Analytical, Nutritional and Clinical Methods
Characterization and fractionation of phenolic compounds extracted from olive oil mill wastewaters

https://doi.org/10.1016/j.foodchem.2006.10.005Get rights and content

Abstract

Liquid–liquid extraction was used in order to recover phenolic compounds from olive mill wastewater (OMWW), a polluting by-product of olive oil production process, and the extraction parameters have been optimized. HPLC analysis showed that hydroxytyrosol is the most abundant biophenol in ethyl acetate extracts from an acidified OMWW. Many other biophenols (tyrosol, caffeic acid, vanillic acid, verbascoside, luteolin-7-glucoside, dialdehydic form of decarboxymethyl oleuropein aglycon, ligstroside, luteolin) were identified.

Fractionation of OMWW phenolic extracts by reversed phase solid phase extraction (RP-SPE) allowed the separation of eight different phenolic fractions, whose radical scavenging activity was measured by the 2,2′-azinobis(3-ethylenbenzothiazoline-6-sulfonic acid) (ABTS) assay. Hydroxytyrosol, in particular, was purified from all the other biophenols, giving a fraction with a very high antioxidant activity.

With the procedure followed in the present study, from one liter of OMWW it seems possible to obtain 1 g of purified hydroxytyrosol.

Introduction

In the olive oil production process, the disposal of olive oil mill waste waters (OMWW) represents the main environmental problem. Continuous three phases extraction systems are still widely used in olive oil mills, especially in Italy, where in most cases they have not yet been replaced by more recent two-phases systems, which involve a reduction of OMWW volumes but an increased concentration in organic matter (Roig, Cayuela, & Sánchez-Monedero, 2006). Three phases extraction systems involve the addition of large amounts of water (up to 50 L/100 kg olive paste), resulting in the worldwide production of more than 30 millions m3 per year of OMWW (Borja, Alba, & Banks, 1997). This represents a great environmental problem, since this by-product is characterized by a high organic load; among the different organic substances found in OMWW, including sugars, tannins, phenolic compounds, polyalcohols, pectins and lipids (D’Annibale, Crestini, Vinciguerra, & Giovannozzi Sermanni, 1998), the toxicity, the antimicrobial activity and the consequent difficult biological degradation of OMWW are mainly due to the phenolic fraction (Bisignano et al., 1999, Borja et al., 1997). The partition coefficients (oil/water) of most olive biophenols, ranging from 6 × 10−4 to 1.5 (Rodis, Karathanos, & Mantzavinou, 2002), are in fact in favour of the water phase: the olive fruit is very rich in phenolic compounds, but only 2% of the total phenolic content of the olive fruit passes in the oil phase, while the remaining amount is lost in the OMWW (approximately 53%) and in the pomace (approximately 45%) (Rodis et al., 2002).

On the other hand, the phenolic compounds, which are very abundant in the OMWW and are the major responsible of their polluting load, are characterized by a strong antioxidant activity (Obied et al., 2005a, Visioli et al., 1999). Extraction of biologically active compounds (namely biophenols) from OMWW may turn a polluting residue into a source of natural antioxidants, object of growing interest in pharmaceutical and food industries since reactive oxygen species are involved in the onset of several human diseases and in the oxidative degradation of food.

The phenolic composition of OMWW has been studied by several recent works (Della Greca et al., 2004, Obied et al., 2005b). Servili et al. (1999) found an OMWW phenolic composition very different from that of olive fruit: while olives are very rich in secoiridoid glucosides, OMWW shows a high concentration of secoiridoid derivates, such as hydroxytyrosol and the dialdehydic form of decarboxymethyl oleuropein aglycon. The OMWW phenolic fraction is characterized by a great complexity, as demonstrated by Bianco et al. (2003), who identified 20 biophenols in OMWW using HPLC-MS-MS.

Among the biophenols present in OMWW, one of the most abundant and very interesting from a nutritional point of view is hydroxytyrosol, which has been widely studied demonstrating its antioxidant and health-beneficial properties as well as its good bioavailability: hydroxytyrosol scavenges free radicals (Visioli et al., 1999), inhibits human low-density lipoprotein (LDL) oxidation (Aruoma et al., 1998), inhibits platelet aggregation (Petroni et al., 1995) and the production of leucotriene for human neutrophyls (De La Puerta, Gutierrez, & Hoult, 1999) and shows in-vitro antimicrobial activity (Bisignano et al., 1999).

However, hydroxytyrosol is not commercially available in high amount as food additive. Several methods have been proposed for the production of hydroxytyrosol by means of chemical (Tuck, Tan, & Hayball, 2000) or enzymatic synthesis (Espin, Soler-Rivas, Cantos, Tomas-Barberan, & Wichers, 2001), but the protocols are usually slow and expensive, resulting in a little number of commercially available products containing pure hydroxytyrosol and in a high cost of them. By-products from processing materials of biological origin, such as wastewaters from olive oil mills, may then become important sources of high added value compounds, such as hydroxytyrosol or other antioxidant biophenols.

Several protocols have been developed for the purification of hydroxytyrosol and other phenolic compounds from OMWW, also with membrane technologies, but they are under patent protection (Crea, 2002, Fernandez-Bolanos et al., 2002, Pizzichini and Russo, 2005, Villanova et al., 2006).

Recent studies have also dealt with the fractionation of phenolic extracts and with the evaluation of the antioxidant activity of purified compounds (Carrasco-Pancorbo et al., 2005, Fki et al., 2005, Morello et al., 2005).

The present study was aimed to verify the efficiency of the liquid–liquid extraction (LLE) for the recovery of biophenols from OMWW; a second purpose was to fractionate the phenolic extracts by Solid Phase Extraction (SPE), in order to obtain purified compounds, and to assess the antioxidant activity of each fraction, in order to determine the relative contribution of the various components to the antioxidant power of the whole phenolic extracts.

Section snippets

Materials

Fresh olive oil mill wastewaters were supplied by a continuous three phases olive processing plant located in province of Benevento (Italy). These samples, obtained from olives collected in November 2003 and immediately processed, were stored at −20 °C.

Ethyl acetate, hexane, methanol, acetonitrile, trifluoroacetic acid (TFA), ethanol and formic acid were HPLC-grade solvents purchased from Carlo Erba (Milan, Italy).

Tyrosol, caffeic acid, vanillic acid and luteolin were purchased from Fluka Co.

Biophenols extraction from OMWW

Among different extraction methods, each one with different efficiency and complexity, the liquid–liquid extraction was preferred for its simplicity and convenience (Obied et al., 2005a). In order to develop an effective (both qualitatively and quantitatively) extraction, different parameters were optimized: solvent nature, pH of OMWW, volumetric ratio between solvent and OMWW, number of extraction stages.

As solvent for the extraction, ethyl acetate was chosen which is frequently used to

Conclusion

In this study, in addition to the extraction and identification of OMWW biophenols, fractionation of phenolic extracts gave promising results. Eight fractions were obtained by RP-SPE which differed as regards the phenolic composition and consequently the radical scavenging activity. Separation of OMWW biophenols with different polarity, structural complexity and antioxidant activity was achieved quite satisfactorily. Hydroxytyrosol, in particular, was easily purified from all the other

Acknowledgement

This work was supported by the Italian Ministry of University and Research (MIUR) and by the Industria Olearia Biagio Mataluni (project “Controllo Qualità ed Innovazione Tecnologica nell’Industria Olearia”, DM 593 del 8/08/2000, Prot. MIUR 1866 del 18/02/2002).

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