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Polyphenolic characterization of olive mill wastewaters, coming fromItalian 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, Italyb Section of Industrial and Food Chemistry, Department of Chemistry, University of Ioannina, Ioannina 45110, Greece
⁎ Corresponding author at: Department of Chemistry, U45110, Greece. Fax: +30 2651008197.
E-mail address: [email protected] (M. Tasioula-Marg1 These authors contributed equally to this work.
The aim of this work was to recover and identify the phenolic compounds from olive mill wastewater (OMWW)samples belonging to two Italian (Cellina and Coratina) and three Greek (Asprolia, Lianolia and Koroneiki) olivecultivars. The OMWWs were processed using membrane technologies to obtain three fractions: microfiltrate(MF), ultrafiltrate (UF) and nanofiltrate (NF). These steps allowed purifying the OMWWs in order to achievefractions with different profile and concentrations of polyphenols. In particular, the amount of polyphenolsranged 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 to1583 μg/mL in NF. Among the cultivars analyzed Coratina followed by Lianolia showed the highest amount ofverbascoside (VB) (308 μg/mL in Coratina versus 145 μg/mL in Lianolia, respectively) in UF fractions.Furthermore, UF fractions that showed adequate purification degree and polyphenol enrichments, were used forthe identification of the phenolic compounds by liquid chromatography/diode array detection/electrospray iontrap tandem mass spectrometry (LC/DAD/ESI–MSn) analysis. Twenty three compounds, belonging to thefollowing classes of constituents: secoiridoids and their derivatives, phenyl alcohols, phenolic acid andderivatives, 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 thatOMWW can be considered as raw material for the isolation of valuable bioactive compounds able to be used infood, cosmetic and pharmaceutical industry.
Olive mill wastewaters (OMWWs) are seasonally generated effluentsin the olive oil extraction industry operating in three-phase mode. Thisagro-industrial waste is produced in huge amounts (6–7 million tons/year) and it is characterized by a strong undesirable smell, an intensebrown to dark color, a pH between 3 and 6 and a highly diverse organicpollutant load (Ginos,Manios, &Mantzavinos, 2006). OMWW, a complexmedium containing polyphenols of different molecular masses, isproduced in Mediterranean countries. This waste is claimed to be oneof the most polluting effluents among those produced by the agro-foodindustries because of its high polluting load and high toxicity to plants,bacteria, and aquatic organisms, owing to its contents (14–15%) oforganic substances and phenols (up to 10 g/L). These latter compounds,
niversity of Ioannina, Ioannina
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l., Polyphenolic characterizaesearch International (2014),
characterized by high specific chemical oxygen demand (COD) and resis-tance to biodegradation, are responsible for its black color, depending ontheir state of degradation and the olives they come from (Capasso,Cristinzio, Evidente, & Scognamiglio, 1992).
For a long time, OMWWhas been regarded as hazardouswaste withnegative impact on the environment and an economic burden on theolive oil industry. Their phytotoxicity is mainly attributed to the highphenolic content (0.5–24 g/L), that, on the other hand, are antioxidantcompounds with potential health-benefits (Obied et al., 2005a). Inlight of these findings, the OMWWs are recognized as a potentiallow-cost starting material rich in bioactive compounds, that can beextracted and applied as natural antioxidants for the food and pharma-ceutical industries.
A typical phenolic substance identified in olive fruit is oleuropein, asecoiridoid glucoside that is absent in OMWW due to enzymatichydrolysis during olive oil extraction, resulting in the formation of sideproducts 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).
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The most abundant biophenols occurring in OMWW arehydroxytyrosol followed by tyrosol. In particular, hydroxytyrosol is themost potent antioxidant phenolic compound occurring in olive oil(Nissiotis & Tasioula-Margari, 2002), and numerous studies have focusedon 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 solubilityof hydroxytyrosol in oil and aqueous media and its high bioavailabilityallow its useful application inmulti-component foods, encouraging pros-pects in commercialization of it in functional foods and natural cosmetics(Bouzid et al., 2005). However, the phenolic composition of OMWWvaries strongly between studies, as it is characterized by a significantcomplexity (Bianco et al., 2003; Obied, Allen, Bedgood, Prenzler, &Robards, 2005b; Obied, Bedgood, Prenzler, & Robards, 2007) andmany compounds are recently identified (Cardoso, Falcão, Peres, &Domingues, 2011). Indeed, hydroxytyrosol acyclodihydroelenolate andp-coumaroyl-6′-secologanoside (comselogoside) were recently identi-fied inOMWWandwere 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 frommatrix such asOMWW, liquid–liquid extraction is employed. This method utilized alarge amount of solvent that has a negative impact for both health andenvironment. Membrane separation has become a promising technolo-gy with several advantages: low power consumption, water-reuse andby-products recovery, stabilization of effluent, absence of organicsolvents. Some studies are already carried on, and the OMWW may betreated efficiently by using microfiltration (MF), ultrafiltration (UF),nanofiltration (NF) and/or reverse osmosis (RO), to obtain a permeatefraction which can be discharged in aquatic systems according tonational or EU regulations or to be used for irrigation (Paraskeva,Papadakis, Tsarouchi, Kanellopoulou, & Koutsoukos, 2007).
Amembrane process for the selective fractionation and total recoveryof polyphenols, water and organic substances from OMWW was alsoproposed by Russo (2007). It was based on the preliminary MF of theOMWW, followed by two UF steps realized with 6 kDa and 1 kDamembranes, 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 MFand UF retentates can be used as fertilizers or in the production of biogasin anaerobic reactors (Garcia-Castello, Cassano, Criscuoli, Conidi, & Orioli,2010).
The current investigation aimed at identifying the phenoliccompounds in the OMWW samples belonging to different Italianand Greek olive cultivars. The OMWWs were processed usingmembrane technologies in order to investigate the impact of differ-ent cultivar in the phenolic profile of the fractions. The fractionswere quantified regarding the main polyphenols present by HPLCanalysis and were deeper studied, using LC/DAD/ESI–MSn analysis,in order to elucidate the identity of phenolic components thatcould also be a characteristic of each OMWW coming from differentolive 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 ofcertified high-performance liquid chromatography (HPLC) grade, andpure 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).
Please cite this article as: D'Antuono, I., et al., Polyphenolic characterizacultivars, after membrane technology, Food Research International (2014),
2.2. OMWW samples
All the OMWWs utilized in this study raised from mills that used athree-phase system. Five fresh OMWW samples (~30 L), among themtwo 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 regionmills and three Greek cultivars, Koroneiki, Lianolia and Asprolia (allorganic), 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 wereprocessed within 7 days after olive oil production. Acetic acid wasadded to pH 5, in the samples, to avoid phenolic compounds oxidation.
The rawmaterial was firstly sieved through a test sieve with 425 μmas porosity. This process allows the removal of large particles andcolloids from the OMWW before the microfiltration process. With thisprocedure, all traces of oil, leaves, seeds, which could then causeproblems of clogging of the membranes, are eliminated.
2.3. Filtration units (MF, UF and NF)
The raw OMWWs coming from the five different cultivars wereprocessed with a laboratory-scale system (Permeare s.r.l., Milano,Italy) present in the laboratory of ISPA-CNR of Bari. This system, thatutilizes 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 byusing a ceramic membranes, PERMAPORE EOV 1046, with cut-off ofabout ~100,000 Da (membrane porosity 0.1 μm). The volume whichcan be processed daily will be limited by the substances contained inthe fluid. In general, it is possible to treat from 200 up to 2000 L perday. This unit can support transmembrane pressure (differencesbetween 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 followingsections: process tank (5–10 L), high pressure pump, pressure vessel formembrane housing. The unit can support operating pressure up to75bar andoperating temperatures that ranges between5 °C asminimumand 60 °C as maximum. Cooling device is supplied for eventually coolingthe solution in order to maintain an acceptable temperature duringprocess. Furthermore, the maximum size of eventual suspended solidsin the feed solution should be less than 3 μm. Pressure vessel formembrane is composed of three AISI316 stainless steel parts: testate,end cup and body for membrane housing with 5 cm of diameter and30.5 cm of length.
This unit performed ultrafiltration (UF) and nanofiltration (NF)processes, utilizing polymeric membranes at different porosities. ForUF, was employed a polyethersulfone membrane, PERMAPORE DGU1812 BS EM with cut-off of 5000 Da, instead for NF, the filtration wasperformed using a polyamide membrane, PERMAPORE AEN 1812 BSwith cut-off of 200 Da. After utilization, the membranes were washedwith alkaline detergent following themanufacture instructions becausetheOMWWcan provoke themembrane clogging during the process. Allthe membrane utilized act as a molecular sieve without any chemicalinteractions with the matrix. In addition, the ceramic membrane wasutilized for MF because they are chemically stable and mechanicallyand biologically inert. In addition, they are available only as limitedrange 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 method100 μL of properly diluted samples, calibration solutions or blank werepipetted into separate test tubes and 100 μL of F–C reagent were addedto each. The mixture was mixed well and was allowed to equilibrate.
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After exactly 2min, 800 μL of a 5% (w/v) sodium carbonate solutionwereadded. The mixture was swirled and put in a temperature bath at 40 °Cfor 20 min. Then, the tubes were rapidly cooled on the rocks and thecolor generated was read at its maximum absorption (750 nm). Theabsorbance was measured in 1-cm cuvette by the Varian Cary 50 ScanUV/visible spectrophotometer. For calibration solutions and blankpreparation, a methanolic solution at the same concentration of sampleswas used (Cicco, Lanorte, Paraggio, Viggiano, & Lattanzio, 2009). Resultswere expressed as micrograms per milliliter (μg/mL) of HT equivalents.
2.5. HPLC-DAD analysis
Analytical-scale HPLC analyses of the OMWW and fractions wereperformed with an Agilent Technologies series 1100 liquid chromatog-raphy (Waldbronn, Germany) equipped with a binary gradient pumpG1312A, a G1315A photodiode array detector, and a G1316Acolumn thermostat set at 45 °C. ChemStation for LC3D (Rev. A. 10.02)software was used for spectra and data processing. An analyticalPhenomenex (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 molecularweight phenolics two solvents were used: A, methanol and B, aceticacid–water (5:95 v/v). The elution profile of the linear gradientwas: 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 ratewas 1 mL/min and the injection volumewas 20 μL.
Please cite this article as: D'Antuono, I., et al., Polyphenolic characterizacultivars, after membrane technology, Food Research International (2014),
2.6. LC–MS analysis
2.6.1. InstrumentationAll LC–MSn experiments were performed on a quadrupole ion trap
mass analyzer (Agilent Technologies, model MSD trap SL) retrofitted toa 1100 binary HPLC system equipped with a degasser, an autosampler,a diode array detector and an electrospray ionization source (AgilentTechnologies, Karlsruhe, Germany). All hardware components werecontrolled by Agilent ChemStation software.
2.6.2. AnalysisA10 μL aliquot of ultrafiltrate fraction of OMWWsampleswasfiltered
(0.45 μm) and injected into the LC–MS instrument. Separation wasachieved on a 25 cm× 4.6mm i.d., 5 μmZorbax Eclipse XDB-C18 analyt-ical column (Alltech, Deerfield, USA), at a flow rate of 1.0 mL/min, usingas 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 OMWWUF fractionswas: 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; and75–80 min, 0–95% A. The UV/vis spectra were recorded in therange of 200–700 nm and chromatograms were acquired at 240,280 and 340 nm.
Both precursor and product (MS2 and MS3) ions scanning of thephenolic compounds were monitored between m/z 50 and m/z 1000in negative polarity. The ionization source conditions were as follows:capillary voltage, 3.5 kV; drying gas temperature, 350 °C; nitrogenflow and pressure, 11 L/min and 50 psi, respectively. Maximum
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Means with a common letter within a column are not significantly different (p ≤ 0.05).
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accumulation time of ion trap and the number of MS repetitions toobtain the MS average spectra were set at 30 and 3 ms, respectively.
2.7. Statistical analysis
For total phenolic amount, statistical differences were determinedby analysis of variance (ANOVA) followed by multiple comparisonprocedure on ranks with Student–Newman–Keuls Method, at 5%significance level, using the software SigmaPlot for Windows (Ver. 12,Systat Software Inc., San Jose, CA 95110 USA).
3. Results and discussion
3.1. Filtration units
During the filtration steps three different fractions were obtainedMF, UF and NF. The MF fraction was recovered using a ceramicmembrane with the aim to stabilize and clarify the OMWW and togive a filtrate free from high molecular weight proteins, enzymes andbacterial component. The yield of the process was from 2 to 4 L/h ofOMWW, it was depending from the wastewaters quality.
The UF fraction, instead, was obtained by a polymeric membrane at5000 Da of cut-off, with yield ranging from 1.5 to 2.4 L/h. This step givesa fraction free from colloidal substances and separates, from the watersolution, organic macromolecules at molecular weight lower thanmembrane cut-off.
Finally, the NF step was performed by amembrane with amolecularweight cut-off of 200Da. The yield of the processwas of about 6 L/h. This
Please cite this article as: D'Antuono, I., et al., Polyphenolic characterizacultivars, after membrane technology, Food Research International (2014),
step is mainly used for separating the polymeric fractions of thepolyphenols from mineral salts.
3.2. OMWW total phenolic contents
The obtained fractions were assayed by a modified Folin–Ciocalteu(FC) method to determine the total phenols content expressed as HTequivalent (Cicco et al., 2009). The results are showed in Table 1.Among Italian cultivars, Cellina showed the higher phenolic content,instead among the Greek, Lianolia was the most abundant. Consideringthe polyphenol contents in MF as 100%, the percentage of polyphenolsrecovered in UF fractions, ranged from 55% to 65%, and in NF fractionfrom 15% to 33% (Table 1). In particular, the amount of polyphenolsranged from 2456 μg/mL to 5284 μg/mL in MF; from 1404 μg/mL to3065 μg/mL in UF and from 373 μg/mL to 1583 μg/mL in NF.
The fractions were also characterized by HPLC analysis, to quantifythe main polyphenols present. As shown in Fig. 3(a, b, c), the main
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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); UFfraction b); NF fraction c).
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phenolics identified were: HT, Tyr, CAA, CUA, VB, IsoVB, caffeoyl-6-secologanoside (SEC), and comselogoside (COM). In all the fractionsanalyzed and in all CVs (Fig. 3a, b, c), the most abundant compoundis HT that represents about 70–80% of the total phenolic concentra-tion, followed by Tyr. On the contrary in MF of Coratina the mainphenolic was VB (Fig. 3a), which in the following step (UF fractionFig. 3b) is reduced with a simultaneous increase of HT, as hydrolysisproduct of VB molecule. VB was lower and in some cases absent, inthe other cultivars. The hydrolytic process affects oleuropein anddemethyloleuropein, hydrolyzing them to hydroxytyrosol, whileverbascoside, 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-
Please cite this article as: D'Antuono, I., et al., Polyphenolic characterizacultivars, after membrane technology, Food Research International (2014),
Hydroxyverbascoside diastereoisomers were detected in traces in the restof cultivars. Even though the OMWWs for the Italian cultivars were storedfor 4 days before the extraction and the respectiveOMWWfromGreek culti-vars were stored for 7 days before the extraction a considerable amount ofverbascosidewas found forCoratinaand Lianolia cultivars. Thedifferentdistri-bution of VB could be considered as a distinguishing element among them.Moreover, besides HT, themost 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-inflammatoryactivity (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.,
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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 molecularweight phenolic compounds, such as HT, Tyr, CAA and CUA. Caffeicacid, 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 thefruit 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 extractiontechnique (De Marco, Savarese, Paduano, & Sacchi, 2007). The endoge-nous enzymes and microbial activities cause a loss of the recoveredphenols. According to Obied, Bedgood, Prenzler et al. (2008) none ofstorage conditions studied (storage at 4 °C, preserve by 40% w/wethanol and 1% w/w acetic acid and storage at 4 °C) could prevent therapid decrease in phenolic concentrations and antioxidant capacity,which happened within the first 24 h.
Among the three fractions obtained, the UF was the best balancebetween the purification degree and the polyphenol enrichments(Garcia-Castello et al., 2010). For this reason the UF fraction was deepercharacterized using LC/DAD/ESI–MSn analysis in order to identify thephenolic profile of each cultivar.
3.3. LC–MS analysis
The LC/DAD/ESI–MSn analysis of the UF fractions from the fiveOMWW cultivars, led to the separation and identification of the major-ity of the constituents. Most of the compounds that were identifiedbelonged to the following classes of constituents: secoiridoids andtheir derivatives, phenyl alcohols, phenolic acids and derivatives and
Fig. 4. Total ion chromatogram and UV chromatograms at 280, 240 and 340 nmof OMWW Coraproduct of decarboxymethyl elenolic acid, 2: 3,4-dihydroxyphenylglycol, 5: HT, 4′: decardecarboxymethyl elenolic acid, 7: Tyr, 9: CAA, 12–13: β-hydroxyverbascoside diastereoisomer
Please cite this article as: D'Antuono, I., et al., Polyphenolic characterizacultivars, after membrane technology, Food Research International (2014),
flavonoids. MS data were acquired in negative ionization mode,because polyphenols contain one or more hydroxyl and/or carboxylicacid groups. Identification was based on accurate mass measurementsof the pseudomolecular [M–H]− ions and their fragmentation pattern,as it has been documented in the literature. In Fig. 4, the total ioncurrent (TIC) chromatogram and UV chromatograms at 280, 240and 340 nm of UF fraction of Coratina is presented (for the rest ofthe UF fractions of different cultivars the respective chromatogramsare given in Supplementary data Supplements 1–4). Data obtainedfrom the ESI-MSn analysis of the UF fractions are summarized inTable 2.
Peak 1 exhibited a base peak [M–H]− atm/z 191, the MS2 spectrumobtained by fragmentation of the ion m/z 191 presented fragment ionsat m/z 127 ([M–CO–2H2O]−) and m/z 173 ([M–H2O]−) whichcorrespond to literature reports for quinic acid (Gouveia & Castilho,2010). Peak 2 exhibited a molecular ion at m/z 169, the fragmentationof this pseudomolecular ion yielded a fragment at m/z 151 probablyproduced by the loss [M–H–H2O]− and was tentatively identified as3,4-dihydroxyphenylglycol, that has previously identified in OMWW(Obied, Bedgood et al., 2007). Peaks 3 and 4 showed similarMS spectra,with a molecular ion at m/z ratio 315, and similar MS/MS spectra, thussuggesting thepresence of two stereoisomers thatwere not distinguish-able by mass spectrometry. The fragmentation pattern revealed twomain fragment ions at the following m/z ratios: 153, which is formedby the loss of a glucose group and 123 corresponding to loss of theCH2OH group. The two isomers were attributed to HT glucoside. Threeisomers of HT glucoside have been identified in Olea europaea, namelyhydroxytyrosol-1-O-glucoside, hydroxytyrosol-3′-O-glucoside andhydroxytyrosol-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 derivedproducts.
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Peak 4′ exhibited base peaks atm/z185 (100%) and atm/z183 (70%),with fragments in its MS2 atm/z 111 (100%) and atm/z 165 (14%). Theion at m/z 183 could probably be assigned to the de(carboxymethyl)elenolic acid derivative ion (De La Torre-Carbot et al., 2005) and theproduct ion at m/z 111 might be caused by the loss of CO and COO ofthe elenolic derivative fragment (m/z 183) in aldehyde forms (Ramoset al., 2013). So this peak can be tentatively identified as anotherelenolic acid derivative.
The spectrum generated for peak 6 yielded a deprotonatedmoleculeatm/z 199, which could be attributed to a derivative of the dialdehydicform of decarboxymethyl elenolic acid. Peak 6was tentatively identifiedas hydroxylated product of dialdehydic form of decarboxymethylelenolic acid, as it presented a fragment at m/z 155 corresponding to aloss of 44 units, which is consistent with the fragmentation pattern ofits nonderivative form (acid group decarboxylation) (Lozano-Sanchezet al., 2011). The mass spectrum of peak 8, displayed an intense peakat 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 tothe 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 theZ fragment of a hexose (loss of 180 Da) (the hexose residue wasattributed to glucose). These results are in agreementwith the presenceof oleoside (Cardoso et al., 2005). Peak 10 showed an intensepseudomolecular peak at m/z 183 in the ESI-MS spectrum whichpresented a fragment at m/z 139, corresponding to a loss of 44 units.This compound was tentatively identified as the dialdehydic form ofdecarboxymethyl elenolic acid, that has been detected before in tableolives (Medina, Brenes, Romero, García, & De Castro, 2007) and inolive oil and wastes generated during the storage of extra virgin oliveoil (Lozano-Sanchez et al., 2011).
The mass spectrum of peak 11 exhibited a base peak atm/z 377 andin its MS2 spectrum showed fragments at m/z 197 and 153, which hasbeen identified before as oleuropein aglycon derivative in varioustautomeric forms (Bouaziz, Jemai, Khabou, & Sayadi, 2010). Theabundant peak at m/z 639, that exhibited peaks 12 and 13, is due tothe molecular ion [M–H]− of two diastereoisomers of the molecule
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β-hydroxyacteoside or β-hydroxyverbascoside (MW 640) (Innocentiet al., 2006). MS/MS fragmentation of molecular ions atm/z 639 yieldedto 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 acatechol unit, m/z 459 corresponding to the loss of a caffeyol group orrhamnose, and m/z 179, assigned to caffeic acid ion. Two diastereoiso-meric structures of the β-hydroxyl derivative of verbascoside andtwo diastereoisomeric structures of the β-hydroxyl derivative ofisoverbascoside have been recently identified in olive mill wastewaterby Cardinali et al. (2012). All of these stereoisomers have the samefragmentation scheme and, therefore, are not distinguishable by massspectrometry. The mass spectrum of peak 16 exhibited a base peak atm/z 609. Its MS2 spectrum showed fragments at m/z 301, an aglyconion, and atm/z 271 confirm the presence of rutin (Savarese, De Marco,& Sacchi, 2007).
Peaks 18 and 19 showed mass spectra with the [M–H]− molecularion species at m/z 241. The MS/MS spectra obtained for the precursorion at m/z 241 gave fragment ions with molecular weights of 139 [M–
CHCHO]− and 165 [M–OH–COOCH3]−, which among others havebeen referred for dialdehydic form of carboxymethyl elenolic acid (DiMaio et al., 2013). These peaks can be tentatively identified as elenolicacid derivatives. The mass spectrum of peak 20 gave a molecular ionat 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/z363 (−18 Da) due to the loss of a H2O unit, at m/z 349 (−32 Da), anion 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 atm/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 fromthe loss of 44 Da, [M–H–CO2]−, the species at m/z 389 representingthe oleoside structure and the ion at m/z 179 is related to the caffeoylgroup (Innocenti et al., 2006). The ESI-MS spectrum of peak 22 showeda pseudomolecular ion at m/z 539 with fragments in its MS2 spectrum
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consistent with the reported fragmentation scheme: the ion atm/z 377arises from cleavage of the glycosyl bond; the ion atm/z 307 is justifiedby the loss of a C4H6O fragment, while the fragment atm/z 275 may de-rive from rearranged fragments. These results confirm the presence ofoleuropein. The spectrum generated for peak 23 yielded a deprotonatedmolecule atm/z at 535was attributed to 6′-p-coumaroyl secologanoside(comselogoside). The ESI-MS2 spectrum of that ion showed the mainfragment at m/z 491 from the loss of 44 Da, [M–H–CO2]− along withthe 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 identifiedas HT, Tyr, CAA, CUA, VB and IsoVB respectively, by direct comparisonof their retention times and UV spectra with those of the standardsand by using their MS, MS2 and MS3 spectra.
Overall twenty three compounds, most of them detected in traceamounts, were identified in almost all the UF fractions of the differentcultivars. Results showed no significant qualitative differences in theHPLC–ESI MS phenolic profile among UF fractions from the OMWWsfrom different cultivars (Table 3). Among the UF fractions from the fiveOMWW cultivars the one coming from Coratina showed twomore com-pounds indicatedwith 1′ and 2′. Peak 1′ exhibited a base peak atm/z 315.ItsMS2 spectrum showed fragments atm/z 151 and atm/z 255. This com-pound could be tentatively identified as cornoside, a quinol glucosideidentified in the vegetation water of olives (Limiroli, Consonni, Ranalli,Bianchi, & Zetta, 1996). Cornoside was thought to derive from oxidationof the glucoside of tyrosol. Peak 2′ exhibited a base peak atm/z 199 thatcould be tentatively identified as another hydroxylated product ofdecarboxymethyl elenolic acid (Kanakis et al., 2013).
Table 3Compounds identified in the OMWW extracts in each different variety. In parenthesis are give340 nm) of UF fractions of OMWW of different cultivars for eight compounds.
20 29.9 Hydroxytyrosol acyclodihydroelenolate21 30.7 SEC22 32.6 Oleuropein23 35.7 COM
a These compounds are detected in traces in the OMWW.b The integral was taken for both compounds 18 and 19.
Please cite this article as: D'Antuono, I., et al., Polyphenolic characterizacultivars, after membrane technology, Food Research International (2014),
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 phenoliccompounds. An attempt was made to compare the integral-areas ofthe main peaks of the chromatograms in the three wavelengths, as thecompounds identified were not available as standards. In Table 3integral-areas are given for eight compounds. Differences wereobserved among the cultivars analyzed for peaks 4′ and 6. In the UFfractions of Lianolia and Cellina the above elenolic acid derivative (P.4′)and the hydroxylated product of dialdehydic form of decarboxymethylelenolic acid (P.6) were most abundant in comparison with the rest ofthe cultivars. Also in the UF fraction of Cellina, followed by Lianolia,hydroxytyrosol glucoside (P.3) was most abundant.
Moreover differenceswere observed in the content of theUF fractionsregarding 3,4-dihydroxyphenylglycol (P.2) and β-hydroxyverbascosidediastereoisomers (P.12 and P.13). Coratina followed by Lianolia hadthe higher content of these compounds. It is noteworthy to mentionthat β-hydroxyverbascoside diastereoisomers were detected in traces inthe rest of cultivars.
Elenolic acid and derivatives constitute the iridoid part of severalimportant secondary olive metabolites, such as oleuropein andligstroside, and are often mentioned as one of their hydrolysisproducts. 3,4-Dihydroxyphenyl glycol is a hydroxylated derivativeof hydroxytyrosol. This substance may be of interest in the fields ofnutrition and pharmacology due to its powerful antioxidantproperties. It is the main metabolite produced by deamination of thehuman neurotransmitter noradrenaline (norepinephrine). Rodríguez,
n the integral-areas in the chromatographic profiles in three wavelengths (280, 240 and
OMWW samples of different varieties
Lianolia Asprolia Koroneiki Coratina Cellina
√(1065)
√(1471)
√(1684)
√(626)
√(1066)
√(1386)
√(744)
√(605)
√(2143)
√(872)
– – – √ –
d – – – √ –
√(1681)
√(207)
√(558)
√(255)
√(2206)
√ √ √ √ √√ √ √ √ √√ √ √ √ √√(15174)
√(3148)
√(1089)
√(8719)
√(9055)
√(9137)
√(6853)
√(2590)
√(5654)
√(16405)
√ √ √ √ √√ – – √ –
√ √ √ √ √√ √ √ √ √√ – – √ √√ √ √ √ √√(507)
√(a)
√(37)
√(983)
√(a)
√(564)
√(a)
√(39)
√(1072)
√(a)
√ √ √ – √√ √ √ √ √– – – √ √√ – – √ –
√ √ √ √ √
√(3805)
√(5860)
√(568)
√(4038)
√(4045)
√ √ √ √ √√ √ √ √ √√ √ √ √ √√ √ √ √ √
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Rodríguez, Fernández-Bolaños, Guillén, and Jiménez (2007) demon-strated that the antioxidant efficiency of 3,4-dihydroxyphenylglycol in water is 2–3 times higher than that of ascorbic acid orhydroxytyrosol, whereas in lipidic medium it is comparable to that ofvitamin E despite its high polarity. Moreover, De Roos et al. (2011)demonstrated that an alperujo extract may protect against plateletactivation, platelet adhesion and possibly have anti-inflammatoryproperties, 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 antioxidantproperties of these fractions from the OMWWs, as they might be ofuse in protecting food and pharmaceutical products by retarding theprocess 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 wereprocessed using membrane technologies. With this system three frac-tions were recovered MF, UF and NF, containing a different percentageof polyphenols. The fractions were characterized by HPLC analysis, toquantify the main polyphenols present: HT, Tyr, CAA, CUA, VB, IsoVB,SEC and COM. The most abundant compound was HT that representabout 70–80% of the total phenolic concentration, followed by Tyr. Onthe contrary, in Coratina MF, the main phenolic compound was VB, thatinstead, was lower and in some cases absent, in all the other cultivars.The UF fractions that represented the best balance between thepurification degree and the polyphenol enrichment were characterizedby LC/DAD/ESI–MSn analysis. Overall twenty three compounds, most ofthem detected in trace amounts, were identified in almost all the UFfractions of the different cultivars. Results showed no significant qualita-tive differences, however, quantitative differences were observed insome of the main phenolic compounds. In detail, differences wereobserved among the different cultivars regarding the presence of elenolicacid derivatives, hydroxytyrosol glucoside, and β-hydroxyverbascosidediastereoisomers. This deeper characterization could help to understandhow the olive cultivars can impact on the phenolic compositions ofOMWW.
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 TerritorialCooperation Programme “Greece–Italy 2007–2013”: “Utilization ofbiophenols from Olea europaea products — Olives, virgin olive oil andolive mill wastewater-BIO-OLEA” project. Special thanks are given tothe Mass Spectrometry Unit of the University of Ioannina for providingaccess to LC–MS/MS facilities.
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