Improvement of bioactive phenol content in virgin olive oil with an olive-vegetation water concentrate produced by membrane treatment

Food Chemistry 124 (2011) 1308–1315

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Improvement of bioactive phenol content in virgin olive oil with an olive-vegetation water concentrate produced by membrane treatment Maurizio Servili , Sonia Esposto, Gianluca Veneziani, Stefania Urbani, Agnese Taticchi, Ilona Di Maio, Roberto Selvaggini, Beatrice Sordini, GianFrancesco Montedoro Dipartimento di Scienze Economico-estimative e degli Alimenti, Sezione di Tecnologie e Biotecnologie degli Alimenti, Università degli Studi di Perugia, Via S. Costanzo, 06126 Perugia, Italy

a r t i c l e

i n f o

Article history: Received 22 February 2010 Received in revised form 5 July 2010 Accepted 13 July 2010

Keywords: Hydrophilic phenols Antioxidants Olive-vegetation water Membranes treatment Crude phenolic concentrate

a b s t r a c t Olive-vegetation water (OVW) is very rich in phenols, which can cause pollution problems, due to their high hydrophilicity and antimicrobial activity. In this study, a three-phase membrane system was applied for the recovery of hydrophilic phenols from fresh OVW in an industrial plant, through a prior enzymatic treatment. This innovation yielded both a crude phenolic concentrate (CPC) and a very high reduction in OVW pollution. Furthermore, the CPC was utilised in a virgin olive oil (VOO) extraction process with the aim of improving VOO phenolic content. The results obtained with four different olive cultivars showed that the CPC extraction increased the phenolic content of the VOOs without any alteration of their aroma profiles. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Virgin olive oil (VOO) is a fundamental constituent of the Mediterranean diet that has been recognised as having an important role in human health. Indeed, the biological activities of VOO (antioxidant, anti-inflammatory, chemopreventive and anti-cancer) have been associated by numerous scientists with the presence of potent antioxidants, i.e., hydrophilic phenols (Beauchamp et al., 2005; Covas, 2009; Fabiani et al., 2008; Hashim et al., 2007; Obied, Allen, Bedgood, Prenzler, & Robards, 2005; Servili et al., 2009; Vissers, Katan, & Zock, 2004). These compounds constitute a group of secondary plant metabolites not generally present in other oils and fats. VOO contains different classes of phenols, such as phenolic acids and alcohols, flavonoids, lignans and secoiridoids (Obied, Prenzler, & Robards, 2008; Servili et al., 2004). From both a qualitative and a quantitative point of view, secoiridoids are the main bioactive compounds in VOO (Servili et al., 2004). Oleuropein, demethyloleuropein, ligstroside and nuzhenide are present in the olive drupes in a glycosidic form. In the final product, their aglyconic derivatives are present as dialdehydic structures of decarboxymethylelenolic acid linked to (3,4-dihydroxyphenyl)ethanol (3,4-DHPEA) or (p-hydroxyphenyl)ethanol (p-HPEA) (3,4-DHPEA-EDA or p-HPEA-EDA), and an isomer of oleuropein aglycone (3,4-DHPEA-EA) and ligstroside aglycone (p-HPEA* Corresponding author. Tel.: +39 075 5857942; fax: +39 075 5857916. E-mail address: [email protected] (M. Servili). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.07.042

EA) (Montedoro et al., 1993; Mulinacci et al., 2001; Owen et al., 2000; Servili et al., 2004, 2009); these substances are also found in VOO by-products such as olive-vegetation water (OVW) (De Marco, Savarese, Paduano, & Sacchi, 2007; Obied et al., 2008; Servili, Baldioli, Selvaggini, Macchioni, & Montedoro, 1999; Servili et al., 2004, 2009). Many authors have previously demonstrated the high biological values of the substances in OVW (Obied et al., 2005, 2008; Obied, Prenzler, & Robards, 2008b; Owen et al., 2004; Visioli et al., 1999; Visioli & Galli, 2003). Both VOO and OVW also contain a hydroxycinnamic acid derivative, verbascoside, which also has a high antioxidant activity (Montedoro et al., 1993; Servili et al., 2004, 2009). Furthermore, recent studies have demonstrated the presence of other novel antioxidant secoiridoids in OVW (Obied, Karuso, Prenzler, & Robards, 2007). The phenolic content of VOO and its by-products show extreme variability, due to the genetic and agronomic factors of olive cultivation and VOO extraction-process technology (Servili et al., 2004, 2009). The majority of VOO in the Mediterranean area is currently extracted by centrifugation; in Italy this technique represents more than 80% of total production (Roig, Cayuela, & Sanchez-Monedero, 2006). The three-phase centrifugation system, largely used in Italy, provides a dilution of the malaxed pastes producing 50–90 l of OVWs/100 kg of olive pastes. These wastes consist of an emulsion composed of oil, mucilage and pectin. Generally, OVW pH ranges from 4.5 to 6 and includes 3–16% organic compounds, of which 1–8% are sugars, 1.2–2.4% are nitrogen-containing compounds

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and 0.34–1.13% are phenols (Niaounakis & Halvadakis, 2004), which have antimicrobial activities (Capasso et al., 1995; Fiorentino et al., 2003; López-Fiuza, Buys, Mosquera-Corral, Omil, & Méndez, 2002). Furthermore, the pollutant potential of OVW is elevated, as its biochemical oxygen demand (BOD5) value generally ranges between 35 and 110 g/l and its chemical oxygen demand (COD) ranges from 40 to 195 g/l (Niaounakis & Halvadakis, 2004). Currently, the resolution of OVW-disposal problems is fundamental, and the idea of its valorisation is very interesting given the high biological value of its abundant secoiridoid content (Roig et al., 2006). The recovery of these substances, in fact, would represent a significant economic value increase of OVW that otherwise would represent only a disposal cost in the oil industry. In this regard, several approaches have been developed in previous studies using physico-chemical or biological treatments for the simultaneous reduction of the OVW pollutant load and the recovery of antioxidant compounds (Agalias et al., 2007; Khoufi, Aloui, & Sayadi, 2008; Kujawski et al., 2004; Paraskeva, Papadakis, Kanellopoulou, Koutsoukos, & Angelopoulos, 2007; Roig et al., 2006; Russo, 2007; Turano, Curcio, De Paola, Calabrò, & Iorio, 2002; Gortzi et al., 2008), although there are many limits for the industrial-scale application of the studied methods. The main problems are related to the complexity of the proposed processes, requiring water pretreatment, or the huge costs for the purchase and the management of the machines. The aim of the present study was to develop a recovery system for the phenolic fraction of fresh OVWs by a membrane filtration at an industrial scale. This recovery system was equipped with three consecutive membrane-filtration steps with decreasing cut-off values, allowing the production of a concentrate enriched in the phenolic fraction (crude phenolic concentrate, CPC) and a permeate purified from phenolic compounds and a major part of the organic fraction, which could thus be also recycled in the VOO-extraction process. In addition to recovering OVW phenolic compounds in the CPC, we studied its use for enriching the antioxidant content of VOO, reusing this natural olive product during the VOO-extraction process. 2. Materials and methods 2.1. Olive-vegetation water (OVW) OVW was obtained from Moraiolo Cv. olives. The drupes were processed by the extraction system of the Società Agricola Trevi ‘‘Il Frantoio” (PG, Italy) equipped with a three-phase decanter-separator system (Rapanelli Spa, Foligno, PG, Italy) with a working capacity of 1.5 ton/h. 2.2. Crude phenolic concentrate (CPC) A crude phenolic concentrate (CPC) was obtained by membrane treatments of OVWs within 24 h of their extraction. After their collection OWVs were enzymatically treated, by depolymerising enzymes, chiefly with pectinase and hemicellulosic activities (O-Max S enzymatic preparation, OE Italia S.r.l., Marsala, Italy). In particular this enzymatic preparation was added at 500 grams per ton of OWV and made to act at room temperature (20 °C) for 12 h. The following membrane treatments were microfiltration, ultrafiltration and reverse osmosis (Fig. 1). All the processes were carried out at controlled temperature (20 °C, using a heat exchanger) and under a N2 atmosphere used for reducing O2 in the headspace of the containers for the OVWcollection and storage during and after the filtration. For the microfiltration, a tubular polypropylene membrane (cut-off 0.1–0.3 lm) with a total area of 8 m2 was employed. The ultrafiltration was carried out using two spiral

OVW Enzymatic treatment Microfiltration (cut-off 0.1-0.3 μm)

concentrate

permeate Ultrafiltration (cut-off 7 kDa) concentrate

permeate reverse osmosis

permeate

CPC

Fig. 1. Flow-chart of CPC production by OVW membrane treatment. Legend: OVW = olive-vegetation water, CPC = crude phenolic concentrate.

membranes made of polyamide and traces of polysulfone with a cut-off of 7 kDa and a total area of 16 m2. In the reverse-osmosis phase, which was the last step of the OVW membrane treatment, we employed a spiral thin-film membrane (TFM) composed of Durasan™ and polysulfone with a total area of 9 m2. This membrane was able to retain molecules characterised by a molecular weight of approximately 100 Dalton. All the membranes were purchased from Permeare S.r.l. (Milano, Italy). 2.3. Virgin olive oil (VOO) VOO was obtained by processing olives from the Peranzana, Ogliarola, Coratina and Moraiolo cultivars harvested at ripening stages of 0.95, 0.98, 0.93 and 1.4, respectively, according to Pannelli, Servili, Selvaggini, Baldioli, and Montedoro (1994). The experiments were performed on an industrial scale using a Rapanelli S.p.A. industrial plant. The crushing operation, for the traditional process, was carried out in a hammer crusher (Model GR 32, Rapanelli S.p.A., Foligno, Italy). The malaxation was performed in a top-covered machine (Rapanelli S.p.A.) at 25 °C for 40 min. At the beginning of each experiment, the initial O2 pressure was 30 kPa, corresponding to the normal ambient air composition (Servili et al., 2008). This value was measured with an oxygen sensor in the malaxer headspace (Mettler Toledo; Model O2 4100). The oil separation was performed using a three-phase decanter (RAMEF model 400 ECO-G, Rapanelli S.p.A.) at low water addition (0.2:1 v:w). For each cultivar, a VOO control and a VOO with CPC addition were prepared; the latter was obtained from pastes malaxed with the addition of CPC (5% v/w) at the beginning of the malaxation. Samples of malaxed pastes were immediately frozen in liquid nitrogen and stored at 70 °C until analysis. VOO samples were filtered and stored in the dark at 13 °C until analysis. Samples of the related by-products (pomaces and OVWs) were immediately frozen in liquid nitrogen and stored at –70 °C until analysis. 2.4. Reference compounds 3,4-DHPEA was obtained from Cayman Chemical Company. (Ann Arbor, MI), while the p-HPEA was obtained from Janssen Chemical Co. (Beerse, Belgium). Oleuropein glucoside was purchased from Extrasynthèse (Genay, France). Demethyloleuropein verbascoside and nüzhenide were extracted from olive fruit

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according to the procedure reported in a previous paper (Servili et al., 1999). Briefly, the phenols were extracted from the (5 g) freeze-dried olive pulp and seeds using a mixture of methanol:water 80:20 v/v at low temperature (50 ml); the extraction procedure was performed three times. 3,4-DHPEA-EDA, p-HPEAEDA, 3,4-DHPEA-EA, the (+)-1-acetoxypinoresinol, and (+)-pinoresinol were extracted from VOO using a procedure previously reported (Montedoro et al., 1993). In short, the phenols were extracted from the oil using methanol:water 80:20 v/v; after solvent evaporation and partial purification of the crude extract obtained from the olive fruit and VOO, the phenolic separation was performed by semi-preparative high-performance liquid chromatography (HPLC) analysis using a 9.4 mm i.d.  500 mm Whatman Partisil 10 ODS-2 semi-preparative column; the mobile phase was 0.2% acetic acid in water (pH 3.1) (A)/methanol (B) at a flow rate of 6.5 ml/min, while the phenols detection was performed using a diode-array detector (DAD) (Montedoro et al., 1993). The purity of all of the substances obtained from direct extraction was tested by HPLC, and their chemical structures were verified by nuclear magnetic resonance spectroscopy (NMR) using the same operative conditions reported in previous papers (Montedoro et al., 1993; Servili et al., 1999). Pure analytical standards of volatile compounds were purchased from Fluka and Aldrich (Milan, Italy).

3. Analytical methods 3.1. COD evaluation The COD on OVW values (mg/l) before and after the membrane treatments were determined according to the official standard methods (Eaton, Clesceri, Rice, & Greenberg, 1992). 3.2. Phenolic compounds extraction and determination 3.2.1. OVW and CPC OVW and CPC phenolic compounds were evaluated by solidphase extraction (SPE) according to Servili, Selvaggini, Taticchi, and Montedoro (2001). The SPE was performed as follows: 1 ml of the OVW and CPC was loaded onto a high-load C18 cartridge (Alltech Italia S.r.l., Sedriano, Italy), and the phenolic compounds were eluted with methanol (50 ml). After solvent removal under vacuum at 30 °C, the residue was redissolved in 5 ml of methanol and then evaporated to dryness under a nitrogen flow. 3.2.2. Malaxed pastes The phenolic extraction from crushed and malaxed olive pastes was carried out according to a modification of the procedure previously described by Servili, Baldioli, Selvaggini, Macchioni, and Montedoro (1999). Olive paste (5 g) was homogenised with 100 ml of 80% methanol containing 20 mg/l sodium diethyldithiocarbamate (DIECA); the extraction was performed in triplicate. After methanol removal, the aqueous extract was used for SPE phenol separation. The SPE procedure was applied by loading 2 ml of the aqueous extract onto a 5 g/25 ml Extraclean high-load C18 cartridge (Alltech Italia S.r.l.); methanol (200 ml) was used as the eluting solvent. After removing the solvent under vacuum at 30 °C, the phenolic extract was recovered and then redissolved in methanol (1 ml). The reversed-phase HPLC analyses of phenolic extracts were conducted with an Agilent Technologies Model 1100 system, consisting of a vacuum degasser, a quaternary pump, an autosampler, a thermostatic column compartment, a diode-array detector (DAD), and a fluorimetric detector (FLD). For evaluation of the phenolic compounds (Selvaggini et al., 2006), a Spherisorb ODS-1 column (250  4.6 mm with a particle size of 5 m, Phase Separa-

tion Ltd., Deeside, UK) held at 25 °C was employed; a 20-ll sample volume was injected. 3.2.3. VOO The extraction of VOO phenols was performed as previously described by Montedoro, Servili, Baldioli, and Miniati (1992), and the HPLC analyses of the phenolic extracts were conducted with the same equipment as reported there, using a C18 column (Spherisorb ODS-1, 250  4.6 mm, with a particle size of 5 lm; Phase Separation Ltd); the injected sample volume was 20 ll. The operating conditions of the chromatographic analysis were identical to those reported previously. 3.2.4. VOO by-products The vegetation waters were treated as described above (see OVW), whereas the treatment of the phenolics of the pomaces was as follows: 20 g of sample were homogenised with 100 ml of 80% methanol containing 20 mg/l DIECA; the extraction was performed in triplicate. After methanol removal, the aqueous extract was used for SPE phenol separation. The SPE procedure was applied by loading 1 ml of the aqueous extract onto an Extraclean High-load C18 cartridge (Alltech Italia S.r.l.) and eluting with methanol (200 ml). After solvent removal under vacuum at 30 °C, the residue was redissolved in 5 ml of methanol and then evaporated to dryness under a nitrogen flow. All the samples above were analysed as described by Selvaggini et al., 2006); they were redissolved in 1 ml of methanol, filtered with a PVDF syringe filter with a cut-off of 0.2 lm, and then analysed by HPLC with an Agilent Technologies Model 1100 system. The mobile phase was composed of 0.2% acetic acid (pH 3.1) in water (solvent A)/methanol (solvent B) at a flow rate of 1 ml/ min. The gradient of solvent B changed as follows: 5% 2 min, 25% for 8 min, 40% for 10 min, 50% for 16 min, and 100% for 14 min. The final composition was maintained for 10 min, then the solvent flow returned to the initial conditions and was held for 13 min for equilibration; the total running time was 73 min. Lignans were detected by an FLD operated at an excitation wavelength of 280 nm and emission at 339 nm (Servili et al., 1999), while the other compounds were detected by DAD at 278 nm. 3.3. Volatile composition Evaluation and quantification of volatile compounds in OVW, CPC, and in VOOs were performed by headspace solid-phase microextraction followed by gas chromatography–mass spectrometry (HS-SPME-GC/MS) according to Servili et al. (2001). Three grams of product were placed into a 10-ml vial. For the sampling of the headspace volatile compounds, solid-phase microextraction (SPME) was applied as follows: all the vials were held at 35 °C, and then, the SPME fibre (a 50/30 lm DVB/Carboxen/ PDMS 1 cm in length, Stableflex; Supelco, Inc., Bellefonte, PA) was exposed to the vapour phase for 30 min to sample the volatile compounds. Afterward, the fibre was inserted into the gas chromatograph (GC) injector, set in splitless mode, using a splitless inlet liner of 0.75 mm i.d. for thermal desorption, where it was left for 10 min. All of the SPME operations were automated through the Varian CP 8410 Autoinjector (Varian, Walnut Creek, CA). 3.3.1. GC-MS analysis A Varian 4000 GC-MS equipped with a 1079 split/splitless injector (Varian, Walnut Creek, CA) was used. A fused-silica capillary column was employed (DB-Wax-ETR, 50 m, 0.32 mm ID, 1 lm film thickness; J&W Scientific, Folsom, CA). The column was operated with helium at a constant flow rate of 1.7 ml/min maintained with an electronic flow controller (EFC). The GC oven heating programme started at 35 °C. This temperature was maintained for

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8 min, then increased to 45 °C at a rate of 1.5 °C/min, increased to 150 °C at a rate of 3 °C/min, increased to 180 °C at a rate of 4 °C/ min, and finally increased to 210 °C at a rate of 3.6 °C/min; this temperature was held for 14.5 min. The total analysis time was 80 min. The injector temperature was maintained at 250 °C, the temperature for the transfer line was fixed at 170 °C, and the mass spectrometer was operated in the electron–ionisation (EI) mode at an ionisation energy of 70 eV, with scanning in the mass range of m/z 25–350 at a scan rate of 0.79 s/scan and a manifold temperature of 150 °C. The GC-MS was operated with the Varian MS Workstation Software, Version 6.6). The volatile compounds were identified by comparison of their mass spectra and retention times with those of authentic reference compounds. Integration of all the chromatographic peaks was performed by choosing the three masses with the highest intensities from among those specific for each compound, to selectively discriminate them from their nearest neighbours. The results of the peak areas were calculated on the basis of the relative calibration curve for each compound and expressed in micrograms per kilogram of oil or micrograms per gram of fresh weight (Servili et al., 2001). 3.4. Statistical analysis A priori one-way analysis of variance, using the Tukey’s honest significant differences test, was performed. All the reported statistical tests were run using the Statgraphics software package, Version 6 (Manugistics, Inc., Rockville, MA, 1992).

Table 1 Phenolic and volatile composition of OVW and CPC. Compound

OVW

Phenols (g/l) 3,4-DHPEAz p-HPEA 3,4-DHPEA-EDA Verbascoside Total phenols

0.01 0.02 4.1 0.7 4.9

CPC (0.01)a (0.04)a (0.1)a (0.1)a (0.2)a

0.03 0.01 16.9 2.4 19.3

(0.003)b (0.001)b (1.7)b (0.2)b (1.7)b

Volatile compounds (lg/l) Aldehydes Hexanalz (E)-2-Pentenal (E)-2-Hexenal

1410 44.0 187

(106)a (2.9)a (14)a

1010 395 3280

(75.5)b (25.6)b (246)b

Alcohols 1-Penten-3-ol 1-Pentanol (E)-2-Penten-1-ol 1-Hexanol (E)-3-Hexen-1-ol (Z)-3-Hexen-1-ol (Z)-2-Hexen-1-ol

677 442 828 2590 350 2840 870

(37.2)a (36.8)a (53.8)a (142)a (19.2)a (156)a (47.8)a

6930 970 5120 9550 910.0 5735.0 1432.0

(381)b (63.1)b (333)b (406)b (65.1)b (315)b (78.8)b

Esters Hexyl acetate (Z)-3-Hexenyl acetate

n.d. n.d.

n.d. n.d.

OVW = olive-vegetation water, CPC = crude phenolic concentrate; n.d. = not detected. z The phenolic and the volatile contents are the mean values of four independent experiments; numbers in parentheses represent ± standard deviation. Values in each row relative to the two methods bearing the same letter next to the value were not significantly different (p < 0.01).

4. Results and discussion 4.1. OVW volume and pollution load The membrane treatments of OVW produced a CPC with final volumes between 20% and 25% of the original OVW. The membrane treatments also yielded a strong reduction of the OVW organic load, with a drawdown of 98%. The initial OVW COD value was 12,900 mg/l; that of the final permeate from reverse osmosis was 2470 mg/l. 4.2. OVW and CPC phenolic and volatile compositions The main phenols found in CPC were the same as those in the OVW (Table 1), but their concentrations, expressed as total phenols, were fourfold higher in the CPC (Table 1). Among these, the most abundant compounds in the CPC were 3,4-DHPEA-EDA and verbascoside, whereas only small amounts of 3,4-DHPEA and p-HPEA were present in both the CPC and OWV. Low concentrations of 3,4-DHPEA in the CPC were due to the immediate OVW treatment, which was performed directly after the mechanical oil-extraction process. Consequently, the hydrolysis of 3,4DHPEA-EDA, which normally causes an increase of 3,4-DHPEA in OVW during storage, did not occur in our case. As shown in Table 1, OVW and CPC did not contain lignans or p-HPEA-EDA, which, in contrast, are generally found in considerable amounts in VOO (Servili et al., 2004). Perhaps most significantly, the phenolic compositions of CPC and OVW were strongly affected by the olive cv. (Servili et al., 2004). Regarding the olive variety, according to our previous results, obtained over the last two years on OVWs from the cvs. Moraiolo and Coratina, the average CPC phenolic concentration was between 16.9 and 31.5 g/l, with the highest values from cv. Coratina. Verbascoside was the most variable phenolic, due to the cultivar (data not shown). Table 1 shows OVW and CPC aroma profiles identified by HSSPME-GC/MS. The most important substances responsible for offflavours in the fermented OVW (Angerosa et al., 2004) were not

present, given the rapid extraction of CPC from fresh OVW without a storage period. All the other substances found in the CPC headspace samples, except hexanal, had higher concentrations than in OVW, but the effect of the process seemed to be selective. In fact, this increase involved only some characteristic VOO substances, such as (E)-2-hexenal, 1-penten-3-ol, 1-hexanol, (E)-2-penten-1ol, (E, Z)-3-hexen-1-ol and (Z)-2-hexen-1-ol. 4.3. CPC addition in the malaxed pastes Preliminary trials with 10% v/w and 5% v/w CPC additions to the pastes before the malaxing phase were performed to examine relationships between CPC volume addition and the phenolic increase in the corresponding VOOs. The results, shown in Table 2, indicated that, while the use of CPC strongly increased phenolic concentrations in the VOO, there were no significant differences between the phenolic content of VOOs extracted from pastes malaxed with 5% v/w CPC and those with 10% v/w. These results led us to hypothesise that the lowest concentration of CPC gave the same results as double that quantity because saturation of phenolic substances occurred in the oil phase of the pastes; thus, excess phenolics were lost in VOO by-products. For that reason, 5% v/w was the volume subsequently chosen to add to pastes of four typical Italian olive cultivars characterised by different phenolic content due to genetic differences. The data reported in Table 3 confirm that the addition of the CPC improved the phenolic concentration in malaxed pastes, with highest differences, in terms of absolute values, for verbascoside and 3,4-DHPEA-EDA in particular, whereas modest variations were shown among the four varieties tested. Moreover, as this phase was performed at low O2 concentration in the headspace of the covered malaxer (an initial partial pressure of 30 kPa), oxidations of phenols by polyphenol oxidase and peroxidase enzymes have been limited (Servili et al., 2008). The release of phenolics from the CPC into the oily phase during malaxation was certainly affected by their original content of

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Table 2 Phenolic composition (mg/kg) of VOOs (cv. Peranzana) from pastes malaxed without CPC addition (control) and with CPC addition at different percentages. Compound

Control

3,4-DHPEAz p-HPEA 3,4-DHPEA-EDA p-HPEA-EDA 3,4-DHPEA-EA (+)-1-Acetoxypinoresinol (+)-Pinoresinol Total phenols

8.7 12.8 100 78.9 220 17.2 20.7 459

CPC addition (10% v/w) (0.9)a (1.1)a (6.9)a (3.2)a (5.9)a (0.8)a (0.9)a (10.1)a

CPC addition (5% v/w)

14.7 12.0 252 73.1 223 18.1 20.2 613

(0.6)b (1.0)a (9.6)b (1.8)a (6.4)a (0.9)a (0.8)a (10.8)b

16.4 11.2 231 76.2 234 17.0 21.1 619

(1.2)b (0.8)a (10.2)b (3.1)a (9.1)a (0.9)a (1.0)a (12.0)b

CPC = crude phenolic concentrate. z The phenolic content is reported as the mean value of four independent experiments; numbers in parentheses represent ± standard deviation. Values in each row relative to the three methods bearing the same letter next to the value were not significantly different (p < 0.01).

Table 3 Phenolic composition (mg/100 g d.w.) of the olive pastes malaxed with and without CPC addition. Compound

3,4-DHPEAz p-HPEA Verbascoside 3,4-DHPE-EDA Oleuropein (+)-1-Acetoxypinoresinol (+)-Pinoresinol Total phenols

cv. Peranzana

cv. Ogliarola

Control

CPC

30.2 7.1 445 160 66.4 11.1 16.0 735

37.0 9.0 479 320 67.5 12.0 18.2 942

(1.2)a (0.3)a (13.9)a (5.3)a (2.6)a (0.4)a (0.6)a (15.2)a

Control (1.4)b (0.3)b (14.5)b (10.7)b (2.6)a (0.5)a (1.4)a (21.7)b

76.0 37.0 782 154 n.d 15.8 21.7 1090

cv. Moraiolo CPC

(2.9)a (1.4)a (17.6)a (4.8)a (0.6)a (0.9)a (18.5)a

85.6 45.7 796 320 n.d 16.1 20.5 1280

Control (3.3)b (1.8)b (18.6)b (9.3)b (0.7)a (0.8)a (21.1)b

54.6 27.5 387 484 169 9.2 10.5 1140

cv. Coratina CPC

(2.1)a (1.1)a (12.4)a (16.1)a (7.2)a (0.4)a (0.4)a (21.7)a

84.4 34.5 419 616 166 9.3 10.1 1340

Control (3.2)b (1.3)b (13.7)b (21.5)b (6.9)a (0.4)a (0.7)a (26.6)b

41.0 36.2 1110 1240 261 18.2 21.2 2730

CPC (1.6)a (1.4)a (25.8)a (41.5)a (10)a (0.7)a (0.8)a (49.9)a

48.3 40.4 1150 1400 254.9 18.3 21.0 2940

(1.9)b (1.6)b (26.8)a (46.8)b (9.8)a (0.8)a (1.6)a (54.9)b

CPC = crude phenolic concentrate; n.d. = not detected. z The phenolic content is reported as the mean value of four independent experiments; numbers in parentheses represent ± standard deviation. Values in each row relative to the two methods for each cultivar bearing the same letter next to the value were not significantly different (p < 0.01).

phenols, depending on the olive variety. For all the varieties, the concentration of these substances significantly increased in all the corresponding VOOs (Table 4); in fact observing the sum of individual phenols reported in Table 4 as total phenols, the increases ranged between 27% and 44% higher than the controls. The highest augmentation was found in the VOOs of cultivars Moraiolo and Ogliarola, which showed the lowest phenolic concentrations in the original malaxed pastes (Table 3), whereas in the Coratina Cv., the phenolic content was the highest in the malaxed pastes and the lowest in the respective VOO. Assuming that in this cultivar, as well as in all the varieties with high phenolic concentrations, the final amount of phenolics dissolved in the oily phase of the paste was close to the saturation value during the entire process, an additional quantity of such compounds would have produced only a marginal effect on the final phenolic content of the

VOO. Regarding the phenolic compositions of the VOOs (Table 4), CPC addition had a selective impact because only two substances, 3,4-DHPEA and the 3,4-DHPEA-EDA, were significantly increased in all the cultivars studied, whereas the ligstroside derivatives (p-HPEA and p-HPEA-EDA) and lignans did not show significant modifications. Furthermore, even if verbascoside was one of the main CPC components, it was not released into the oily phase. Furthermore, Table 5 shows the phenolic distributions between VOO by-products; the high water solubility of 3,4-DHPEA-EDA, 3,4-DHPEA and verbascoside already described by Rodis, Karathanos, and Mantzavinou (2002) was confirmed by their elevated quantities in the water phase, while the phenolic concentrations in the pomaces hardly changed with CPC addition. The preferential release of these hydrophilic phenols from malaxed pastes with CPC in the corresponding vegetation waters emphasises the opportunity

Table 4 Phenolic composition (mg/kg) of the VOOs from olive pastes malaxed with and without CPC addition. Compound

cv. Peranzana Control

3,4-DHPEAz p-HPEA 3,4-DHPEA-EDA p-HPEA-EDA 3,4-DHPEA-EA (+)-1-Acetoxypinoresinol (+)-Pinoresinol Total phenols

2.6 4.5 69.6 48.4 148 17.7 19.5 311

cv. Ogliarola CPC

(0.1)a (0.2)a (3.3)a (2.4)a (7.4)a (0.9)a (0.9)a (8.6)a

5.2 5.1 173 52.1 152 17.1 19.9 425

Control (0.3)b (0.2)b (8.2)b (2.6)a (7.6)a (0.9)a (0.9)a (11.6)b

1.7 9.1 56.9 72.3 183 12.5 22.1 357

cv. Moraiolo CPC

(0.1)a (0.4)a (2.7)a (3.6)a (12.2)a (0.6)a (1.1)a (10.3)a

5.5 7.5 138 80.2 213 15.0 25.8 485

Control (0.3)b (0.4)a (6.6)b (4.01)a (15.2)a (0.8)b (2.6)a (17.3)b

6.5 10.3 114 103 136 13.2 15.0 393

cv. Coratina CPC

(0.32)a (0.5)a (5.4)a (7.2)a (6.8)a (0.9)a (1.1)a (11.4)a

11.0 11.7 252 119 141 15.4 17.4 567

Control (0.6)b (0.9)a (12)b (8.9)a (7.1)a (1.1)a (1.2)a (16.7)b

1.9 6.3 282 216 278 13.2 18.4 816

CPC (0.1)a (0.4)a (13.4)a (10.8)a (13.9)a (0.7)a (1.2)a (22.2)a

2.9 5.3 481 220 297 14.4 18.8 1040

(0.2)b (0.5)a (39.1)b (19.9)a (24.1)a (1.2)a (1.3)a (50.1)b

CPC = crude phenolic concentrate. z The phenolic content is reported as the mean value of four independent experiments; numbers in parentheses represent ± standard deviation. Values in each row relative to the two methods for each cultivar bearing the same letter next to the value were not significantly different (p < 0.01).

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M. Servili et al. / Food Chemistry 124 (2011) 1308–1315 Table 5 Phenolic composition (mg/100 g d.w.) of pomaces and vegetation waters from olive pastes malaxed with and without CPC addition. Compound

cv. Peranzana

cv. Ogliarola

Control Pomaces 3,4-DHPEAz p-HPEA Verbascoside 3,4-DHPE-EDA Total phenols

30.5 3.4 392 208 634

Vegetation waters 3,4-DHPEA 54.4 p-HPEA 16.9 Verbascoside 1600 3,4-DHPE-EDA 1260 Total phenols 2940

CPC

cv. Moraiolo

Control

CPC

cv. Coratina

Control

CPC

Control

CPC

(1.2)a (0.1)a (15.1)a (8.1)a (17.1)a

30.8 5.4 421 323 781

(1.2)a (0.2)b (15.9)a (12.4)b (20.2)b

72.4 14.6 416 378 882

(2.8)a (0.5)a (16)a (14.7)a (21.9)a

74.7 21.0 428 485 1010

(2.82)a (0.8)b (16.2)a (18.7)b (24.9)b

54.9 12.5 3615 650 1080

(2.1)a (0.4)a (13.9)a (25.3)a (28.9)a

55.8 17.3 384 747 1200

(2.1)a (0.6)b (14.5)a (28.7)b (32.2)b

44.1 14.5 867 1410 2330

(1.7)a (0.5)a (33.3)a (43)a (54.4)a

46.4 16.9 878 1520 2460

(1.7)a (0.6)b (33.1)a (47.4)b (57.9)b

(2.1)a (0.6)a (61.7)a (49.2)a (78.9)a

55.0 18.1 1760 2060 3890

(2.1)a (0.7)a (66.4)b (79.2)b (103)b

151 148 2300 1130 3730

(5.8)a (5.3)a (88.5)a (43.8)a (99)a

156 151 2410 1920 4650

(5.9)a (5.5)a (91.1)b (74.1)b (117.7)b

102 66.5 1449.6 3815.0 5433.3

(3.9)a (2.4)a (55.8)a (148.4)a (158.6)a

111 68.5 1604.6 4595.1 6413.8

(4.2)a (2.5)a (60.6)b (176.7)b (186.9)b

54.4 16.9 1600 1260 2940

(2.9)a (2.5)a (61.9)a (51.4)a (80.5)a

55.0 18.1 1760 2060 3890

(2.9)a (2.6)a (87.7) (98.6)b (132)b

CPC = crude phenolic concentrate. z The phenolic content is reported as the mean value of four independent experiments; numbers in parentheses represent ± standard deviation. Values in each row relative to the two methods for each cultivar bearing the same letter next to the value were not significantly different (p < 0.01).

Table 6 Volatile composition (lg/kg) of the VOOs from olive pastes malaxed with and without CPC addition. Control

CPC

Control

cv. Peranzana

CPC

cv. Ogliarola

Aldehydes Hexanalz (E)-2-Pentenal (E)-2-Hexenal

1280 405 89,700

(74.4)a (30.5)a (7170)a

1250 356 75,700

(68.9)a (28.6)a (5830)a

2260 299 99,400

(131)a (17.3)a (5770)a

2020 320 89,300

(111)a (17.6)a (4910)a

Alcohols 1-Penten-3-ol 1-Pentanol (E)-2-Penten-1-ol 1-Hexanol (E)-3-Hexen-1-ol (Z)-3-Hexen-1-ol (Z)-2-Hexen-1-ol

686 23.5 457 480 10.0 367 867

(39.8)a (1.4)a (23.5)a (31.8)a (0.6)a (26.3)a (51.9)a

684 29.8 480 575 17.5 467 1030

(37.6)a (1.6)b (26.4)a (45.6)b (1)b (36.7)b (67.2)b

263 18.0 242 1350 19.5 193 1160

(18.2)a (1.0)a (17.1)a (78.2)a (1.3)a (11.2)a (78.6)a

317 16.0 276 1060 15.5 200 1330

(23.4)b (0.9)a (20.2)a (58.3)b (1.1)a (13)a (100)a

Esters Hexyl acetate (Z)-3-Hexenyl acetate

1880 2130

(147)a (138)a

1650 2690

(107)a (183.8)b

25.5 15.0

(1.5)a (0.9)a

35.5 27.5

(2.0)b (1.5)b

cv. Moraiolo

cv. Coratina

Aldehydes Hexanalz (E)-2-Pentenal (E)-2-Hexenal

884 187 103,000

(70.7)a (11.2)a (4110)a

921 205 97,800

(69.1)a (15.3)a (3420)a

943 185 119,000

(54.7)a (10.7)a (6930)a

782 210 127,000

(42.8)b (11.5)a (6990)a

Alcohols 1-Penten-3-ol 1-Pentanol (E)-2-Penten-1-ol 1-Hexanol (E)-3-Hexen-1-ol (Z)-3-Hexen-1-ol (Z)-2-Hexen-1-ol

471 15.5 340 1230 15.0 967 1820

(29.2)a (1.1)a (20.4)a (73.6)a (1.4)a (58.0)a (180)a

545 23.3 406 1700 21.0 860 2140

(38.1)a (1.6)a (30.4)b (127)b (1.7)b (64.7)a (161)a

521 27.3 377 509 15.3 199 1170

(30.2)a (1.6)a (21.8)a (29.5)a (1.2)a (11.6)a (67.7)a

551 13.3 392 524 10.3 223 1040

(32.3)a (0.7)b (21.6)a (28.8)a (0.8)b (12.3)a (57.3)a

Esters Hexyl acetate (Z)-3-Hexenyl acetate

36.5 181

(2.4)a (11.7)a

32.8 176

(2.5)a (13.2)a

11.8 28.5

(0.7)a (1.7)a

8.5 22.0

(0.5)b (1.9)b

CPC = crude phenolic concentrate. z The volatile content is reported as the mean value of four independent experiments; numbers in parentheses represent ± standard deviation. Values in each row relative to the two methods for each cultivar bearing the same letter next to the value were not significantly different (p < 0.01).

for their recovery by membrane treatments, perhaps in a continuous process including a direct CPC recycle in the VOO-extraction process. The volatile compositions of the VOOs were determined for a complete profile of the effect of CPC addition on the VOO aroma. As stated, the CPC did not contain volatile substances associated with negative sensory impact (Table 1) and, thus, its addition to the olive pastes did not cause any off flavours to arise in the corre-

sponding VOOs (Table 6). However, the presence of several typical VOO impact compounds in the CPC, such as hexanal, (E)-2-hexenal, 1-hexanol, and (E)-2-penten-1-ol (Angerosa et al., 2004; GarcíaGonzález, Tena, & Aparicio, 2007) could cause some alterations of the characteristic VOO aromas. In conclusion, we observed that the major volatile compounds characterising the typical VOO flavour (Angerosa et al., 2004; García-González et al., 2007) did not show significant modifications,

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as their concentrations in the CPC were low. These results highlight the very important selective impact of CPC addition on the hydrophilic phenolic content of VOO without consequent alteration of the aroma profile. In this regard, as also demonstrated by the various clinical studies mentioned above, reductions of LDL cholesterol oxidation observed when including VOO in the diet led to the conclusion that the phenolic concentration of VOO is a critical point regarding health benefits from consumption of this product. However, because the content of these phenolic compounds are highly variable due to various agronomic and technological factors (Servili et al., 2004), the use of CPC addition during the VOO extraction could be an important opportunity in the optimisation of VOO secoiridoid levels. Several olive cultivars, for example, are genetically characterised by a low fruit phenolic content; hence, it is very probable that the corresponding VOOs cannot guarantee some health advantages. In cases like these, the addition of CPC could improve the important health aspects of VOO while preserving the characteristic varietal flavour.

5. Conclusions CPC produced from OVWs by membrane treatment could be considered a novel product extracted from a natural matrix; one of its potential uses was demonstrated. In other applications, prior to its further purification, it could be used in food processing (e.g., to create functional foods and dietary supplements enriched with the bioactive phenols found exclusively in olives). This study is the first report of the direct use of a CPC recovered from fresh OVW in an industrial setting and on its recycling in the same food process to increase VOO phenolic concentration and consequently improve the health properties of this product. Therefore, this application could be considered a new approach in OVW exploitation, and the results are very interesting in terms of VOO improvement because of increased concentrations of the main antioxidants 3,4DHPEA-EDA and 3,4-DHPEA-EA. These compounds have previously been demonstrated to have definite relationships with the improvement of VOO oxidative stability, sensory properties (pungency and bitterness), and human health (i.e., cancer and cardiovascular disease prevention). Acknowledgments This work was supported by the FAR project ‘‘Sistema per l’estrazione e purificazione di sostanze antiossidanti naturali nell’acqua di vegetazione” of the Italian Ministry of University and Research (MIUR) (D.M. 8/8/2000, no 293) and by the Consorzio Olivicolo Italiano UNAPROL. The authors wish to thank, the Società Agricola ‘‘Il Frantoio”, Trevi (PG) and Mr. Falaluna Sergio, Mr. Giglioni Michele and Mr. Santibacci Roberto for technical assistance in the industrial implant and in the lab. References Agalias, A., Magiatis, P., Skaltsounis, A., Mikros, E., Tsarbopoulos, A., Gikas, E., et al. (2007). A new process for the management of olive oil mill waste water and recovery of natural antioxidants. Journal of Agricultural and Food Chemistry, 55, 2671–2676. Angerosa, F., Servili, M., Selvaggini, R., Taticchi, A., Esposto, S., & Montedoro, G. F. (2004). Volatile compounds in virgin olive oil: Occurrence and their relationship with the quality. Journal Chromatography A, 1054, 17–31. Beauchamp, G. K., Keast, R. S. J., Morel, D., Lin, J., Pika, J., Han, Q., et al. (2005). Ibuprofen-like activity in extra-virgin olive oil. Nature, 437, 45–46. Capasso, R., Evidente, A., Schivo, L., Orru, G., Marcialis, M. A., & Cristinzio, G. (1995). Antibacterial polyphenols from olive oil mill wastewaters. Journal of Applied Bacteriology, 79, 393–398.

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