Food Research International 65 (2014) 301–310

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Food Research International

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Polyphenolic characterization of olive mill wastewaters, coming from

Italian and Greek olive cultivars, after membrane technology
Isabella D'Antuono a,1, Vassiliki G. Kontogianni b,1, Kali Kotsiou b, Vito Linsalata a, Antonio F. Logrieco a,
Maria Tasioula-Margari b,⁎, Angela Cardinali a
a
Institute of Sciences of Food Production, ISPA National Research Council of Italy, Via G. Amendola, 122/O, 70126 Bari, Italy
b
Section of Industrial and Food Chemistry, Department of Chemistry, University of Ioannina, Ioannina 45110, Greece

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

Article history: The aim of this work was to recover and identify the phenolic compounds from olive mill wastewater (OMWW)
Received 26 June 2014 samples belonging to two Italian (Cellina and Coratina) and three Greek (Asprolia, Lianolia and Koroneiki) olive
Received in revised form 19 September 2014 cultivars. The OMWWs were processed using membrane technologies to obtain three fractions: microfiltrate
Accepted 26 September 2014
(MF), ultrafiltrate (UF) and nanofiltrate (NF). These steps allowed purifying the OMWWs in order to achieve
Available online 18 October 2014
fractions with different profile and concentrations of polyphenols. In particular, the amount of polyphenols
Keywords:
ranged from 2456 μg/mL to 5284 μg/mL in MF; from 1404 μg/mL to 3065 μg/mL in UF and from 373 μg/mL to
Olive mill wastewaters 1583 μg/mL in NF. Among the cultivars analyzed Coratina followed by Lianolia showed the highest amount of
Olive wastewater membrane fractionation verbascoside (VB) (308 μg/mL in Coratina versus 145 μg/mL in Lianolia, respectively) in UF fractions.
Olive wastewaters biophenols Furthermore, UF fractions that showed adequate purification degree and polyphenol enrichments, were used for
Olive wastewaters chemical composition the identification of the phenolic compounds by liquid chromatography/diode array detection/electrospray ion
trap tandem mass spectrometry (LC/DAD/ESI–MSn) analysis. Twenty three compounds, belonging to the
following classes of constituents: secoiridoids and their derivatives, phenyl alcohols, phenolic acid and
derivatives, and flavonoids, were identified in almost all the UF fractions of the different cultivars. Finally,
differences were observed among the cultivars regarding the presence of elenolic acid derivatives,
hydroxytyrosol glucoside, and β-hydroxyverbascoside diastereoisomers. The results obtained showed that
OMWW can be considered as raw material for the isolation of valuable bioactive compounds able to be used in
food, cosmetic and pharmaceutical industry.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction
Olive mill wastewaters (OMWWs) are seasonally generated effluents
in the olive oil extraction industry operating in three-phase mode. This
agro-industrial waste is produced in huge amounts (6–7 million tons/
year) and it is characterized by a strong undesirable smell, an intense
brown to dark color, a pH between 3 and 6 and a highly diverse organic
pollutant load (Ginos, Manios, & Mantzavinos, 2006). OMWW, a complex
medium containing polyphenols of different molecular masses, is
produced in Mediterranean countries. This waste is claimed to be one
of the most polluting effluents among those produced by the agro-food
industries because of its high polluting load and high toxicity to plants,
bacteria, and aquatic organisms, owing to its contents (14–15%) of
organic substances and phenols (up to 10 g/L). These latter compounds,
⁎ Corresponding author at: Department of Chemistry, University of Ioannina, Ioannina
45110, Greece. Fax: +30 2651008197.
E-mail address: mtasioul@cc.uoi.gr (M. Tasioula-Margari).
1
These authors contributed equally to this work.
characterized by high specific chemical oxygen demand (COD) and resis-
tance to biodegradation, are responsible for its black color, depending on
their state of degradation and the olives they come from (Capasso,
Cristinzio, Evidente, & Scognamiglio, 1992).
For a long time, OMWW has been regarded as hazardous waste with
negative impact on the environment and an economic burden on the
olive oil industry. Their phytotoxicity is mainly attributed to the high
phenolic content (0.5–24 g/L), that, on the other hand, are antioxidant
compounds with potential health-benefits (Obied et al., 2005a). In
light of these findings, the OMWWs are recognized as a potential
low-cost starting material rich in bioactive compounds, that can be
extracted and applied as natural antioxidants for the food and pharma-
ceutical industries.
A typical phenolic substance identified in olive fruit is oleuropein, a
secoiridoid glucoside that is absent in OMWW due to enzymatic
hydrolysis during olive oil extraction, resulting in the formation of side
products such as hydroxytyrosol and elenolic acid. Other phenolics iden-
tified in OMWW are verbascoside, tyrosol, catechol, 4-methylcatechol,
p-hydroxybenzoic acid, vanillic acid, syringic acid, and gallic acid
(Capasso et al., 1992; Visioli et al., 1999).

http://dx.doi.org/10.1016/j.foodres.2014.09.033
0963-9969/© 2014 Elsevier Ltd. All rights reserved.
302 I. D'Antuono et al. / Food Research International 65 (2014) 301–310
The most abundant biophenols occurring in OMWW are
hydroxytyrosol followed by tyrosol. In particular, hydroxytyrosol is the
most potent antioxidant phenolic compound occurring in olive oil
(Nissiotis & Tasioula-Margari, 2002), and numerous studies have focused
on its many other health-beneficial effects (Obied, Allen, Bedgood,
Prenzler, Robards, et al., 2005a) among them in inhibition of low-
density lipoprotein oxidation (EFSA, 2011). Moreover, the good solubility
of hydroxytyrosol in oil and aqueous media and its high bioavailability
allow its useful application in multi-component foods, encouraging pros-
pects in commercialization of it in functional foods and natural cosmetics
(Bouzid et al., 2005). However, the phenolic composition of OMWW
varies strongly between studies, as it is characterized by a significant
complexity (Bianco et al., 2003; Obied, Allen, Bedgood, Prenzler, &
Robards, 2005b; Obied, Bedgood, Prenzler, & Robards, 2007) and
many compounds are recently identified (Cardoso, Falcão, Peres, &
Domingues, 2011). Indeed, hydroxytyrosol acyclodihydroelenolate and
p-coumaroyl-6′-secologanoside (comselogoside) were recently identi-
fied in OMWW and were examined for their antioxidant and antiprolifer-
ative activities (Obied, Karuso, Prenzler, & Robards, 2007; Obied, Prenzler,
Konczak, Rehman, & Robards, 2009).
Traditionally, to isolate and recover polyphenols from matrix such as
OMWW, liquid–liquid extraction is employed. This method utilized a
large amount of solvent that has a negative impact for both health and
environment. Membrane separation has become a promising technolo-
gy with several advantages: low power consumption, water-reuse and
by-products recovery, stabilization of effluent, absence of organic
solvents. Some studies are already carried on, and the OMWW may be
treated efficiently by using microfiltration (MF), ultrafiltration (UF),
nanofiltration (NF) and/or reverse osmosis (RO), to obtain a permeate
fraction which can be discharged in aquatic systems according to
national or EU regulations or to be used for irrigation (Paraskeva,
Papadakis, Tsarouchi, Kanellopoulou, & Koutsoukos, 2007).
A membrane process for the selective fractionation and total recovery
of polyphenols, water and organic substances from OMWW was also
proposed by Russo (2007). It was based on the preliminary MF of the
OMWW, followed by two UF steps realized with 6 kDa and 1 kDa
membranes, respectively, and a final RO treatment. The RO retentate,
containing enriched and purified low molecular weight polyphenols,
was proposed for food, pharmaceutical or cosmetic industries, while MF
and UF retentates can be used as fertilizers or in the production of biogas
in anaerobic reactors (Garcia-Castello, Cassano, Criscuoli, Conidi, & Orioli,
2010).
The current investigation aimed at identifying the phenolic
compounds in the OMWW samples belonging to different Italian
and Greek olive cultivars. The OMWWs were processed using
membrane technologies in order to investigate the impact of differ-
ent cultivar in the phenolic profile of the fractions. The fractions
were quantified regarding the main polyphenols present by HPLC
analysis and were deeper studied, using LC/DAD/ESI–MSn analysis,
in order to elucidate the identity of phenolic components that
could also be a characteristic of each OMWW coming from different
olive variety.
2. Material and methods
2.1. Chemicals
Extraction and chromatography solvents, methanol (MeOH), acetoni-
trile (MeCN), glacial acetic acid (AcOH), and ethanol (EtOH), were of
certified high-performance liquid chromatography (HPLC) grade, and
pure standard of hydroxytyrosol (HT), tyrosol (Tyr), caffeic acid (CAA),
coumaric acid (CUA), verbascoside (VB), isoverbascoside (IsoVB),
were obtained from PhytoLab GmbH & Co. KG (Vestenbergsgreuth,
Germany). Folin–Ciocalteu reagent was purchased from Sigma-Aldrich
(Milano, Italy).
2.2. OMWW samples
All the OMWWs utilized in this study raised from mills that used a
three-phase system. Five fresh OMWW samples (~30 L), among them
two Italian cultivars: Cellina and Coratina obtained respectively from:
Cooperativa Agricola Nuova Generazione Srl (Martano, Lecce, Italy)
and Oleificio Di Molfetta (Bisceglie, Bari, Italy) from Apulia region
mills and three Greek cultivars, Koroneiki, Lianolia and Asprolia (all
organic), collected from Greek mills.
Italian samples were processed within 4 days after olive oil produc-
tion, Greek samples were collected and shipped 2 days later, so were
processed within 7 days after olive oil production. Acetic acid was
added to pH 5, in the samples, to avoid phenolic compounds oxidation.
The raw material was firstly sieved through a test sieve with 425 μm
as porosity. This process allows the removal of large particles and
colloids from the OMWW before the microfiltration process. With this
procedure, all traces of oil, leaves, seeds, which could then cause
problems of clogging of the membranes, are eliminated.
2.3. Filtration units (MF, UF and NF)
The raw OMWWs coming from the five different cultivars were
processed with a laboratory-scale system (Permeare s.r.l., Milano,
Italy) present in the laboratory of ISPA-CNR of Bari. This system, that
utilizes a continuous parallel flow, is consisting of two different units:
Pilot Plant N022/N256C and N021/N256C (Figs. 1 and 2).
The first (Fig. 1), filled by external tank, performed microfiltration
(MF) process with continuous recirculation of the sample and by
using a ceramic membranes, PERMAPORE EOV 1046, with cut-off of
about ~ 100,000 Da (membrane porosity 0.1 μm). The volume which
can be processed daily will be limited by the substances contained in
the fluid. In general, it is possible to treat from 200 up to 2000 L per
day. This unit can support transmembrane pressure (differences
between the inlet pressure and outlet pressure) up to 6 bars, at 25 °C.
The Pilot Plant N021/N256C unit (Fig. 2) is composed of the following
sections: process tank (5–10 L), high pressure pump, pressure vessel for
membrane housing. The unit can support operating pressure up to
75 bar and operating temperatures that ranges between 5 °C as minimum
and 60 °C as maximum. Cooling device is supplied for eventually cooling
the solution in order to maintain an acceptable temperature during
process. Furthermore, the maximum size of eventual suspended solids
in the feed solution should be less than 3 μm. Pressure vessel for
membrane is composed of three AISI316 stainless steel parts: testate,
end cup and body for membrane housing with 5 cm of diameter and
30.5 cm of length.
This unit performed ultrafiltration (UF) and nanofiltration (NF)
processes, utilizing polymeric membranes at different porosities. For
UF, was employed a polyethersulfone membrane, PERMAPORE DGU
1812 BS EM with cut-off of 5000 Da, instead for NF, the filtration was
performed using a polyamide membrane, PERMAPORE AEN 1812 BS
with cut-off of 200 Da. After utilization, the membranes were washed
with alkaline detergent following the manufacture instructions because
the OMWW can provoke the membrane clogging during the process. All
the membrane utilized act as a molecular sieve without any chemical
interactions with the matrix. In addition, the ceramic membrane was
utilized for MF because they are chemically stable and mechanically
and biologically inert. In addition, they are available only as limited
range of porosity and, for this reason, mainly utilized for microfiltration.
2.4. Determination of total phenolic content
The total phenolic content of the OMWW and fractions was deter-
mined using a modified Folin–Ciocalteu spectrophotometric method
100 μL of properly diluted samples, calibration solutions or blank were
pipetted into separate test tubes and 100 μL of F–C reagent were added
to each. The mixture was mixed well and was allowed to equilibrate.
 I. D'Antuono et al. / Food Research International 65 (2014) 301–310
Fig. 1. Microfiltration: A: Feed, B: Microfiltration membrane, C: Permeate, D: Drain, E: Switchboard.
After exactly 2 min, 800 μL of a 5% (w/v) sodium carbonate solution were
added. The mixture was swirled and put in a temperature bath at 40 °C
for 20 min. Then, the tubes were rapidly cooled on the rocks and the
color generated was read at its maximum absorption (750 nm). The
absorbance was measured in 1-cm cuvette by the Varian Cary 50 Scan
UV/visible spectrophotometer. For calibration solutions and blank
preparation, a methanolic solution at the same concentration of samples
was used (Cicco, Lanorte, Paraggio, Viggiano, & Lattanzio, 2009). Results
were expressed as micrograms per milliliter (μg/mL) of HT equivalents.
2.5. HPLC-DAD analysis
Analytical-scale HPLC analyses of the OMWW and fractions were
performed with an Agilent Technologies series 1100 liquid chromatog-
raphy (Waldbronn, Germany) equipped with a binary gradient pump
G1312A, a G1315A photodiode array detector, and a G1316A
column thermostat set at 45 °C. ChemStation for LC3D (Rev. A. 10.02)
software was used for spectra and data processing. An analytical
Phenomenex (Torrance, CA) Luna C18 (5 μm) column (4.6 × 250 mm)
was used throughout this work. The solvent system consisted of
(A) methanol and (B) acetic acid/water (5:95, v/v). For low molecular
weight phenolics two solvents were used: A, methanol and B, acetic
acid–water (5:95 v/v). The elution profile of the linear gradient
was: 0–21 min, 15–40% A; 21–30 min, 40% A (isocratic); 30–45 min,
40–63% A; 45–47 min, 63% A (isocratic); and 47–51 min, 63–100% A
(Lattanzio, 1982). The flow rate was 1 mL/min and the injection volume
was 20 μL.
303
2.6. LC–MS analysis
2.6.1. Instrumentation
All LC–MSn experiments were performed on a quadrupole ion trap
mass analyzer (Agilent Technologies, model MSD trap SL) retrofitted to
a 1100 binary HPLC system equipped with a degasser, an autosampler,
a diode array detector and an electrospray ionization source (Agilent
Technologies, Karlsruhe, Germany). All hardware components were
controlled by Agilent ChemStation software.
2.6.2. Analysis
A 10 μL aliquot of ultrafiltrate fraction of OMWW samples was filtered
(0.45 μm) and injected into the LC–MS instrument. Separation was
achieved on a 25 cm × 4.6 mm i.d., 5 μm Zorbax Eclipse XDB-C18 analyt-
ical column (Alltech, Deerfield, USA), at a flow rate of 1.0 mL/min, using
as solvent A (water/acetic acid, 99.9: 0.1 v/v) and solvent B (acetonitrile/
methanol 1:1). The gradient used for the analysis of OMWW UF fractions
was: 0–20 min, 95–70% A; 20–25 min, 70–65% A; 25–45 min, 65–60% A;
45–60 min, 60–30% A; 60–65 min, 30–0% A; 65–75 min, 0% A; and
75–80 min, 0–95% A. The UV/vis spectra were recorded in the
range of 200–700 nm and chromatograms were acquired at 240,
280 and 340 nm.
Both precursor and product (MS2 and MS3) ions scanning of the
phenolic compounds were monitored between m/z 50 and m/z 1000
in negative polarity. The ionization source conditions were as follows:
capillary voltage, 3.5 kV; drying gas temperature, 350 °C; nitrogen
flow and pressure, 11 L/min and 50 psi, respectively. Maximum
304 I. D'Antuono et al. / Food Research International 65 (2014) 301–310

Fig. 2. Ultra and nanofiltration: A: Feed tank, B: Membrane, C: Permeate, D: Concentrate, E: Switchboard.

accumulation time of ion trap and the number of MS repetitions to step is mainly used for separating the polymeric fractions of the
obtain the MS average spectra were set at 30 and 3 ms, respectively. polyphenols from mineral salts.

3.2. OMWW total phenolic contents

2.7. Statistical analysis
The obtained fractions were assayed by a modified Folin–Ciocalteu
For total phenolic amount, statistical differences were determined
(FC) method to determine the total phenols content expressed as HT
by analysis of variance (ANOVA) followed by multiple comparison
equivalent (Cicco et al., 2009). The results are showed in Table 1.
procedure on ranks with Student–Newman–Keuls Method, at 5%
Among Italian cultivars, Cellina showed the higher phenolic content,
significance level, using the software SigmaPlot for Windows (Ver. 12,
instead among the Greek, Lianolia was the most abundant. Considering
Systat Software Inc., San Jose, CA 95110 USA).
the polyphenol contents in MF as 100%, the percentage of polyphenols
recovered in UF fractions, ranged from 55% to 65%, and in NF fraction
3. Results and discussion from 15% to 33% (Table 1). In particular, the amount of polyphenols
ranged from 2456 μg/mL to 5284 μg/mL in MF; from 1404 μg/mL to
3.1. Filtration units 3065 μg/mL in UF and from 373 μg/mL to 1583 μg/mL in NF.
The fractions were also characterized by HPLC analysis, to quantify
During the filtration steps three different fractions were obtained the main polyphenols present. As shown in Fig. 3(a, b, c), the main
MF, UF and NF. The MF fraction was recovered using a ceramic
membrane with the aim to stabilize and clarify the OMWW and to Table 1
give a filtrate free from high molecular weight proteins, enzymes and Total phenolic content and relative percentage of five OMWW cultivars after filtration
process. The data are expressed as HT equivalent (μg/mL).
bacterial component. The yield of the process was from 2 to 4 L/h of
OMWW, it was depending from the wastewaters quality. OMWW CVs MF fraction UF fraction NF fraction
The UF fraction, instead, was obtained by a polymeric membrane at μg/mL % μg/mL % μg/mL %
5000 Da of cut-off, with yield ranging from 1.5 to 2.4 L/h. This step gives a a a
Asprolia 3488 ± 500 100 2270 ± 475 65 976 ± 110 28
a fraction free from colloidal substances and separates, from the water Koroneiki 2456 ± 328b 100 1404 ± 221b 57 373 ± 43b 15
solution, organic macromolecules at molecular weight lower than Lianolia 4623 ± 236c 100 2559 ± 353c 55 1402 ± 135c 30
membrane cut-off. Coratina 4768 ± 289c 100 2755 ± 321c 58 1583 ± 121c 33
Finally, the NF step was performed by a membrane with a molecular Cellina 5284 ± 265c 100 3065 ± 431c 58 1423 ± 134c 27

weight cut-off of 200 Da. The yield of the process was of about 6 L/h. This Means with a common letter within a column are not significantly different (p ≤ 0.05).
 I. D'Antuono et al. / Food Research International 65 (2014) 301–310 305

Fig. 3. Phenolic composition of OMWW fractions, expressed as relative percentage of each compound respect to the total phenolics quantified by HPLC-DAD analysis. MF fraction a); UF
fraction b); NF fraction c).

phenolics identified were: HT, Tyr, CAA, CUA, VB, IsoVB, caffeoyl-6-
secologanoside (SEC), and comselogoside (COM). In all the fractions
analyzed and in all CVs (Fig. 3a, b, c), the most abundant compound
is HT that represents about 70–80% of the total phenolic concentra-
tion, followed by Tyr. On the contrary in MF of Coratina the main
phenolic was VB (Fig. 3a), which in the following step (UF fraction
Fig. 3b) is reduced with a simultaneous increase of HT, as hydrolysis
product of VB molecule. VB was lower and in some cases absent, in
the other cultivars. The hydrolytic process affects oleuropein and
demethyloleuropein, hydrolyzing them to hydroxytyrosol, while
verbascoside, depending on the storage time, was affected to a lower extent.
Coratina followed by Lianolia had the higher content of these compounds
(308 μg/mL in Coratina versus 145 μg/mL in Lianolia, respectively). β-
Hydroxyverbascoside diastereoisomers were detected in traces in the rest
of cultivars. Even though the OMWWs for the Italian cultivars were stored
for 4 days before the extraction and the respective OMWW from Greek culti-
vars were stored for 7 days before the extraction a considerable amount of
verbascoside was found for Coratina and Lianolia cultivars. The different distri-
bution of VB could be considered as a distinguishing element among them.
Moreover, besides HT, the most studied polyphenol for its biological proper-
ties is VB. Recent studies have assessed the biological activities of VB, consis-
tent with disease prevention including antioxidant and anti-inflammatory
activity (Cardinali et al., 2010, 2012; Esposito et al., 2010; Korkina, 2007;
Speranza et al., 2010).
Interesting is the presence of comselogoside and caffeoyl-6-
secologanoside, already identified in OMWW (Obied, Bedgood et al.,
306 I. D'Antuono et al. / Food Research International 65 (2014) 301–310

2007) and olive fruits (Kanakis et al., 2013; Obied, Karuso et al., 2007)
that exhibit antioxidant activity comparable to other compounds
(Obied, Bedgood, Prenzler, & Robards, 2007).
The last step of filtration, NF (Fig. 3c), showed only low molecular
weight phenolic compounds, such as HT, Tyr, CAA and CUA. Caffeic
acid, although present at very low concentration (from 3 to 5 μg/mL)
it is reported to have high antioxidant activity (Obied, Prenzler et al.,
2008).
The occurrence of specific biophenols in OMWW depends on the
fruit cultivar and maturity (Mulinacci et al., 2001; Obied, Bedgood,
Mailer, Prenzler, & Robards, 2008), and storage time (Obied, Bedgood,
Prenzler, & Robards, 2008) in addition to the processing extraction
technique (De Marco, Savarese, Paduano, & Sacchi, 2007). The endoge-
nous enzymes and microbial activities cause a loss of the recovered
phenols. According to Obied, Bedgood, Prenzler et al. (2008) none of
storage conditions studied (storage at 4 °C, preserve by 40% w/w
ethanol and 1% w/w acetic acid and storage at 4 °C) could prevent the
rapid decrease in phenolic concentrations and antioxidant capacity,
which happened within the first 24 h.
Among the three fractions obtained, the UF was the best balance
between the purification degree and the polyphenol enrichments
(Garcia-Castello et al., 2010). For this reason the UF fraction was deeper
characterized using LC/DAD/ESI–MSn analysis in order to identify the
phenolic profile of each cultivar.
3.3. LC–MS analysis
The LC/DAD/ESI–MSn analysis of the UF fractions from the five
OMWW cultivars, led to the separation and identification of the major-
ity of the constituents. Most of the compounds that were identified
belonged to the following classes of constituents: secoiridoids and
their derivatives, phenyl alcohols, phenolic acids and derivatives and
flavonoids. MS data were acquired in negative ionization mode,
because polyphenols contain one or more hydroxyl and/or carboxylic
acid groups. Identification was based on accurate mass measurements
of the pseudomolecular [M–H]− ions and their fragmentation pattern,
as it has been documented in the literature. In Fig. 4, the total ion
current (TIC) chromatogram and UV chromatograms at 280, 240
and 340 nm of UF fraction of Coratina is presented (for the rest of
the UF fractions of different cultivars the respective chromatograms
are given in Supplementary data Supplements 1–4). Data obtained
from the ESI-MSn analysis of the UF fractions are summarized in
Table 2.
Peak 1 exhibited a base peak [M–H]− at m/z 191, the MS2 spectrum
obtained by fragmentation of the ion m/z 191 presented fragment ions
at m/z 127 ([M–CO–2H2O]−) and m/z 173 ([M–H2O]−) which
correspond to literature reports for quinic acid (Gouveia & Castilho,
2010). Peak 2 exhibited a molecular ion at m/z 169, the fragmentation
of this pseudomolecular ion yielded a fragment at m/z 151 probably
produced by the loss [M–H–H2O]− and was tentatively identified as
3,4-dihydroxyphenylglycol, that has previously identified in OMWW
(Obied, Bedgood et al., 2007). Peaks 3 and 4 showed similar MS spectra,
with a molecular ion at m/z ratio 315, and similar MS/MS spectra, thus
suggesting the presence of two stereoisomers that were not distinguish-
able by mass spectrometry. The fragmentation pattern revealed two
main fragment ions at the following m/z ratios: 153, which is formed
by the loss of a glucose group and 123 corresponding to loss of the
CH2OH group. The two isomers were attributed to HT glucoside. Three
isomers of HT glucoside have been identified in Olea europaea, namely
hydroxytyrosol-1-O-glucoside, hydroxytyrosol-3′-O-glucoside and
hydroxytyrosol-4′-O-glucoside (Obied, Bedgood et al., 2007). Romero,
Brenes, Garcia, and Garrido (2002), found that hydroxytyrosol-4-β-D-
glucoside was the most abundant isomer in olive fruits and derived
products.

Fig. 4. Total ion chromatogram and UV chromatograms at 280, 240 and 340 nm of OMWW Coratina UF fraction. Main compounds of extract: 1: quinic acid, 1′: cornoside, 2′: hydroxylated
product of decarboxymethyl elenolic acid, 2: 3,4-dihydroxyphenylglycol, 5: HT, 4′: decarboxymethyl-elenolic acid derivative, 6: hydroxylated product of dialdehydic form of
decarboxymethyl elenolic acid, 7: Tyr, 9: CAA, 12–13: β-hydroxyverbascoside diastereoisomer, 15: Ver, 18–19: elenolic acid derivative, 21: SEC, 23: COM.
 I. D'Antuono et al. / Food Research International 65 (2014) 301–310 307

Table 2
Main ions identified by HPLC-DAD–MSn in the OMWW extracts and their proposed structures.

Peak Rt (min) [M–H]− −

MS2 [M–H]- (m/z) (%) -MS3 [base peak]− (m/z) (%) Compounds
(m/z)

1 2.5 191 111 (100), 173 (26), 127 (13) – Quinic acid
2 4.1 169 151 (100) – 3,4-Dihydroxyphenylglycol
1' 5.5 315 151 (100), 255 (76) – Cornoside
2' 7.8 199 – – Hydroxylated product of decarboxymethyl elenolic acid
3 9.8 315 153 (100), 123 (20) 123(100) Hydroxytyrosol glucoside
4 10.1 315 153 (100), 123 (20) – Hydroxytyrosol glucoside
5 10.5 153 123 (100) – HT
3' 11.2 407 389 (100), 375 (88), 357 (72), 313 (58) 313 (100), 357 (74), 161 (6) Unknown
4' 12.1 183 111 (100), 165 (14) – Decarboxymethyl-elenolic acid derivative
6 12.8 199 111 (100), 155 (78), 181 (51) – Hydroxylated product of daldehydic form of
decarboxymethyl elenolic acid
7 14.3 137 – – Tyr
5' 15.2 353 191 (100) – Unknown
8 16.3 389 345 (100), 209 (47), 165 (26) – Oleoside
9 18.2 179 135 (100) – CAA
10 18.6 183 139 (100), 95 (58) – Decarboxymethyl elenolic acid (HyEDA)
11 19.6 377 197 (100), 153 (16) 153 (100) Oleuropein aglycon derivative
12 20.9 639 621 (100), 529 (12), 459 (11), 179 (3) 459 (100), 469 (13), 179 (10) β-Hydroxyverbascoside diastereoisomer
13 21.3 639 621(100), 529(5), 459(9) 459 (100), 469 (13), 179 (10) β-Hydroxyverbascoside diastereoisomer
14 22.9 163 119(100) – CUA
15 25.6 623 421(100) 135(100), 315(92), 297(16), 161(16) Ver
16 26.1 609 301(100), 271(6), 343(5) – Rutin
17 27.4 623 421(100) 135 (100), 315 (92), 297 (16), 161 (16) IsoVB
18 27.7 241 139(100), 127(58), 95(52), 165(26) 95 (100) Elenolic acid derivative
19 28.2 241 139(100), 127(67), 95(43), 165(30), 207(18) 95 (100) Elenolic acid derivative
20 29.9 381 331 (100), 349 (96), 363 (92), 151 (44) 151 (100), 195 (9), 287 (8) Hydroxytyrosol acyclodihydroelenolate
21 30.7 551 507(100), 281(36), 179(26), 389(25) 161 (100), 345 (44), 393 (25), 281 (20) SEC
22 32.6 539 275 (100), 307 (95), 377 (68) 139 (100), 95 (68), 245 (11) Oleuropein
23 35.7 535 491 (100), 265 (28), 389 (27) 145 (100), 345 (77), 265 (36), 307 (26) COM

Peak 4′ exhibited base peaks at m/z 185 (100%) and at m/z 183 (70%),
with fragments in its MS2 at m/z 111 (100%) and at m/z 165 (14%). The
ion at m/z 183 could probably be assigned to the de(carboxymethyl)
elenolic acid derivative ion (De La Torre-Carbot et al., 2005) and the
product ion at m/z 111 might be caused by the loss of CO and COO of
the elenolic derivative fragment (m/z 183) in aldehyde forms (Ramos
et al., 2013). So this peak can be tentatively identified as another
elenolic acid derivative.
The spectrum generated for peak 6 yielded a deprotonated molecule
at m/z 199, which could be attributed to a derivative of the dialdehydic
form of decarboxymethyl elenolic acid. Peak 6 was tentatively identified
as hydroxylated product of dialdehydic form of decarboxymethyl
elenolic acid, as it presented a fragment at m/z 155 corresponding to a
loss of 44 units, which is consistent with the fragmentation pattern of
its nonderivative form (acid group decarboxylation) (Lozano-Sanchez
et al., 2011). The mass spectrum of peak 8, displayed an intense peak
at m/z 389 which formed two major fragments in the MS2 spectrum,
one at m/z 345 and the other at m/z 209. The former corresponded to
the loss of 44 Da, which can be justified by the elimination of a CO2
molecule of a carboxylic group, and the latter can be attributed to the
Z fragment of a hexose (loss of 180 Da) (the hexose residue was
attributed to glucose). These results are in agreement with the presence
of oleoside (Cardoso et al., 2005). Peak 10 showed an intense
pseudomolecular peak at m/z 183 in the ESI-MS spectrum which
presented a fragment at m/z 139, corresponding to a loss of 44 units.
This compound was tentatively identified as the dialdehydic form of
decarboxymethyl elenolic acid, that has been detected before in table
olives (Medina, Brenes, Romero, García, & De Castro, 2007) and in
olive oil and wastes generated during the storage of extra virgin olive
oil (Lozano-Sanchez et al., 2011).
The mass spectrum of peak 11 exhibited a base peak at m/z 377 and
in its MS2 spectrum showed fragments at m/z 197 and 153, which has
been identified before as oleuropein aglycon derivative in various
tautomeric forms (Bouaziz, Jemai, Khabou, & Sayadi, 2010). The
abundant peak at m/z 639, that exhibited peaks 12 and 13, is due to
the molecular ion [M–H]− of two diastereoisomers of the molecule
β-hydroxyacteoside or β-hydroxyverbascoside (MW 640) (Innocenti
et al., 2006). MS/MS fragmentation of molecular ions at m/z 639 yielded
to the main daughter ion at m/z 621, corresponding to the water loss,
and three minor fragments at m/z 529, corresponding to the loss of a
catechol unit, m/z 459 corresponding to the loss of a caffeyol group or
rhamnose, and m/z 179, assigned to caffeic acid ion. Two diastereoiso-
meric structures of the β-hydroxyl derivative of verbascoside and
two diastereoisomeric structures of the β-hydroxyl derivative of
isoverbascoside have been recently identified in olive mill wastewater
by Cardinali et al. (2012). All of these stereoisomers have the same
fragmentation scheme and, therefore, are not distinguishable by mass
spectrometry. The mass spectrum of peak 16 exhibited a base peak at
m/z 609. Its MS2 spectrum showed fragments at m/z 301, an aglycon
ion, and at m/z 271 confirm the presence of rutin (Savarese, De Marco,
& Sacchi, 2007).
Peaks 18 and 19 showed mass spectra with the [M–H]− molecular
ion species at m/z 241. The MS/MS spectra obtained for the precursor
ion at m/z 241 gave fragment ions with molecular weights of 139 [M–
COOH–COOCH3]−, 127 [M–COOH–C4H5O]−, 95 [M–COOH–COOCH3–
CHCHO]− and 165 [M–OH–COOCH3]−, which among others have
been referred for dialdehydic form of carboxymethyl elenolic acid (Di
Maio et al., 2013). These peaks can be tentatively identified as elenolic
acid derivatives. The mass spectrum of peak 20 gave a molecular ion
at m/z 381, attributed to hydroxytyrosol acyclodihydroelenolate,
identified in OMWW extracts for the first time by Obied, Karuso et al.
(2007). In its MS2 spectrum, it formed the following fragments, at m/z
363 (−18 Da) due to the loss of a H2O unit, at m/z 349 (− 32 Da), an
ion related to the cleavage of the secoiridoid function of the molecule,
occurring due to the respective loss of its CH3OH moiety, at m/z 331
(−50 Da) [M–H–H2O–CH3OH]− and at m/z 151. Peak 21, with a molec-
ular weight at m/z 551, was attributed to caffeoyl-6-secologanoside.
Fragmentation of this ion originated an intense signal at m/z 507 from
the loss of 44 Da, [M–H–CO2]−, the species at m/z 389 representing
the oleoside structure and the ion at m/z 179 is related to the caffeoyl
group (Innocenti et al., 2006). The ESI-MS spectrum of peak 22 showed
a pseudomolecular ion at m/z 539 with fragments in its MS2 spectrum
308 I. D'Antuono et al. / Food Research International 65 (2014) 301–310

consistent with the reported fragmentation scheme: the ion at m/z 377
arises from cleavage of the glycosyl bond; the ion at m/z 307 is justified
by the loss of a C4H6O fragment, while the fragment at m/z 275 may de-
rive from rearranged fragments. These results confirm the presence of
oleuropein. The spectrum generated for peak 23 yielded a deprotonated
molecule at m/z at 535 was attributed to 6′-p-coumaroyl secologanoside
(comselogoside). The ESI-MS2 spectrum of that ion showed the main
fragment at m/z 491 from the loss of 44 Da, [M–H–CO2]− along with
the ionic species at m/z 389 corresponding to the oleoside ion (Obied,
Karuso et al., 2007). Finally peaks 5, 7, 9, 14 15 and 17 were identified
as HT, Tyr, CAA, CUA, VB and IsoVB respectively, by direct comparison
of their retention times and UV spectra with those of the standards
and by using their MS, MS2 and MS3 spectra.
Overall twenty three compounds, most of them detected in trace
amounts, were identified in almost all the UF fractions of the different
cultivars. Results showed no significant qualitative differences in the
HPLC–ESI MS phenolic profile among UF fractions from the OMWWs
from different cultivars (Table 3). Among the UF fractions from the five
OMWW cultivars the one coming from Coratina showed two more com-
pounds indicated with 1′ and 2′. Peak 1′ exhibited a base peak at m/z 315.
Its MS2 spectrum showed fragments at m/z 151 and at m/z 255. This com-
pound could be tentatively identified as cornoside, a quinol glucoside
identified in the vegetation water of olives (Limiroli, Consonni, Ranalli,
Bianchi, & Zetta, 1996). Cornoside was thought to derive from oxidation
of the glucoside of tyrosol. Peak 2′ exhibited a base peak at m/z 199 that
could be tentatively identified as another hydroxylated product of
decarboxymethyl elenolic acid (Kanakis et al., 2013).
In this UF fraction was additionally found and quantified, IsoVB,
which was also detected in traces in Lianolia cultivar. However,
quantitative differences were observed in some of the main phenolic
compounds. An attempt was made to compare the integral-areas of
the main peaks of the chromatograms in the three wavelengths, as the
compounds identified were not available as standards. In Table 3
integral-areas are given for eight compounds. Differences were
observed among the cultivars analyzed for peaks 4′ and 6. In the UF
fractions of Lianolia and Cellina the above elenolic acid derivative (P.4′)
and the hydroxylated product of dialdehydic form of decarboxymethyl
elenolic acid (P.6) were most abundant in comparison with the rest of
the cultivars. Also in the UF fraction of Cellina, followed by Lianolia,
hydroxytyrosol glucoside (P.3) was most abundant.
Moreover differences were observed in the content of the UF fractions
regarding 3,4-dihydroxyphenylglycol (P.2) and β-hydroxyverbascoside
diastereoisomers (P.12 and P.13). Coratina followed by Lianolia had
the higher content of these compounds. It is noteworthy to mention
that β-hydroxyverbascoside diastereoisomers were detected in traces in
the rest of cultivars.
Elenolic acid and derivatives constitute the iridoid part of several
important secondary olive metabolites, such as oleuropein and
ligstroside, and are often mentioned as one of their hydrolysis
products. 3,4-Dihydroxyphenyl glycol is a hydroxylated derivative
of hydroxytyrosol. This substance may be of interest in the fields of
nutrition and pharmacology due to its powerful antioxidant
properties. It is the main metabolite produced by deamination of the
human neurotransmitter noradrenaline (norepinephrine). Rodríguez,

Table 3
Compounds identified in the OMWW extracts in each different variety. In parenthesis are given the integral-areas in the chromatographic profiles in three wavelengths (280, 240 and
340 nm) of UF fractions of OMWW of different cultivars for eight compounds.

Peak Rt (min) Compounds OMWW samples of different varieties

Lianolia Asprolia Koroneiki Coratina Cellina

1 2.5 Quinic acid √ √ √ √ √

(240 nm) (1065) (1471) (1684) (626) (1066)
2 4.1 3,4-Dihydroxyphenylglycol √ √ √ √ √
(280 nm) (1386) (744) (605) (2143) (872)
1′ 5.5 Cornoside – – – √ –
2′ 7.8 Hydroxylated product of decarboxymethyl elenolic acid – – – √ –
3 9.8 Hydroxytyrosol glucoside √ √ √ √ √
(280 nm) (1681) (207) (558) (255) (2206)
4 10.1 Hydroxytyrosol glucoside √ √ √ √ √
5 10.5 HT √ √ √ √ √
3′ 11.2 Unknown √ √ √ √ √
4′ 12.1 Decarboxymethyl-elenolic acid derivative √ √ √ √ √
(240 nm) (15174) (3148) (1089) (8719) (9055)
6 12.8 Hydroxylated product of dialdehydic form of √ √ √ √ √
(240 nm) decarboxymethyl elenolic acid (9137) (6853) (2590) (5654) (16405)
7 14.3 Tyr √ √ √ √ √
5′ 15.2 Unknown √ – – √ –
8 16.3 Oleoside √ √ √ √ √
9 18.2 CAA √ √ √ √ √
10 18.6 Decarboxymethyl elenolic acid (HyEDA) √ – – √ √
11 19.6 Oleuropein aglycon derivative √ √ √ √ √
12 20.9 β-Hydroxyverbascoside diastereoisomer √ √ √ √ √
(340 nm) (507) (a) (37) (983) (a)
13 21.3 β-Hydroxyverbascoside diastereoisomer √ √ √ √ √
(340 nm) (564) (a) (39) (1072) (a)
14 22.9 CUA √ √ √ – √
15 25.6 Ver √ √ √ √ √
16 26.1 Rutin – – – √ √
17 27.4 IsoVB √ – – √ –
18b 27.7 Elenolic acid derivative √ √ √ √ √
(240 nm)
19b 28.2 Elenolic acid derivative √ √ √ √ √
(240 nm) (3805) (5860) (568) (4038) (4045)
20 29.9 Hydroxytyrosol acyclodihydroelenolate √ √ √ √ √
21 30.7 SEC √ √ √ √ √
22 32.6 Oleuropein √ √ √ √ √
23 35.7 COM √ √ √ √ √
a
These compounds are detected in traces in the OMWW.
b
The integral was taken for both compounds 18 and 19.
 I. D'Antuono et al. / Food Research International 65 (2014) 301–310
Rodríguez, Fernández-Bolaños, Guillén, and Jiménez (2007) demon-
strated that the antioxidant efficiency of 3,4-dihydroxyphenyl
glycol in water is 2–3 times higher than that of ascorbic acid or
hydroxytyrosol, whereas in lipidic medium it is comparable to that of
vitamin E despite its high polarity. Moreover, De Roos et al. (2011)
demonstrated that an alperujo extract may protect against platelet
activation, platelet adhesion and possibly have anti-inflammatory
properties, referring that a combination of hydroxytyrosol and 3,4-
dihydroxyphenylglycol were, at least partly, responsible for this effect.
So it would be interesting to study in depth the in vitro antioxidant
properties of these fractions from the OMWWs, as they might be of
use in protecting food and pharmaceutical products by retarding the
process of oxidation and deterioration during processing and storage.
Also further studies of other bioactivities associated with the non-
phenolic secoiridoids (elenolic acid and its derivatives) such as antimi-
crobial activity could be investigated.
4. Conclusion
In this study OMWWs coming from Italian and Greek cultivars were
processed using membrane technologies. With this system three frac-
tions were recovered MF, UF and NF, containing a different percentage
of polyphenols. The fractions were characterized by HPLC analysis, to
quantify the main polyphenols present: HT, Tyr, CAA, CUA, VB, IsoVB,
SEC and COM. The most abundant compound was HT that represent
about 70–80% of the total phenolic concentration, followed by Tyr. On
the contrary, in Coratina MF, the main phenolic compound was VB, that
instead, was lower and in some cases absent, in all the other cultivars.
The UF fractions that represented the best balance between the
purification degree and the polyphenol enrichment were characterized
by LC/DAD/ESI–MSn analysis. Overall twenty three compounds, most of
them detected in trace amounts, were identified in almost all the UF
fractions of the different cultivars. Results showed no significant qualita-
tive differences, however, quantitative differences were observed in
some of the main phenolic compounds. In detail, differences were
observed among the different cultivars regarding the presence of elenolic
acid derivatives, hydroxytyrosol glucoside, and β-hydroxyverbascoside
diastereoisomers. This deeper characterization could help to understand
how the olive cultivars can impact on the phenolic compositions of
OMWW.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.foodres.2014.09.033.
Acknowledgments
The authors want to thank the INTERREG IV European Territorial
Cooperation Programme “Greece–Italy 2007–2013”: “Utilization of
biophenols from Olea europaea products — Olives, virgin olive oil and
olive mill wastewater-BIO-OLEA” project. Special thanks are given to
the Mass Spectrometry Unit of the University of Ioannina for providing
access to LC–MS/MS facilities.
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