.58 0.02
86.83 0.14b
85.63 0.06c
a

b*

3.59 0.02
6.14 0.56b
25.11 0.13c
a

C*

9.63 0.02
12.39 0.29b
11.49 0.37c
a

hab*

10.27 0.02
13.83 0.48b
27.62 0.19c
a

69.53 0.08a
63.66 1.65b
24.59 0.72c

All values are shown as the mean standard deviation (n = 3). In each column, different superscript letters indicate significant differences (p 0.05).
values expressed in angle degrees.
RWV, red wine vinegar; PV, pomegranate vinegar; C* = (a*2 + b*2 ) ; h* = tan 1 b*
a*

journal of functional foods 8 (2014) 161168

the red wine vinegars; the L* values of the three products were
very close to the upper limit of white. Pomegranate vinegar had
a much higher positive a* value corresponding to a higher intensity of the red colour. No differentiation between the examined vinegars could be found with respect to b* value that
indicates the presence of orange-yellow constituents. On the
other hand, interesting differences were found after calculation of the chroma (C*) and the hue angle (h*) values. In particular, pomegranate vinegar had much more saturated colour
than the red wine vinegars on the basis of its higher C* values.
In addition, its colour was evaluated as the closest to redorange (h* ~ 25) while those of the commercial red wine
products were characterized as closer to orange-yellow
(h* = 6370).

4.

Conclusions

The results of this study pointed out the potential of pomegranate vinegar as functional condiment on the basis of its composition and content in phenolic constituents, moderate
antioxidant activity and attractive red colour together with the
typical quality requirements (acetic acid and residual ethanol
content) for commercialization.

Acknowledgements
The authors are indebted to P. Drogoudi (Pomology Institute,
Imathia, Greece) for providing sampling allowance and guidance on genotype selection as well as to V. de Freitas (Department of Chemistry, Univ. of Porto, Portugal) for LC-MS analysis.

Appendix: Supplementary material

Supplementary data to this article can be found online at
doi:10.1016/j.jff.2014.03.015

REFERENCES

Ameyapoh, Y., Leveau, J. Y., Karou, S. D., Bouix, M., Sossou, S. K., &
De Souza, C. (2010). Vinegar production from Togolese variety
Mangovi of mango Mangifera indica Linn (Anacardiaceae).
Pakistan Journal of Biological Sciences, 13, 132137.
Andlauer, W., Stumpf, C., & Furst, P. (2000). Influence of the
acetification process on phenolic compounds. Journal of
Agricultural and Food Chemistry, 48, 35333536.
Association of Official Analytical Chemists (1998). Official method
of analysis, (a) no. 932.14, (b) no. 942.15, and (c) no. 930.35J, K.
(16th ed.). Gaithersburg, USA.
Blanco-Gomis, D., Gutierrez-Alvarez, M. D., Mangas-Alonso, J. J., &
Noval-Vallina, A. (1988). Determination of sugar and alcohols
in apple juice and cider by high performance liquid
chromatography. Chromatographia, 25, 701706.
Chen, X., Zhang, G., Mao, F., Zhang, Q., Li, Y., & He, Y. (2012).
Method for preparing pomegranate fruit vinegar. International
Patent CN102618430.

167

de Ory, I., Romero, L. E., & Cantero, D. (1998). Modelling the

kinetics of growth of Acetobacter aceti in discontinuous
culture: Influence of the temperature of operation. Applied
Microbiology and Biotechnology, 49, 189193.
Drogoudi, P. D., Tsipouridis, C., & Michailidis, Z. (2005). Physical
and chemical characteristics of pomegranates. Hortscience: A
Publication of the American Society for Horticultural Science, 40,
12001203.
Faria, A., & Calhau, C. (2011). The bioactivity of pomegranate:
Impact on health and disease. Critical Reviews in Food Science
and Nutrition, 51, 626634.
Fernandes, I., Faria, A., Calhau, C., de Freitas, V., & Mateus, N.
(2013). Bioavailability of anthocyanins and derivatives. Journal
of Functional Foods, doi:10.1016/j.jff.2013.05.010.
Filannino, P., Azzi, L., Cavoski, I., Vincentini, O., Rizzello, C. G.,
Gobbetti, M., & Di Cagno, R. (2013). Exploitation of the
health-promoting and sensory properties of organic
pomegranate (Punica granatum L.) juice through lactic acid
fermentation. International Journal of Food Microbiology, 163,
184192.
Fischer, U. A., Carle, R., & Kammerer, D. R. (2011). Identification
and quantification of phenolic compounds from pomegranate
(Punica granatum L.) peel, mesocarp, aril and differently
produced juices by HPLC-DADESI/MSn. Food Chemistry, 127,
807821.
Fischer, U. A., Carle, R., & Kammerer, D. R. (2013). Thermal
stability of anthocyanins and colourless phenolics in
pomegranate (Punica granatum L.) juices and model solutions.
Food Chemistry, 138, 18001809.
Garca-Parrilla, M. C., Ayala, F., Echvarri, J. F., Troncoso, A. M.,
Negueruela, A., I, & Heredia, F. J. (1998). Measurement of wine
vinegars colour: Application of the characteristic vector
method. Journal of Agricultural and Food Chemistry, 46, 4238
4241.
Gil, M. I., Toms-Barbern, F. A., Hess-Pierce, B., Holcroft, D. M., &
Kader, A. A. (2000). Antioxidant activity of pomegranate juice
and its relationship with phenolic composition and
processing. Journal of Agricultural and Food Chemistry, 48, 4581
4589.
Gonzalez, A., & De Vuyst, L. (2009). Vinegars from tropical Africa.
In L. Solieri & P. Giudici (Eds.), Vinegars of the world (pp. 209
222). Milan: Springer-Verlag.
Grewal, H. S., Tewari, H. K., & Kalra, K. L. (1988). Biological Waste,
26, 914.
Hidalgo, C., Mateo, E., Cerezo, A. B., Torija, M. J., & Mas, A. (2010).
Technological process for production of persimmon and
strawberry vinegars. International Journal of Wine Research, 2,
5561.
Ivanov, S. A., Nomura, K., Malfanov, I. L., & Ptitsyn, L. R. (2012).
Glutinoin, a novel antioxidative ellagitannin from Alnus
glutinosa cones with glutinoic acid dilactone moiety. Natural
Product Research, 26, 18061816.
Joshi, V. K., & Sharma, S. (2009). Cider vinegar: Microbiology,
technology and quality. In L. Solieri & P. Giudici (Eds.),
Vinegars of the world (pp. 197208). Milan: Springer-Verlag.
Madrigal-Carballo, S., Rodriguez, G., Krueger, C. G., Dreher, M., &
Reed, J. D. (2009). Pomegranate (Punica granatum) supplements:
Authenticity, antioxidant and polyphenol composition. Journal
of Functional Foods, 1, 324329.
Mena, P., Calani, L., DallAsta, C., Galaverna, G., Garca-Viguera, C.,
Bruni, R., Crozier, A., & Del Rio, D. (2012a). Rapid and
comprehensive evaluation of (poly)phenolic compounds in
pomegranate (Punica granatum L.) juice by UHPLC-MSn.
Molecules, 17, 1482114840.
Mena, P., Girons-Vilaplana, A., Mart, N., & Garca-Viguera, C.
(2012b). Pomegranate varietal wines: Phytochemical
composition and quality parameters. Food Chemistry, 133, 108
115.

168

journal of functional foods 8 (2014) 161168

Mena, P., Vegara, S., Mart, N., Garca-Viguera, C., Saura, D., &
Valero, M. (2013). Changes on indigenous microbiota, colour,
bioactive compounds and antioxidant activity of pasteurised
pomegranate juice. Food Chemistry, 141, 21222129.
Mousavi, Z. E., Mousavi, S. M., Razavi, S. H., Emam-Djomeh, Z., &
Kiani, H. (2011). Fermentation of pomegranate juice by
probiotic lactic acid bacteria. World Journal of Microbiology and
Biotechnology, 27, 123128.
Naziri, E., Mantzouridou, F., & Tsimidou, M. Z. (2011). Enhanced
squalene production by wild-type Saccharomyces cerevisiae
strains using safe chemical means. Journal of Agricultural and
Food Chemistry, 59, 99809989.
Nenadis, N., Kyriakoudi, A., & Tsimidou, M. Z. (2013). Impact of
alkaline or acid digestion to antioxidant activity, phenolic
content and composition of rice hull extracts. LWT Food
Science and Technology, 54, 207215.
Obn, J. M., Daz-Garca, M. C., & Castellar, M. R. (2011). Red fruit
juice quality and authenticity control by HPLC. Journal of Food
Composition and Analysis, 24, 760771.
Pathare, P. B., Opara, U. L., & Al-Said, F. A. J. (2013). Colour
measurement and analysis in fresh and processed foods: A
review. Food and Bioprocess Technology, 6, 3660.
Ribreau-Gayon, P., Glories, Y., Maujean, A., & Dubourdieu, D.
(2000). Phenolic compounds. In P. Ribreau-Gayon, Y. Glories,
A. Maujean, & D. Dubourdieu (Eds.), Handbook of enology, the
chemistry of wine, stabilization and treatments (Vol. 2, pp. 145
147, 173174, 189). Chichester: Wiley.
Rubio-Fernndez, H., Salvador, M. D., & Fregapane, G. (2004).
Influence of fermentation oxygen partial pressure on
semicontinuous acetification for wine vinegar production.
European Food Research and Technology, 219, 393397.
Sakanaka, S., & Ishihara, Y. (2008). Comparison of antioxidant
properties of persimmon vinegar and some other commercial
vinegars in radical-scavenging assays and on lipid oxidation
in tuna homogenates. Food Chemistry, 107, 739744.
Silva, M. E., Torres Neto, A. B., Silva, W. B., Silva, F. L. H., &
Swarnakar, R. (2007). Cashew wine vinegar production:
Alcoholic and acetic fermentation. Brazilian Journal of Chemical
Engineering, 24, 163169.

Somers, T. C., & Verette, E. (1988). Phenolic composition of

natural wine types. In H. F. Linskens & J. F. Jackson (Eds.), Wine
analysis, modern methods of plant analysis (Vol. 6, pp. 219257).
Berlin: Springer-Verlag.
Su, M. S., & Chien, P. J. (2007). Antioxidant activity, anthocyanins,
and phenolics of rabbiteye blueberry (Vaccinium ashei) fluid
products as affected by fermentation. Food Chemistry, 104, 182
187.
Ubeda, C., Callejn, R. M., Hidalgo, C., Torija, M. J., Troncoso, A. M.,
& Morales, M. L. (2012). Employment of different processes for
the production of strawberry vinegars: Effects on antioxidant
activity, total phenols and monomeric anthocyanins. LWT
Food Science and Technology, 27, 313321.
Wang, B., Gao, H., Cheng, N., & Deng, J. (2012). Production
methods for pomegranate fruit vinegar and pomegranate
vinegar beverage. International Patent CN102604812.
Wu, D., Ma, X., & Tian, W. (2013). Pomegranate husk extract,
punicalagin and ellagic acid inhibit fatty acid synthase and
adipogenesis of 3T3-L1 adipocyte. Journal of Functional Foods, 5,
633641.
Yae, M. J., Lee, G.-H., Nam, K. H., Jang, S.-Y., Woo, S. M., & Jeong, Y.
J. (2007). Establishment of quality control standardization for
pomegranate vinegar. Journal of the Korean Society of Food
Science and Nutrition, 36, 14251430.
Yao, W. (2012). Pomegranate vinegar. International Patent
CN102676362.
Ye, G., Peng, H., Fan, M., & Huang, C.-G. (2007). Ellagic acid
derivatives from the stem bark of Dipentodon sinicus. Chemistry
of Natural Compounds, 43, 125127.
Yu, X., Yu, Z., & Yu, Z. (2012). Preparation method of pomegranate
fruit vinegar. International Patent CN102776113.
Zhou, Z. (2012). Pomegranate fruit vinegar drink and production
process thereof. International Patent CN102551163.
Zhuang, H., Du, J., & Wang, Y. (2011). Antioxidant capacity
changes of 3 cultivar Chinese pomegranate (Punica granatum
L.) juices and corresponding wines. Journal of Food Science, 76,
606611.