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Food Control 78 (2017) 132e137

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Food fraud detection in commercial pomegranate molasses syrups by


UVeVIS spectroscopy, ATR-FTIR spectroscopy and HPLC methods
Nada El Darra a, *, 1, Hiba N. Rajha b, 1, Fatima Saleh a, Rami Al-Oweini c,
Richard G. Maroun b, Nicolas Louka b
a
Beirut Arab University, Faculty of Heath Sciences, Tarik El Jedidah, Beirut, P.O.Box: 115020, Riad EL Solh, 1107 2809, Lebanon
b
Unit
e de Recherche Technologies et Valorisation Agro-alimentaire, Centre d’Analyses et de Recherche, Facult e des Sciences, Universit
e Saint-Joseph, B.P. 11-
514, Riad El Solh, Beirut 1107 2050, Lebanon
c
Department of Chemistry, Faculty of Science, Beirut Arab University, P.O. Box 11 50 20, Riad El Solh, 1107 2809, Beirut, Lebanon

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

Article history: Food fraud is a serious ethical and economic problem affecting the food industry everywhere. As
Received 20 September 2016 pomegranate molasses’ consumption continues to increase due to its unique taste and antioxidant ac-
Received in revised form tivity, its adulteration is taking several forms. Customers are deluded by the “100% pomegranate content”
13 February 2017
label present on most of the commercial pomegranate molasses. The purpose of this study was to detect,
Accepted 16 February 2017
for the first time, the adulteration of commercial pomegranate molasses with date molasses. To distin-
guish pomegranate molasses from the date syrup, we determined different parameters that could signal
adulteration, such as total acidity content, polyphenol yield, anthocyanins concentration, colour intensity
Keywords:
Fraud
and antiradical activity. UVeVIS spectroscopy was used as a screening method to detect fraud and High
Pomegranate molasses Performance Liquid Chromatography (HPLC) was conducted for a quantitative analysis. Additionally,
Date syrup Attenuated Total Reflectance Fourier-Transform Infrared (ATR-FTIR) spectroscopic analysis was con-
Polyphenols ducted to compare the resulting spectra of commercial pomegranate molasses, natural pomegranate
ATR-FTIR molasses and date syrup. Our findings support the hypothesis that some of the commercialized pome-
granate molasses in the Middle East area are adulterated with cheaper date syrup.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction antitumoral (Seidi, Jahanban-Esfahlan, Abasi, & Abbasi, 2016),


antimicrobial (Duman, Ozgen, Dayisoylu, Erbil, & Durgac, 2009),
Pomegranate Molasses (PM) is a thick syrup obtained from anti-inflammatory (Lee, Chen, Liang, & Wang, 2010) and antidia-
pomegranate (Punica granatum L.) juice concentration. It is a betic properties (Xu, Zhu, Kim, Yamahara, & Li, 2009). PM was also
slightly astringent, sweet-sour condiment that gained a crucial shown to reduce the risk of cardiovascular diseases. Being an
interest as a medicinal and nutritional product (Johanningsmeier & expensive functional food, cheaper fruit juices are deceptively
Harris, 2011). PM has many health-related beneficial effects, espe- added to the pomegranate molasses to reduce its production cost.
cially in the prevention and treatment of several illnesses. They The added juices are not declared in the labeling, which subjects
decelerate the progress of chronic diseases due to their strong allergic end consumers to dangerous risks (Boggia, Casolino,
antioxidant (Cam, Hisil, & Durmaz, 2009; Faria & Calhau, 2010; Hysenaj, Oliveri, & Zunin, 2013). Different types of juices were
Viuda-Martos, Ferna ndez-Lo pez, & Pe 
rez-Alvarez, 2010), used to adulterate pomegranate juices such as grapes, apples,
cherry, sour cherry and strawberry (Zhang et al., 2009). As far as the
commercial pomegranate molasses were concerned, all the com-
* Corresponding author. Department of Nutrition & Dietetics, Faculty of Health panies claimed on the package that none of them contained un-
Sciences, P.O. Box: 11 5020, Beirut, Lebanon. declared added juices. The detection of pomegranate juice
E-mail addresses: n.aldarra@bau.edu.lb, healthsciences@bau.edu.lb (N. El Darra), adulteration was established by different advanced analytical ap-
hiba.rajha@net.usj.edu.lb (H.N. Rajha), f.saleh@bau.edu.lb (F. Saleh), roweini@bau.
proaches like FITR spectroscopy (Vardin, Tay, Ozen, & Mauer, 2008).
edu.lb (R. Al-Oweini), richard.maroun@usj.edu.lb (R.G. Maroun), nicolas.louka@
usj.edu.lb (N. Louka). Zhang et al. (2009) authenticated pomegranate juice by developing
URL: http://www.bau.edu.lb an algorithm to assess the chemical profile of the juice including
1
Both Nada El Darra and Hiba N. Rajha equally contributed to this work.

http://dx.doi.org/10.1016/j.foodcont.2017.02.043
0956-7135/© 2017 Elsevier Ltd. All rights reserved.
N. El Darra et al. / Food Control 78 (2017) 132e137 133

phenolic diversity, sugar concentration and organic acids content. 2.5. Anthocyanin concentration
High performance liquid chromatography coupled with Mass
Spectrometry (HPLC-MS) was also used to discern authentic Anthocyanin concentration was conducted based on their
pomegranate juices from the adulterated ones with grapes and discoloration by SO2 (Ribe reau-Gayon, Glories, Maujean, &
apples through the detection of the organic acids profiles (citric, Dubourdieu, 2006). 1 mL of each sample was added to 20 mL of
malic, quinic and tartaric) (Ehling & Cole, 2011). The main aim of HCl (2%) and 1 mL of ethanol (0.1% HCl). The mixture was split in
this study was to test the adulteration of commercial pomegranate two tubes containing 4 mL of distilled water and 4 mL of sodium
molasses with date molasses by developing a rapid, easy, qualita- bisulfite (15%) respectively. Bleaching occurs instantaneously in the
tive and affordable technique. In order to distinguish pomegranate tube containing SO2. The optical density was measured (after
molasses from the date syrup, different parameters were 20 min) at 520 nm against distilled water. Anthocyanin concen-
compared: total acidity content, polyphenol and anthocyanins tration (A) (mg/L) was calculated as follows:
concentrations, colour intensity and antiradical activity. UVeVIS mg   
spectroscopy was used to analyze the different samples of com- A ¼ 875  DOtube 1 in water  DOtube 2 in bisulfite
mercial pomegranate molasses suspected of adulteration by date L
molasses. This method is a first screening step, qualitative and
with 875 being the slope of the calibration curve obtained from
quantitative tool to detect the presence of date syrup in commercial
malvidin-3-glucoside.
pomegranate molasses. Each analyzed sample was described as a
vector of absorbance proposed a fingerprint. A second-step com-
parison between UVeVIS spectroscopy and High Performance 2.6. UVeVIS spectroscopy
Liquid Chromatography coupled with Diode Array Detector (HPLC-
DAD) was conducted, in order to confirm the validity of the Absorption spectra in the ultraviolet and visible regions were
developed technique. Moreover, ATR-FTIR spectroscopy, which is obtained in the range of 190e1100 nm using a UVeVIS spectro-
both an easy and a nondestructive technique, was utilized as a photometer V-530 (Jasco Inc.) with 1 nm resolution. The cells were
qualitative control to distinguish between the different molecular rectangular quartz cuvettes with 1 cm path length. MilliQ water
information of the pomegranate molasses liquid samples’ func- was used as a blank. For each sample the spectrum was collected at
tional groups. To our knowledge, no study has yet shown that date room temperature in duplicate and the results were averaged
syrup is a potential raw material to adulterate commercial pome- (Boggia et al., 2013).
granate molasses. Moreover, this study suggests that the biological
activity of a product, such as its radical scavenging capacity, along
with a simple combination of spectrophotometric analyses (colour 2.7. HPLC-DAD analysis
intensity, anthocyanin content and UVeVIS screening) can prove
the authenticity of pomegranate molasses. Polyphenol analyses were performed using a Jasco HPLC system
(PV-2089) equipped with an autosampler, an L-2130 pump, a Jet-
stream column oven and an L-2450 diode array detector. The sep-
2. Materials and methods aration was carried out with a Column C18, 25  0.46 mm. The
diode array detector was set at an acquisition range of
2.1. Samples preparation 200e600 nm. Trans-cinnamic acid, caffeic acid, hydroxybenzoic
acid, chlorogenic acid, catechin, rutin, quercetin, protocatechin,
Two natural samples of authentic pomegranate molasses (NPM1 gallic acid, epigallocatechin, kaempferol, catechin gallate and
and NPM 2) (Punica granatum L.), 3 commercial pomegranate myrecetin standards were used for identification and quantification
molasses (CPM1, CPM2 and CPM3) and 1 natural date syrup (DS) purposes with HPLC-DAD, respectively. All these phenolic stan-
samples were studied. A 50 times dilution was conducted (w/v) dards were purchased from Sigma Laboratories.
prior to analyses.

2.7.1. HPLC protocol for anthocyanin detection


2.2. Chemicals The mobile phase consisted of 5% (v/v) formic acid in water
(eluent A) and of formic acid, water and methanol (10/10/80, v/v/v;
All chemicals were purchased from Fluka Chemie GmbH (Buchs, eluent B). The flow rate was 0.4 mL/min and the gradient program
Switzerland) or from SigmaeAldrich (Steinheim, Germany). was optimized as follows: 10e14% B (5 min), 14e23% B (11 min),
23e35% B (5 min), 35e40% B (14 min), 40e100% B (3 min), 100% B
2.3. Total acidity measurement isocratic (3 min), 100e10% B (3 min), 10% B isocratic (4 min). The
total run time was 48 min. The injection volume of all samples was
The total acidity measurement was conducted by an acid/base 10 mL. Monitoring was performed at 520 nm (Fischer, Carle, &
(0.1 N NaOH) titration with an indicator dye (bromothymol blue Kammerer, 2011).
(4 g/L)).
2.7.2. HPLC protocol for non-anthocyanin detection
2.4. Colour intensity and total phenolic index The mobile phase consisted of 2% (v/v) acetic acid in water
(eluent A) and of 0.5% acetic acid in water and methanol (10/90, v/v;
Colour intensity (CI) was monitored through the absorbance eluent B). The flow rate was 0.4 mL/min, and the gradient program
measurement of appropriately diluted samples at 420 nm (yellow), was optimized as follows: 0e2% B (13 min), 2e5% B (5 min), 5e10%
520 nm (red), and 620 nm (blue) and was expressed as the sum of B (5 min), 10e25% B (20 min), 25e50% B (10 min), 50e100% B
the three values (Glories, 1984). The total phenolic index (TPI) was (5 min), 100% B isocratic (5 min), 100e0% B (3 min), 0% B isocratic
determined from the absorbance at 280 nm after a 100-fold dilu- (5 min). The total run time was 71 min. The injection volume for all
tion. A UVeVIS spectrophotometer V-530 (Jasco Inc.) with 1 cm samples was 15 mL. Monitoring was performed at 280 nm and
path length rectangular quartz cuvettes was used. 320 nm ((Fischer et al., 2011).
134 N. El Darra et al. / Food Control 78 (2017) 132e137

2.8. Radical scavenging activity 3.1. UVeVIS spectral profiles of natural pomegranate molasses,
commercial pomegranate molasses and date syrup
According to (Kallithraka, Mohdaly, Makris, & Kefalas, 2005), the
free radical scavenging activity was measured by the capacity of the Fig. 1 shows the UVeVIS spectral profiles examined for the
polyphenols to reduce DPPH (2,2-diphenyl-picrylhydrazyl), a stable different studied samples; natural (NPM1 and NPM2) and com-
free radical. 50 mL of extracts were added to 450 mL of Tris-HCl mercial (CPM1, CPM2 and CPM3) pomegranate molasses; that were
buffer solution (50 mM, pH 7.4) and 1.5 mL of DPPH solution compared to date syrup (DS). Authentic and commercial pome-
(0.1 mM). Absorbance at 517 nm was measured after 30 min of granate molasses as well as date syrup were characterized by a
incubation at room temperature using pure methanol as a blank. maximal absorption at 280 nm wavelength. Natural pomegranate
The inhibition percentage of the DPPH free radical is calculated as molasses’ samples showed the highest peak amplitudes with
follows: Inhibition Percentage ¼ [(absorbance of negative almost similar maximal optical density values ranging from 1.01 to
control  absorbance of sample)/absorbance of negative 1.14 for NPM1 and NPM2 respectively. On the other hand, com-
control]  100. The free radical scavenging activity was evaluated mercial pomegranate molasses and date syrup had significantly
by the decrease in the peak area of the DPPH radical which exhibits lower peak amplitudes equal to 0.41, 0.35, 0.42 and 0.34 for CMP1,
a deep purple colour with maximum absorption at 517 nm. Anti- CPM2, CPM3 and DS respectively. This dissimilarity can be attrib-
oxidant molecules can quench DPPH free radicals, provoking a uted to a different concentration and/or diversity of the absorbing
discoloration of the latter and its conversion into a colorless molecules of phenolic compounds (Boggia et al., 2013). This result
product. shows an extreme resemblance between the spectra of natural
pomegranate molasses that clearly differ from those obtained with
commercial pomegranate molasses and date syrup.
2.9. ATR-FTIR spectroscopy

The Attenuated Total Reflectance Fourier-Transform Infrared 3.2. Characterization and identification of polyphenols by HPLC
(ATR-FTIR) spectra were recorded on a Nicolet™ 4700 FTIR spec- (280 nm)
trometer equipped with a multi-bounce ZnSe crystal ATR accessory,
and OMNIC software. The spectra were collected over 32 scans in Fig. 2 shows the retention times and the corresponding peak
the 4000e650 cm1 range with a resolution of 4 cm1. The heights of the major phenolic compounds detected by HPLC. Nat-
following abbreviations were used to assign the peak intensities: ural pomegranate molasses had two major peaks with similar
w ¼ weak; m ¼ medium; s ¼ strong; sh ¼ shoulder; br ¼ broad. heights detected at identical retention times (31.412 and
68.497 min) suggesting the same diversity and quantity of the two
2.10. Statistical analysis polyphenols in NPM1 and NPM2. These Two phenolic compounds
were identified as rutin (retention time: 31.412) and gallic acid
All experiments were repeated at least three times for the (retention time: 68.497) (Fig. 2). Rutin concentrations in NPM1 and
calculation of the standard deviations. Variance analyses (ANOVA) NPM2 were 18 mg/mL; whereas those of CPM1, CPM2, CPM3 and
and Least Significant Difference test (LSD) were carried out to DS were 3 mg/mL. The highest gallic acid concentration was found
assess the significant differences between the results. STAT- in NPM1 and NPM2 with 0.31 and 0.28 mg/mL, respectively.
GRAPHICS® Centurion XV (StatPoint Technologies, Inc.) was used to Commercial pomegranate molasses CPM1, CPM2 and CPM3
conduct the statistical analysis. showed a similar gallic acid content (0.14 mg/mL) to that obtained
with date syrup (significantly lower peak heights than NPM1 and
NPM2). HPLC results showed a significant correlation with UVeVIS
3. Results and discussion spectral profile. On the other hand, three different polyphenols
with similar peak heights were exclusively detected (retention
Fraud detection through polyphenol diversity, quantity and times: 11.285, 8.95 and 7.658) in commercial pomegranate
bioactivity

Fig. 1. UVeVIS spectral profiles for the different samples NPM1, NPM2, CPM1, CPM2, Fig. 2. HPLC results (280 nm) for the different samples NPM1, NPM2, CPM1, CPM2,
CPM3, and DS. CPM3, and DS.
N. El Darra et al. / Food Control 78 (2017) 132e137 135

molasses and date syrup. Therefore, our results indicate the adul- concentration of the two natural pomegranate molasses (NPM1
teration of commercial pomegranate molasses with date syrup. The and NPM2) is around 118 and 113 mg/L respectively; whereas the
comparison of HPLC phenolic profile of a sample could therefore be commercial pomegranate molasses show lower yields of 87.5 mg/L.
suggested as a simple tool to ensure the authenticity of molasses. On the other hand, DS presents an anthocyanin concentration of
The similarity in the diversity and quantity of the phenolic com- 131.25 mg/L (Fig. 3a). A similar tendency was obtained by HPLC
pounds between commercial pomegranate molasses and date analysis (Fig. 3b). Since a similar final texture and viscosity are
syrup highlights the possible addition of date syrup to natural required for both natural and commercial molasses, a boiling
pomegranate molasses to obtain commercial pomegranate phenomenon was suspected to have occurred after mixing the
molasses. natural pomegranate molasses with date syrup to obtain the same
final characteristics of the fraudulent molasses as the natural ones.
Anthocyanins, being heat-sensitive polyphenols, are likely to be the
3.3. Comparison between the anthocyanins concentration of the
most affected with this boiling phenomenon (Sadilova, Stintzing, &
natural and commercial pomegranate molasses and the
Carle, 2006), which explains lower anthocyanin yields for CPM1,
pomegranate juice by bisulfite discoloration and HPLC
CPM2 and CPM3 as compared to NPM1, NPM2 and DS. To validate
the heat degradation process of anthocyanins, pomegranate juices
Noting that, there is universal agreement of the presence of a
were compared to pomegranate molasses that endured a boiling
highly constant group of six anthocyanins (delphinidin-3,5-
phenomenon throughout the industrial process. A mean anthocy-
diglucoside, delphinidin-3-glucoside, cyanidin-3,5-diglucoside,
anin content of 650 mg/L was obtained for pomegranate juices,
cyanidin-3-glucoside, pelargonidin-3,5-diglucoside, and pelargo-
with an HPLC surface area of 517 737 (Data not shown), as opposed
nidin-3-glucoside) characterizing pomegranate juice polyphenols;
to 113 mg/L and 130 000 respectively, for pomegranate molasses.
anthocyanin profiles can be used to detect the adulteration of the
A high correlation was observed between the anthocyanin
commercial pomegranate molasses (Zhang et al., 2009). Based on
concentration determination by the bisulfite discoloration method
the hypothesis that natural pomegranate molasses are mixed with
and that obtained by area calculation in HPLC analyses (R2 ¼ 0.99);
date syrup to prepare commercial molasses, the analysis of an-
suggesting both methods can be used to compare the authenticity
thocyanins was conducted. Fig. 3a shows the anthocyanin yield
of different samples (Fig. 3c). Similar to the anthocyanin content
obtained by the bisulfite spectrophotometric method and the
results, the highest colour intensity was obtained for date syrup 9.7
anthocyanin area obtained by HPLC (Fig. 3b). The anthocyanin
followed by natural PM 8 and then CPM around 7 (Fig. 3d). A strong
correlation was noted between anthocyanin concentration and
colour intensity. This was also shown by many previous studies
(Boulton, 2001; Preys et al., 2006).

3.4. Radical scavenging activity of natural pomegranate molasses,


commercial pomegranate molasses and date syrup

Fig. 4 shows the radical scavenging capacity of NPM1, NPM2,


CPM1, CPM2, CPM3 and DS. The highest inhibition percentage of
the DPPH. radical (25%) was obtained with date syrup; whereas the
lowest values were obtained for NPM1 and NPM2 (8.5 and 6.8%,
respectively). The high antioxidant capacity of date syrup was re-
ported in many previous studies (Al-Farsi et al., 2007; Vayalil,
2002). Commercial pomegranate molasses (CPM1, CPM2 and

Fig. 3. Anthocyanin concentration determined by bisulfite discoloration method (a),


HPLC peak areas of anthocyanins (b) determined for samples NPM1, NPM2, CPM1,
CPM2, CPM3, and DS, correlation between the spectrophotometric and HPLC methods
(c) and colour intensity (d). Different letters (a, b, c) indicate significant statistical Fig. 4. Antiradical activity (DPPH inhibition percentage) of the different samples
difference between means (p < 0.05). (For interpretation of the references to colour in NPM1, NPM2, CPM1, CPM2, CPM3, and DS (at 50 mg/mL). Different letters (a, b, c)
this figure legend, the reader is referred to the web version of this article.) indicate significant statistical difference between means (p < 0.05).
136 N. El Darra et al. / Food Control 78 (2017) 132e137

Fraud detection through acidity

3.5. Acidity measurements

Fig. 5 shows that the total acidity in citric acid (g per 100 mL) is
the highest for the commercial pomegranate molasses (CPM1,
CPM2 and CPM3), with values between 3.2 and 3.52 g per 100 mL.
Lower values were observed for NPM1, NPM2 (1.92 g per 100 mL)
and DS (0.96 g per 100 mL). This difference in acidity between
natural and commercial pomegranate molasses was suspected to
be due to citric acid addition to the commercial PM without always
declaring it on the labels. Therefore, commercial pomegranate
molasses should not be declared 100% natural. It is worth noting
that adding citric acid to commercial pomegranate molasses
adulterated with date syrup is to mask the sweet taste of date by
enhancing the sour taste.
Fraud detection through qualitative molecular vibration
identification
Fig. 5. Acidity of the different samples NPM1, NPM2, CPM1, CPM2, CPM3, and DS.
Different letters (a, b, c, d) indicate significant statistical difference between means 3.6. ATR-FTIR spectroscopic analysis
(p < 0.05).

ATR-FTIR spectra of natural pomegranate NPM1, commercial


pomegranate molasses CPM1, CPM2 and CPM3, as well as date
CPM3) represented intermediate inhibition percentages of 15.9,
syrup DS are shown in Fig. 6a and b. Particularly, Fig. 6a shows the
16.1 and 17% respectively. These observations support the possi-
spectra of the natural pomegranate in comparison with the DS
bility of date syrup addition to natural pomegranate molasses to
sample; then Fig. 6b correspond to the spectra of the commercial
form cheaper commercial pomegranate molasses. On the other
pomegranate molasses CPM1, CPM2 and CPM3 respectively in
hand, the higher radical scavenging capacity induced by date syrup
comparison with both the natural pomegranate and the date syrup;
addition to natural pomegranate molasses suggests that it is
and finally Fig. 6b combines the spectra of CPM1, CPM2 and CPM3
beneficial to industrials to declare this procedure and avoid fraud.
for the sake of comparison, which also reflects their close similarity

Fig. 6. ATR-FTIR spectra of NPM1 (upper) and DS (lower) 1 (a), ATR-FTIR spectra of DS, NPM1, CPM1, CPM2 and CPM3 from top to bottom, respectively (b).
N. El Darra et al. / Food Control 78 (2017) 132e137 137

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