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Effect of Fermentation of Pomegranate

Juice by Lactobacillus plantarum
and Lactobacillus acidophilus on the
Antioxidant Activity and Metabolism
of Sugars, Organic Acids and Phenolic
Compounds
a a
Zeinab E. Mousavi , Seyed Mohammad Mousavi , Seyed Hadi
a a a
Razavi , Mehri Hadinejad , Zahra Emam-Djomeh & Mojgan
a
Mirzapour
a
Laboratory of Bioprocess Engineering, Department of Food Science
and Technology, University of Tehran, Tehran, Iran

To cite this article: Zeinab E. Mousavi , Seyed Mohammad Mousavi , Seyed Hadi Razavi , Mehri
Hadinejad , Zahra Emam-Djomeh & Mojgan Mirzapour (2013): Effect of Fermentation of Pomegranate
Juice by Lactobacillus plantarum and Lactobacillus acidophilus on the Antioxidant Activity and
Metabolism of Sugars, Organic Acids and Phenolic Compounds, Food Biotechnology, 27:1, 1-13

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 Food Biotechnology, 27:113, 2013
Copyright Taylor & Francis Group, LLC
ISSN: 0890-5436 print / 1532-4249 online
DOI: 10.1080/08905436.2012.724037

Effect of Fermentation of
Pomegranate Juice by
Lactobacillus plantarum and
Lactobacillus acidophilus on
Downloaded by [Zeinab Mousavi] at 14:21 08 February 2013

the Antioxidant Activity and

Metabolism of Sugars, Organic
Acids and Phenolic
Compounds
Zeinab E. Mousavi, Seyed Mohammad Mousavi,
Seyed Hadi Razavi, Mehri Hadinejad, Zahra Emam-Djomeh,
and Mojgan Mirzapour
Laboratory of Bioprocess Engineering, Department of Food Science and Technology,
University of Tehran, Tehran, Iran
The effect of lactic acid bacterial fermentation on sugars, organic acids, bio-transforma-
tion of phenolic compounds (anthocyanins and ellagic acid), and antioxidant activity
was investigated in pomegranate juice. L. plantarum and L. acidophilus were used
as probiotic starter organisms. Both bacteria were able to grow in the juice and their
viable cells reached to 3.9108 CFU/mL after 72 h of fermentation. Fructose and glu-
cose of the juice were significantly consumed by both probiotic starter cultures, and
L. plantarum utilized more sugars in comparison with L. acidophilus. Glucose degra-
dation rate was higher than fructose. The concentration of citric acid, as the main acid
found in the juice, was significantly reduced by both bacteria through the first 48 h of
the process (P < 0.05). Lactic acid was detected as the most abundant acidic metabo-
lite (6.1 g/L) produced within the fermentation, especially by L. plantarum. LC/MS
analysis of different anthocyanins, revealed that these compounds (except pelargonidin
3-glucoside) were significantly decreased in the pomegranate juice after fermenta-
tion. DPPH Radical scavenging studies showed that fermentation of pomegranate
juice using selected probiotic starters increased the antioxidant activity significantly

Address correspondence to Zeinab E. Mousavi, Food Science and Technology,

Laboratory of Bioprocess Engineering, University of Tehran, Tehran, Iran; E-mail:
zeinab.moussavi@gmail.com
 2 Z. E. Mousavi et al.

(P < 0.05). In addition, L. acidophilus improved the antioxidant activity of the juice
more extensively than L. plantarum. The results of this study showed that fermentation
of pomegranate juice by probiotic bacteria would enhance the health benefits of the juice.
Key Words: pomegranate juice; fermentation; Lactobacillus; anthocyanins; antioxidant
activity

INTRODUCTION
Pomegranate juice has been reported to possess numerous health benefits
which are attributed to a wide range of phenolic compounds in the juice includ-
Downloaded by [Zeinab Mousavi] at 14:21 08 February 2013

ing punicalagin isomers, ellagic acid derivatives and anthocyanins (delphini-

din, cyanidin, pelargonidin 3- and 3,5-diglucosides) (Pokorny & Schmidt, 2003).
The beneficial health proprieties are ascribed to their antioxidant activity, as
scavengers of free-radicals, protective effect against cardiovascular disease and
anti-infiammatory and anticarcinogenesis activity (Fanali et al., 2011). Some
researchers attributed the potent biological activity of these components to free
hydroxyl groups in their structural criteria (Miguel et al., 2006).
Anthocyanidins in fresh pomegranate juice are usually found glycosylated
with sugars (anthocyanins) while fermentation of the juice and sugar consump-
tion by micro-organisms would eventually result in the release of free hydroxyl
groups and relevant aglycones which may lead to the increase of bioavailability
of phenolic compounds and antioxidant activity of pomegranate juice (Kawaii
and Lansky, 2004).
Several authors have studied the fermentation of different juices using
probiotic lactic acid bacteria. Lactic acid bacteria are among the most impor-
tant probiotic microorganisms typically associated with the human gastroin-
testinal tract (Klewicka et al., 2004; Mousavi et al., 2011; Yoon et al., 2004,
2005, 2006). Since fruit juices are rich sources of sugars, they may serve as a
suitable medium to cultivate probiotic lactic acid bacteria to enhance the health
benefits of the food product. In addition, fruit juice are suitable for consumers
that are lactose intolerant and are prohibited using probiotic dairy products
(Nualkaekul and Charalampopoulos, 2011).
The aim of this study was to evaluate the growth and substrate consump-
tion of probiotic lactic acid bacteria in pomegranate juice and determine the
antioxidant activity of the juice as affected by fermentation in order to exam-
ine the possibility of producing a fermented pomegranate drink with improved
health benefits.

MATERIAL AND METHODS

Microbial Strains
Probiotic lactic acid bacteria (Lactobacilus acidophilus DSMZ 20079,
Lactobacillus plantarum DSMZ 20174) were supplied by DSMZ, Germany.
 Effect of Fermentation of Pomegranate Juice 3

All bacterial cultures were stored frozen at-20 C in MRS medium (Merck,
Germany) containing 20% glycerol. The strains were reactivated by means of
double passage on MRS when needed.

Fermentation Process
The commercial concentrated pomegranate juice (ZamZam Iran Co.,
Teheran, Iran) with 62 Brix was diluted to 20 degree Brix with distilled water
and then pasteurized for 5 min at 80 C. Selected lactic acid bacteria were grown
in the MRS broth incubated at 30 C for 24 h. For obtaining an initial cell den-
Downloaded by [Zeinab Mousavi] at 14:21 08 February 2013

sity of 107 CFU/mL in the final juice, 15 mL of the cultivated MRS broth
was centrifuged at 4000 rpm for 10 min and the biomass was washed with
saline solution twice and was introduced into the juice (150 mL). The inocu-
lated juice was then incubated at 30 C for 72 h, and sampling was taken every
24 h for microbiological and chemical analysis. Viable cells were determined by
the standard plate count method using MRS agar medium and expressed as
colony-forming units per millilitre of sample (CFU/mL).

Sugars and Organic Acid Analysis by HPLC

The concentration of fructose and glucose was determined by HPLC
(Knauer, Germany) equipped with a K-2310 refractive index (RI) detector.
Standard samples were supplied by Merck, Germany. An isocratic separa-
tion was performed using a separation column (Eurokat H 250 30 mm)
with 2.25 mM sulphuric acid, as mobile phase at flow rate of 0.4 mL/min.
Column temperature was kept at 45 C during the operation. The volume of
the injected sample for each run was 20 l. Sugar content was reported using
external standards (0.254%). All organic acid standards (Formic, lactic, cit-
ric, acetic, and propionic acid) were supplied by Merck, Germany. Organic
acid determination was carried out by HPLC apparatus (Knauer, Deutchland)
equipped with a K-2600 UV-visible detector set at 210 nm. An isocratic sep-
aration was performed using a separation column (Ultrasep ES-FS special,
250 30 mm) set at room temperature, 0.0045 N sulphuric acid as mobile
phase with injection volume of 20 L and flow rate of 0.2 mL/min. Each com-
pound was quantified by comparing its peak area against the standard curve
obtained specifically for the reference solutions containing that compound.
To obtain the standard curves, five different concentrations of formic acid
(0.0050.04% w/v), lactic acid (0.0150.12% w/v), citric acid (0.252% w/v),
acetic acid (0.00150.012% w/v), and propionic acid (0.01250.1% w/v) were
injected.
All the samples were filtered through a Nylon syringe filter (Whatman,
0.2 m, Germany) before injection. Samples were analysed in triplicate
and the results are represented as the average of these three independent
measurements.
 4 Z. E. Mousavi et al.

Determination of Anthocyanins and Ellagic Acid Using LC-MS

The samples were filtered through a 0.45 m PTFE filter (Aijiren, Zhejiang
Aijiren Technology Co., Ltd., Zhejiang China) into testing vials. The samples
were analyzed by an LC-MS instrument using a Waters 2690 HPLC system
equipped with a Micromass triple quadruple Quattro-Ultima mass spectrom-
eter in series, consisting of a HPLC quaternary pump, an auto sampler, and
a vacuum degasser (Waters, Milford, Mass., USA). The system was controlled
by Micromass-Mass Lynx software (version 3.5). Chromatographic separations
were carried out on a reverse phase XTerra MS C18 column (150 4.6 mm;
particle size 5 m) with a guard column containing the same stationary phase
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(Safeguard holder 5001-CS) was used. Mobile phase consisted of solvent A

(2.5%, v/v solution of acetic acid in water) and solvent B (2.5%, v/v solution
of acetic acid in methanol) at a flow rate of 0.3 mL/min on a linear gradient
mode. For determination of ellagic acid, pure deionised water and methanol
were used as solvent A and B respectively. The solvent linear gradient started
with 90% (A), 5 min 90% (A), 7 min 30% (A), 13 min 30% (A), 15 min 90%
(A), 18 min 90% (A). The solute was inserted into the mass spectrometer using
electro spray ionization (ESI) probe in the positive mode for anthocyanins and
negative mode for ellagic acid. The high selectivity identity of the compound
was obtained using the multiple reaction monitoring (MRM) method accord-
ing to their mother and daughter ions. The mother ion (precursor ion) was
fragmented by argon using the same collision energy. The specific LC-MS con-
ditions for each of these analytes were for anthocyanins: ESI capillary voltage
(kV) = 3.5, source temperature ( C) = 120, desolvation temperature ( C) = 200,
cone gas flow (l/h) = 40, desolvation gas flow (l/h) = 425, multiplier (V) = 525;
and for ellagic acid, ESI capillary voltage (kV) = 3.0, source temperature ( C) =
120, desolvation temperature ( C) = 220, cone gas flow (l/h) = 40, desolvation
gas flow (L/h) = 425, multiplier (V) = 525.
Each compound was quantified by comparing its peak area against the
standard curve obtained specifically for the reference solutions containing
that compound. To obtain the standard curves, five different concentrations
of ellagic acid (1 10 g/100 L) and five different concentrations (1 40
g/100 L) of each anthocyanin type, including cyanidin 3-glucoside (Cyan.
3-G), cyanidin 3,5-diglucosides (Cyan. 3,5-DG), delphinidin 3-glucoside (Del.
3-G), pelargonidin 3-glucoside (Pelar. 3-G), pelargonidin 3,5-diglucosides (Pelar.
3,5-DG) were injected.

Free Radical-Linked Antioxidant Assay

The capacity to scavenge the 2, 2-diphenyl picryl hydrazyl (DPPH, Sigma
Co., USA) free radical was monitored according to the method of Hatano et al.
(1989). A volume of 0.3 mL of various concentrations of filtered juice samples
were mixed with 2.7 mL of methanolized solution of DPPH radical (6105
 Effect of Fermentation of Pomegranate Juice 5

mol/L). The mixtures were shaken vigorously and kept in the dark until sta-
ble absorption values were obtained. The reduction of the DPPH radicals was
measured by continuously monitoring the decrease of absorption at 517 nm by
spectrophotometer equipment (CEILE CE 2502, 2000 series, England). DPPH
scavenging effect was calculated using the following equation:
 
ADPPH As
% Scavenging effect = 100 (1)
ADPPH

where As is the absorbance of the solution when the sample has been added
Downloaded by [Zeinab Mousavi] at 14:21 08 February 2013

at a particular level and ADPPH is the absorbance of the DPPH solution. The
concentration providing 50% antioxidant activity (EC50 ) was obtained through
interpolation of quadratic polynomial equation analysis (Matlab 7.11.0 r2010b,
Mathworks, USA). EC50 (mg/mL) of the non-fermented juice, fermented juice
by L. plantarum and fermented juice by L. acidophilus, was calculated accord-
ing to Eqs., (2), (3), and (4), respectively:

y = 28.87x2 + 93.71x 7.055 (2)

y = 73.18x2 + 157.3x 10.81 (3)

y = 98.06x2 + 199.2x 13.07 (4)

Statistical Analysis
Samples were analyzed in triplicate and analysis of variance (ANOVA)
was performed using SAS version 9.1. Mean analysis using Duncans multiple
range test at significance level of p < 0.05 was performed if needed.

RESULTS AND DISCUSSION

Bacterial Population Growth

The kinetics of cell growth of L. plantarum and L. acidophilus during 96 h
under microaerophilic cultivation conditions at 30 C was studied. As displayed
in Figs. 1 and 2, a slow drop in the microbial population was observed for
both strains during the first 24 h of fermentation. A potential cause for this
slight decline in the cell number was pH differences between preculture and
the fermentation medium. The pH of MRS broth, as the preculture medium,
was 5.6 while the initial pH of the pomegranate juice was considerably lower
at about 3.09. This pH difference put the bacteria in a stressful condition and
decreased the microbial growth at the early stage of the fermentation process
(lag phase). Similar observations have been reported by Yez et al. (2008)
 6 Z. E. Mousavi et al.

4E+08 9
8

Concentration (g L1)
Microbial population
3E+08 7
6
(CFU/ mL1)

5
2E+08
4

Growth 3
1E+08 2
Fructose
1
Glucose
0E+00 0
Downloaded by [Zeinab Mousavi] at 14:21 08 February 2013

0 24 48 72
Time (h)

Figure 1: Kinetics of growth and sugar consumption of L. plantarum and during 72 h of

fermentation of pomegranate juice with initial Brix = 20 and pH = 3.1 at 30 C.

4E+08 9
8

Concentration (g L1)
7
Microbial population

3E+08
6
(CFU mL1)

5
2E+08
4

Growth 3
1E+08 2
Fructose
1
Glucose
0E+00 0
0 24 48 72
Time (h)

Figure 2: Kinetics of growth and sugar consumption by L. acidophilus during 72 h of fermenta-

tion of pomegranate juice with initial Brix = 20 and pH = 3.1 at 30 C.

showing that a low pH of the medium may decrease the growth rate, resulting
in an extended length of the lag phase. Upon transition from the lag phase
to logarithmic growth stage (from 24 to 48 h), the microbial population of
L. plantarum and L. acidophilus increased to maximum levels of 3.07108 and
3.9108 CFU/mL, respectively. Further increase in the fermentation process
time (from 48 h to 72 h) did not change the viable cells number of L. plan-
tarum and L. acidophilus significantly (p > 0.05). The results revealed that the
selected strains were able to grow in pomegranate juice successfully. Bialonska
et al. (2010) stated that the addition of a byproduct of pomegranate juice can
stimulate the growth of Bifidobacterium breve and Bifidobacterium infantis in
the human gut.
 Effect of Fermentation of Pomegranate Juice 7

Kinetics of Sugars Consumption

Changes in the concentration of glucose and fructose in the pomegranate
juice fermented with L. plantarum and L. acidophilus is displayed in Figs. 1
and 2. Regardless of the type of probiotic starter organism, the concentration
of both sugars decreased as the fermentation time increased to 72 h. In com-
parison with fructose, the rate of glucose consumption was significantly higher
(p 0.05), during the exponential growth phase but the degradation rate of
both sugars slowed down after this stage. L. plantarum reduced the level of
glucose and fructose from 7.5 and 6.29 g/L, to 5.49 and 5.27 g/L, respectively,
after 48 h of fermentation. Compared to L. plantarum, the level of both sugars
Downloaded by [Zeinab Mousavi] at 14:21 08 February 2013

consumed by L. acidophilus was significantly lower (P < 0.05). This observa-

tion indicated that L. plantarum utilized these substrates more efficiently than
L. acidophilus. The reports of other authors also proved that metabolism of
carbohydrates by Lactobacillus varies from strain to strain and depends on the
substrate and also on the fermentation time (Hou et al., 2000).

Kinetics of Organic Acid Production

Metabolism of organic acids in the pomegranate juice during the fermen-
tation using L. plantarum and L. acidophilus are shown in Figs. 3 and 4.
During the first 24 h of fermentation period (lag phase) the metabolism of
nutrient in the juice was relatively weak. Therefore, insignificant change in
the organic acids level was observed. However, with increasing fermentation
time to 48 h, the logarithmic phase of growth commenced; therefore, signifi-
cant variations in the amount of organic acids in the pomegranate juice were
detected.
In accordance with the results reported by Tezcan et al. (2009), citric acid
was the main organic acid in pomegranate juice measured by HPLC. The initial

7 70
Organic acid concentration

Citric acid concentration

6 60

5 Lactic acid 50
Formic acid
( g L1)

(g L1)

4 40
Acetic acid
3 30
Pripionic acid
2 20
Citric acid
1 10

0 0
0 24 48 72
Time (h)

Figure 3: Kinetic of consumption and production of different organic acids by L. plantarum

during 72 h of fermentation of pomegranate juice with initial Brix = 20 and pH = 3.1 at 30 C.
 8 Z. E. Mousavi et al.

6 70
Organic acid concentration

Citric acid concentration

5 60

Lactic acid 50
4
(g L1)

(g L1)
Formic acid 40
3
Acetic acid 30
2
Pripionic acid 20
1 Citric acid 10

0 0
0 24 48 72
Downloaded by [Zeinab Mousavi] at 14:21 08 February 2013

Time (h)

Figure 4: Kinetic of consumption and production of different organic acids by L. acidophilus

during 72 h of fermentation of pomegranate juice with initial Brix = 20 and pH = 3.1 at 30 C.

concentration of citric acid in the juice was 60 g/L, but its level decreased sig-
nificantly (P < 0.05) by L. acidophilus and L. plantarum to 15.5 g/L and 13 g/L,
respectively, within 48 h of fermentation. The results revealed that the selected
probiotic lactic acid bacteria were strongly capable of metabolizing citric acid
as a carbon source accessible at high level in the juice.
Other organic acids, including lactic, acetic, formic, and propionic, were
only detected after fermentation. Results from previous studies by other inves-
tigators revealed that in the presence of carbohydrate source, lactic acid bac-
teria produced short fatty acids such as as lactic, acetic, propionic, and formic
acid, possessing anti-microbial and nutritional benefits (Davidson et al., 2000).
During the fermentation of pomegranate juice, the production of lactic acid
was more evident compared with other organic acids. Lactic acid level reached
its highest level of 6.1 g/L and 4.9g/L by L. plantarum and L. acidophilus,
respectively, after 48 h fermentation. In addition, the amount of lactic acid
produced by L. plantarum was significantly (P < 0.05) higher than that synthe-
sized by L. acidophilus. In other similar studies, this organic acid has also been
reported as the main short fatty acid produced through lactic acid fermentation
(Bergqvist et al., 2005; Kun et al., 2008).
After lactic acid, formic and propionic acids were the next main organic
acid metabolites produced by both lactic acid bacteria in terms of quantity
(1.1 and 0.88 g/L, respectively). Results indicated that the selected probiotic
strains produced more considerable amounts of propionic acid rather than
acetic acid. Finally, acetic acid constituted the least proportion of the pro-
duced organic acids (0.35 g/L) by both L. plantarum and L. acidophilus. Other
investigators showed in their research that L. plantarum 299v was capable of
producing acetic and propionic acid that induce mucin secretion in intestinal
epithelial cells and has inhibitory effect on a wide range of harmful micro-
organisms such as Salmonella, Escherichia coli, Clostridium and Helicobacter
 Effect of Fermentation of Pomegranate Juice 9

(Mack et al., 1999; Saarela et al., 2000). Other studies showed that acetate
produced by probiotics increased cholesterol synthesis after being absorbed in
the body while propionate inhibited cholesterol synthesis. For health benefit
purposes, a lower acetate: propionate ratio is preferred (Wong et al., 2006).

Changes in Glycosylated Anthocyanins and Ellagic

Acid Concentration
Anthocyanins and ellagic acids have been recognized as the main phenolic
compounds present in pomegranate juice with the ability to scavenge free radi-
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cals (Gil et al., 2000). Therefore, in this study, the concentration change of these
groups of phenolic compounds during fermentation was studied. As displayed
in the Fig. 5, a significant decrease (P < 0.05) in evaluated phenolic com-
pounds, except pelargonidin, 3-glucoside was detected in the juice fermented by
both strains compared with the non-fermented juice. The results indicated that
these compounds were metabolized by these starter organisms. Other studies
reported that some bacteria possess an enzymatic system capable of metabo-
lizing anthocyanins. As an instance, it is shown that species of Bifidobacteria
and Lactobacillus had -glucosidase activity and participated in the hydrol-
ysis of plant -glycosides (vila et al., 2009). Reduction of the concentration
of glycosylated anthocyanins results in increasing their aglycone form (antho-
cyanidin). Aglycone form of anthocyanins does not contain sugar moieties. The
sugar moiety could be metabolized and consumed as carbohydrate source by
the selected probiotic lactic acid bacteria resulting in the increase of aglycone
form during the fermentation.

Non-fermented juice a
Concentration (g mL1)

50
Fermented by L. plantarum
40 a
Fermented by L. acidophilus
30 b a b
b c
20
b b a
10
a b. b. b b
0
Del. 3G E. A. Cyan. 3G Cyan, 3,5 Pelarg. 3, 5
DG DG
Phenolic compounds

Figure 5: Changes in anthocyanins and ellagic acid concentration in fermented pomegranate

Juice after 72 h using L. plantarum and L. acidophilus (Del. 3-G: Delphinidine 3-Glucoside;
E.A. : Ellagic. acid; Cyan. 3,5-DG: cyanidin 3,5-diglucosides; Pelar. 3,5-DG: pelargonidin 3,5-
diglucosides; N.F.J.: Non fermented juice; F.J.: fermented juice).The level of each phenolic
compound in the juices fermented by both bacteria and nonfermented juice was compared
according to according to Duncans multiple test range. Values with different superscripts (a-c)
are significantly different (p < 0.05).
 10 Z. E. Mousavi et al.

Changes in the Radical Scavenging Properties of Pomegranate

Juice During Fermentation
DPPH assay has been widely used to determine the free radical scavenging
activity of various plant extracts (Hatano et al., 1989). Antioxidant molecules
in the sample scavenge the free radical DPPH and the color from the DPPH
assay solution becomes light yellow resulting in a decrease of the absorbance
at 517 nm. As shown in Fig. 6, the DPPH scavenging effect of pomegranate
juice, regardless of the starter organism employed for fermentation, increased
as the concentration increased up to 1 mg/mL. Further increase in the dosage
level did not significantly increase the DPPH-scavenging effect. By compar-
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ing the three graphs in Fig. 6, it is perceived that EC50 of fermented juice
by both strains was significantly lower (p < 0.05) than that of the nonfer-
mented juice, and was 0.5 mg/mL and 0.41 mg/mL, for L. plantarum and
L. acidophilus, respectively. In addition, statistical analysis revealed that the
samples fermented by L. acidophilus, had a significantly higher antioxidant
activity compared to L. plantarum (analysed at concentration of 1 mg/mL).
It can be concluded from these results that lactic acid fermentation
performed by both probiotic strains improved the antioxidant activity of
pomegranate juice considerably. According to the results described in previous
sections, the concentration of anthocyanin compounds, as strong antioxidant
ingredients in pomegranate juice, was decreased after fermentation. Some
probable explanation can be provided for the contrast detected between the
decreased concentration of phenolics and the increased antioxidant activity
after fermentation. The majority of the anthocyanins studied were conjugated
through hydroxyl groups with sugars and glycosides. The glucose molecule

100
90
DPPH-scavenging effect (%)

a a a
80
70 b b b
60 a
c c c
50
a
40
Non- fermented juice
30 b Fermented by L. acidophilus
20
a a Fermented by L. plantarum
10
0 b
0.1 0.5 1 2 3
Concentration (mg mL1)

Figure 6: DPPH radical-scavenging effects of the pomegranate juice fermented by L. plan-

tarum and L. acidophilus. The comparisons were between values of the same concentration
and were performed according to Duncans multiple test range. Values with different super-
scripts (a-c) are significantly different (p < 0.05).
 Effect of Fermentation of Pomegranate Juice 11

available in the anthocyanin structure was likely depleted by bacteria as an

energy source, resulting in the production of related aglycones with higher
radical scavenging effect, which led to some metabolites with antioxidant
activity being produced during the fermentation. Laparra and Sanz (2010)
reported that metabolized conjugated polyphenols by gut microbiota were
better absorbed in the intestine and their bioavailability was improved signifi-
cantly. A research carried on by Lee et al. (2008) showed that some organisms
were capable of producing -galactosidase during the black bean fermentation,
catalysing the release of phenolic compounds from the bounded sugar. This
process led to an increase in the antioxidant activity of black bean after fer-
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mentation. Other researchers found same justification for the increase in the
antioxidant activity of fermented samples by some micro-organisms (6ila et al.,
2009; Pokorny and Schmidt, 2003).
However, other metabolites produced by the selected strains through fer-
mentation may also have considerable impact on the antioxidant activity of
pomegranate juice (Kim et al., 2011). Therefore, in future studies the iden-
tification of aglycones and other key compounds in the juice and studies on
their metabolic pathways appears to be crucial for a better analysis of the
antioxidant activity in fermented pomegranate juice.

CONCLUSION
The results from this investigation showed that L. plantarum and L. aci-
dophilus are capable of growing in pomegranate juice and metabolizing sugars,
organic acids, and phenolic compounds. The production of lactic acid, followed
by formic and acetic acids as the main organic acids produced, was much higher
by L. plantarum. Fructose and glucose were utilized as energy source by both
bacteria and a higher rate of consumption was detected for glucose. The free
radical scavenging effect of the juice was increased through fermentation; how-
ever, the enhanced effect of fermentation varied with the employed bacteria
since L. acidophilus exhibited higher improvement in the antioxidant activity
of the juice. Improvements in the radical scavenging effect can be related to the
increase in the free form of phenolic compounds and the production of other by-
products through fermentation. These findings highlight the beneficial effect
of pomegranate juice fermentation by the incorporation of probiotic bacteria
able to produce organic acids and improve the antioxidant activity of the juice.
Further research is needed to investigate other key compounds produced dur-
ing fermentation such as aglycones and their effect on the antioxidant activity
of the fermented juice.

ACKNOWLEDGMENTS
The authors would like to appreciate ZamZam Iran Co. (Tehran, Iran) for
the beneficial scientific and financial support granted to this research. We are
 12 Z. E. Mousavi et al.

also thankful to Mr. H Kiani for his helps for discussions and analysis to the
results of this research and Dr. Ahmadkhani in Razi Research Institute for his
assistance in LC/Mass analysis.

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