Stability and antioxidant potential of purified olive mill wastewater extracts

Food Chemistry 131 (2012) 1312–1321

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Stability and antioxidant potential of purified olive mill wastewater extracts Jingren He a,b,⇑, Michelle Alister-Briggs c, Ted de Lyster c, Graham P. Jones b,⇑ a

College of Food Science and Engineering, Wuhan Polytechnic University, No. 68 South Xuefu Road, Changqing Garden, Wuhan 430023, Hubei, PR China School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, PMB1, Glen Osmond, South Australia 5064, Australia c Australian Olive Oil Brokerage Pty. Ltd., Legoe Road, Buckland Park, South Australia 5120, Australia b

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Article history: Received 27 September 2010 Received in revised form 27 July 2011 Accepted 26 September 2011 Available online 4 October 2011 Keywords: Olive mill wastewaters Resin Secoiridoids Total phenols Stability Antioxidant activity Storage conditions

a b s t r a c t A purified olive extract (POE) rich in phenolic and oleosidic compounds was prepared from olive mill wastewaters by adsorption onto an amphoteric polymer resin with the yield of 2.2% (w/v). In addition to complex and simple phenolic compounds found in the POE, the highly hydrophilic elenolic acid and DEDA (dialdehydic form of decarboxymethyl elenolic acid, a non-phenolic secoiridoids) were also present in high concentration (45%, w/w). Among the phenolic constituents, the major compounds are hydroxytyrosol and its glucoside, DEDA linked to hydroxytyrosol (Hy-DEDA), p-coumaric acid and caffeic acid, verbascoside and some flavonoids. The high content (23%, w/w in gallic acid equivalent) and structural diversity of these phenolic compounds confer superior antioxidant potential of POE with an IC50 less than 8.5 lg/ml determined by DPPH assay. The stability of bioactive components during storage of the extract and changes in antioxidant properties were studied under different conditions. Determination of kinetics of degradation revealed that air/oxygen is the determinant factor which influences the stability of POE under low temperature storage conditions, while exposure to sunlight did not have significant effect on their stability. Whilst an increase in storage temperature decreased the total content of phenolic compounds (20–24% reduction) and non-phenolic secoiridoid aglycons, a high antioxidant capacity was still observed particularly in the nitrogen-protected POE samples (93–95% of the initial value). This was believed to be due to the transformation of hydroxytyrosol-containing complex phenols, especially the secoiridoid ester Hy-DEDA, into free phenolic monomer. This contributed significantly to the maintenance of a high antioxidant potency of the whole extract during storage. The high antioxidant, radical scavenging activity and stability of the extract suggest potential applications in the food and pharmaceutical industries. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction One of the main by-products produced from three-phase centrifugal olive oil mills is the aqueous liquid known as olive mill wastewater (OMWW), whose disposal is considered a significant environmental issue in olive oil-producing countries. Olive mill waste is rich in a diverse range of biophenols and typically contains about 98% of the phenols present in olive fruit, either in the wastewater (approx. 53%) or in the pomace (approx. 45%) (Rodis, Karathanos, & Mantzavinou, 2002). To date, more than 50 biophenols and related compounds have been identified by hyphenated liquid chromatography in olive mill waste (Obied, Bedgood, Prenzler, & Robards, 2007). Among them, the prevalent classes of hydrophilic ⇑ Corresponding authors. Addresses: College of Food Science and Engineering, Wuhan Polytechnic University, No. 68 South Xuefu Road, Changqing Garden, Wuhan 430023, Hubei, PR China. Tel./fax: +86 27 83924790 (J. He); School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, PMB1, Glen Osmond, South Australia 5064, Australia. Tel./fax: +61 8 83036648 (G. Jones). E-mail addresses: [email protected], [email protected] (J. He), [email protected] (G.P. Jones). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.09.124

components include phenyl alcohols, phenolic acids, secoiridoids, and flavonoids. Of these, hydroxytyrosol is the main natural phenolic compound in OMWW and is characterized by a high bio-antioxidant activity. Together with other simple phenols and flavonoids, it also exhibits diverse biological activities including potential cardioprotective and cancer-preventing activities in humans as reviewed by Obied et al. (2005a). Secoiridoids are another group of important bioactive compounds found in OMWW. Oleuropein and demethyloleuropein are the predominant phenolic secoiridoids of olive fruits (Ryan, Robards, & Lavee, 1998; Ryan, Robards, & Lavee, 1999; Servili, Baldioli, Selvaggini, Macchioni, & Montedoro, 1999a), while oleosidic compounds (oleaceae-specific non-phenolic secoiridoids) generated by the hydrolysis of oleuropein and its derivatives by certain enzymes were found in high concentrations in OMWW (Lo Scalzo & Scarpati, 1993; Servili, Baldioli, Selvaggini, Macchioni, & Montedoro, 1999b). It is estimated that during olive oil extraction, almost 80% of all olive-derived oleuropein is degraded upon crushing the olives (Vasquez-Roncero, Janer del Valle, & Janer del Valle, 1976).

J. He et al. / Food Chemistry 131 (2012) 1312–1321

It has been demonstrated that oleosidic compounds such as elenolic acid and its derivatives, together with phenolic acids, were the main antimicrobials in olive oil and OMWW, exerting a more potent antibacterial activity than the well-studied oleuropein and many other simple phenols (Medina, Brenes, Romero, Garcia, & de Castro, 2007). The useful bioactive properties of olive oil mill effluent provide a stimulus for the extraction of compounds of biologically interest from this source. Indeed, developing such products as natural antioxidants and antimicrobials provides a means of increasing the value of the problematic waste. Over recent years, preparations of OMWW extracts have been obtained by liquid–liquid extraction (LLE) with ethyl acetate (Visioli et al., 1999), membrane filtration (Canepa, Marignetti, Rognini, & Clagari, 1988; Mameri et al., 2000), adsorption onto XAD resin (Agalias et al., 2007; Visioli et al., 1999) and Sephadex LH-20 gel filtration chromatography (Visioli et al., 1999). LLE was selective for low and medium molecular weight phenols; however its extraction efficiency was very low especially for some highly polar compounds such as hydroxytyrosol glucoside (Lesage-Messen et al., 2001; Obied, Allen, Bedgood, Prenzler, & Robards, 2005b). Although membrane filtration has been proposed for the treatment of OMWW, some technological difficulties related to the presence in OMWW of gelling substances that gave rise to fouling which strongly reduced the membrane efficiency have limited this particular application. Recently, Agalias et al. (2007) developed a pilot scale system using two adsorbent resins for the treatment of OMWW and the recovery of high value-added polyphenols. Subsequent purification of hydroxytyrosol was achieved by fast centrifuge partition chromatography of the enriched extract. The compositional changes resulting from the storage of raw OMWW have been investigated. Prolonged storage resulted in significant accumulation of hydroxytyrosol, while other complex monomeric and oligomeric phenolic components decreased markedly (Feki, Allouche, Bouaziz, Gargoubi, & Sayadi, 2006). Obied et al. (2005b) found that addition of 2% sodium metabisulfite to a solvent-derived crude extract of OMWW enhanced its stability. Additionally, storage of the stabilized methanolic crude extract of two-phase olive mill waste at 20 °C and 5 °C showed similar patterns of change in the total phenolic pools and also an increase in hydroxytyrosol after 30 days of storage. Antioxidant biophenols are reactive compounds, which are capable of scavenging free radicals and increasing shelf life of foodstuffs by retarding the process of oxidation and deterioration. The present work investigated the preparation and characterization of a purified extract from OMWW which was rich in phenolics and oleosidic compounds. The stability of this extract during storage under different conditions through the monitoring of its antioxidant and free radical scavenging capacities was measured. Additionally, relationships between the compositional changes of the extract and its radical scavenging activities during storage are discussed.

2. Material and methods 2.1. Chemicals and reagents Hydroxytyrosol was purchased from Cayman (Ann Arbor, MI); Verbascoside, oleuropein, apigenin, apigenin-7-O-gluoside, luteolin, luteolin-7-O-glucoside and tyrosol were from Extrasynthese (Genay, France); 5-caffeoylquinic acid, rutin, isoquercitrin, caffeic acid, vanillic acid, p-coumaric acid, gallic acid, 3,4-dihydroxyphenyl acetic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 1,1-diphenyl-2-picrylhydrazyl (DPPH) and

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Folin–Ciocalteu reagent were purchased from Sigma–Aldrich (St. Louis, MO, USA). All other chemicals used were of analytical grade. 2.2. Samples Fresh OMWW samples (100 l) were obtained from a continuous three-phase olive processing mill in South Australia. These samples were generated from the Barnea olive cultivar in the 2008 harvest season (May of 2008) and stored at 20 °C. The commercial grape extract GrapEX obtained from Tarac Technologies Australia was a concentrated extract prepared by aqueous extraction of grape marc and sold in Australia to modulate colour and sensory qualities of wine. This product has high levels of antioxidants such as anthocyanins, proanthocyanidins and flavanols. The content of total polyphenols of this extract was 301 mg/ g of gallic acid equivalent (GAE). The olive leaf extract was obtained from Naturalinbio Resource Co., Ltd. (China). The total phenolic content of this material was 153 mg GAE/g and contained about 20% oleuropein. 2.3. Preparation of purified bioactive extracts from OMWW The purified OMWW extract (POE) was produced from the waste water of olive oil mill by a series of clarification and fractionation processes. Ten litres of raw OMWW was centrifuged at 5000 rpm to remove the suspended solids and then passed through a sintered glass Buchner filtration apparatus containing a proprietary filter medium to remove any residual solids and oil. The filtered waste water was then subjected to chromatographic fractionation on a proprietary adsorption resin column. Wastewater was fed into the resin column (55 mm diameter and 90 cm height) at a flow rate of 1.5 l/h. The column was washed with sufficient water to give a colourless eluant so as to remove any nonadsorbed compounds such as inorganic salts, proteins and sugars, followed by 1.5 l of 5% aqueous ethanol to remove residual non-active impurities. Three litres of 70% ethanol was then used to recover the biophenols and other bioactive components. The ethanolic fraction was collected and concentrated on the rotary evaporator at 30 °C; the residues were subsequently lyophilized and stored at 20 °C for further analysis. 2.4. Determination of total phenol content Total phenols were determined according to the modified Folin–Ciocalteu (FC) method (Fogliano, Verde, Rndazzo, & Ritieni, 1999). A 50 ll aliquot of sample solution was added to a 10-ml volumetric flask containing 5 ml of deionized water. FC reagent (0.25 ml) was added and the contents were mixed thoroughly for 1 min. Then 0.5 ml of saturated sodium carbonate solution was added, and the flask was made up to 10 ml with water and mixed thoroughly again. The controls contained all reactants except the sample. After 60 min of reaction time the absorbance was measured at 750 nm and compared against a gallic acid calibration curve. The content of total phenols was expressed by reference to a six-point regression curve as gallic acid equivalent (GAE). All the measurements were repeated on triplicate samples and the average values are presented with the standard deviation less than 5%. 2.5. Reducing sugar assay The amount of total reducing sugar was measured by using the DNS (2,4-dinitrosalicylic acid) reagent (dissolve by stirring 1 g dinitrosalicylic acid, 200 mg crystalline phenol and 50 mg sodium sulphite in 100 ml 1% NaOH) against standard glucose solution at 550 nm according to the method of Wood and Bhat (1988).

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Appropriate dilutions are used for each test sample. Hundred microliter of sample solution was incubated with 900 ll of citrate buffer (pH 4.8, 50 mM) and 1.5 ml DNS. The reaction mixture was kept for 10 min at 100 °C. All tests and analyses were run in triplicate and the average values are presented with the standard deviation less than 5%. 2.6. Radical-scavenging activity assay Free radical-scavenging activity in the POE was evaluated using the stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) (Cos et al., 2002). Aqueous solution of sample extracts (0.5 ml) at different concentrations was mixed with 1.0 ml methanolic DPPH (6  105 mol/l). The mixture was shaken vigorously and left to stand in the dark for 60 min after which time the reduction of DPPH absorbance was measured at 517 nm. DPPH radical-scavenging activity was calculated using the equation:

Scavenging effect ð%Þ ¼ ½ðAcontrol  Asample Þ=Acontrol   100; where Acontrol is the absorbance of the DPPH solution without sample extract and Asample is the absorbance of the solution when the test sample has been added. A calibration curve was constructed by measuring the reduction of DPPH absorbance in the presence of different concentrations of Trolox (0–200 mM). The radical scavenging activity of the extracts was determined using the Trolox standard curve and results were expressed as mM Trolox equivalent antioxidant capacity (TEAC). The values presented are means of triplicate analyses that did not differ by more than 5%. BHT and other commercial extracts were used for positive control and comparison. Scavenging activity of the extracts was also estimated based on the percentage of the DPPH reduction by calculating the IC50 values (concentration in ug/ml that caused 50% inhibition of DPPH radicals) from the graph that plotted inhibition percentage against extract concentration.

Usually the identification of the major biophenols and oleosidic compounds in the OMWW samples and the prepared POE was based on the data from HPLC-DAD and LC-MS analyses, by comparison and combination of their retention times and UV–vis spectra and mass spectral data with the corresponding standards or literature data (Christophoridou, Dais, Tseng, & Spraul, 2005; Feki et al., 2006; Medina et al., 2007; Mulinacci et al., 2001; Visioli et al., 1999). For quantitative analysis, hydroxytyrosol and its glucoside, tyrosol, 3,4-dihydroxyphenyl acetic acid were determined at 280 nm using respective reference compounds; verbascoside, chlorogenic acid, caffeic acid, coumaric acid and other cinnamic derivatives were evaluated at 320 nm using respective reference compounds; nonphenolic oleosidic compounds (elenolic acid and its derivatives) were evaluated at 240 nm while decarboxymethyl oleuropein aglycon and other oleuropein derivatives (phenolic secoiridoids) were evaluated at 280 nm using oleuropein as reference with a multiplication factor to account for their respective molecular weights. Flavonoids compounds were quantified using the corresponding standards evaluated at 360 nm. All of the data reported in tables and figures represent an average of triplicate analysis. 2.8. Stability of the POE in aqueous solution during storage conditions Eight aliquots each of approximately 5% (w/v) aqueous samples of POE were freshly prepared and stored in 50 ml screw-capped glass vials. Half of the vials with 50 ml each of POE samples were placed under vacuum and then treated with nitrogen before sealing. The others with 25 ml each of POE samples were sealed with ambient air and no vacuum. Each treatment was either exposed to sunlight with storage temperature around 38 °C or protected from light and stored at 4 °C, 23 °C and 45 °C in refrigerator, temperature-controlled room and oven, respectively. The same treatments were performed for all samples sealed after each sampling. 3. Results and discussion

2.7. Chromatographic analysis and identification of extract components

3.1. Preparation and physicochemical characterization of the purified extracts

HPLC-DAD was performed using a HP 1100 liquid chromatography equipped with a DAD detector (Hewlett–Packard, Palo Alto, CA). The analytical column was a 4.6  250 mm Luna C-18 column, 5 lm (Phenomenex, Lane Cove, NSW, Australia) attach to a SecurityGuard guard cartridge (Phenomenex), maintained at 30 °C and detection was carried out using a diode array detector recorded in the 190–600 nm range. The solvents were (A), aqueous 0.1% trifluoroacetic acid and (B), acetonitrile. The gradient consisted of: 6–70% B over 70 min, 70–100% B over 5 min, and then isocratic for 10 min at a flow rate of 0.8 ml/min (Mulinacci et al., 2001). The chromatograms were acquired at 240, 280, 320 and 360 nm. The LC-MS analysis was performed using a Finnigan Surveyor series liquid chromatograph, equipped with a 150  4.6 mm i.d., 5 lm LicroCARTÒ reversed-phase C18 column thermostated at 35 °C. Mass detection was carried out by a Finnigan LCQ DECA XP MAX (Finnigan Corp., San José, Calif., USA) mass detector with an API (Atmospheric Pressure Ionization) source of ionization and an ESI (ElectroSpray Ionization) interface. The solvents were A: H2O/HCOOH (99:1), and B: HCOOH/H2O/CH3CN (0.5:19.5:80). The gradient consisted of: 6–70% B over 70 min, 70–100% B over 5 min, and then isocratic for 10 min at a flow rate of 0.8 ml/min. The capillary voltage and temperature were 4 V and 190 °C, respectively. Spectra were recorded in negative ion mode between m/z 120 and 1500. The mass spectrometer was programmed to do a series of scans and MS-MS spectra were registered using relative collision energies of 30–60 V.

Among different extraction methods, selective resin adsorption and separation has been widely used in pharmaceutical applications for plant extract preparation due to its procedural simplicity, high efficiency and ease in up-scaling. The method developed to obtain purified olive extract (POE) consisted essentially of three steps. First, the raw OMWW was clarified by centrifugation, secondly filtration on a selective filter medium was used to remove remaining suspended solids and any residual olive oil and thirdly the POE was recovered by chromatographic fractionation on selective adsorption resins. OMWW is a complex matrix consisting of diverse range of hydrophilic phenolic and non-phenolic bioactive compounds. Chemical screening of OMWW at various single wavelengths plus three-dimensional plotting of the photodiode array data revealed its complexity and the difficulty of representing the large number of compounds at a single detection wavelength. The HPLC profiles of the OMWW sample at 280, 350, 320, 230 and 510 nm are shown in Fig. 1, where hydroxytyrosol with its glucoside and dialdehydic form of decarboxymethyl elenolic acid (DEDA) are important member of two classes (phenolic and non-phenolic) of highly polar compounds are among the compounds highlighted. These are often overlooked in solid-phase extraction and cleanup procedures using different adsorption media to those used in the present study, or by solvent extraction. Therefore, resin selection is mainly based on its retention of highly hydrophylic phenolic and oleosidic (non-phenolic secoiridoids) compounds. In the present study, up to eight times

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resin bed volume of OMWW can be loaded onto the resin column where the entire amount of bioactive compounds were successfully adsorbed with over 99% of hydroxytyrosol and 98% DEDA remaining bound on the resin. After the removal of all the residues and non-bioactive impurities, a purified extract rich in phenolic and oleosidic compounds was recovered and lyophilized with the average yield of 2.2% (w/v) on the basis of original OMWW. The gross characterization of the extract is summarized in Table 1. The prepared POE was a yellow brownish powder with a characteristic odour of processed olives. The total polyphenol content of the POE was 23.5% (235 mg GAE/g ext.). The chromatographic profile of the purified extract was shown in Fig. 2A. As expected, all the

Table 1 Characteristics and chemical composition of the purified OMWW extract (all of the data reported represent an average of triplicate analysis with the percentage standard deviation ranging within 2–5%). Description

Characteristic/composition (w/w)

Appearance Odour Solubility (5% w/v in water) pH Total sugar Protein Fat Total phenols TEAC (DPPH)

Yellow brownish powder Processed olives 98% 4.0 13.6% 1.4% 0.5% 23.5% 120.4 mmol Trolox equivalent/g

important phenolic and oleosidic compounds with high to low polarities were recovered successfully from OMWW samples and the pigmented polymeric complexes responsible for the broad hump typically observed in Fig. 1 have also been significantly reduced. The concentration of major compounds identified in the extract is shown in Table 2. Five groups of compounds were present: phenyl alcohols, flavones, flavonols, secoiridoids, and phenolic acids. Elenolic acid, DEDA and their derivatives (non-phenolic secoiridoids) were the most abundant compounds in the POE (45% of the total amount), which is in agreement with previous findings (Mulinacci et al., 2001; Visioli et al., 1999), followed by hydroxytyrosol and its glucoside, hydroxytyrosol linked to DEDA (Hy-DEDA), two unknown cinnamic acid derivatives and p-coumaric acid. The total concentrations of the remaining constituents was less than 10 mg/g of the extract, among which, the amount of glucosidic flavonoids was relatively higher than that of other simple phenolic monomers. The chemical structures of the main compounds are shown in Fig. 3. Antioxidant capacity is widely used as a parameter of importance in the food and pharmaceutical industry to describe bioactive components. The activity of the POE against other synthetic products or natural commercially available extracts is shown in Fig. 4. The POE exhibited a concentration-dependent DPPH radical scavenging activity with a linear relationship at concentrations less than 20 lg/ml. The IC50 value of POE (8.2 lg/ml) was much lower than that of positive control BHT (34.2 lg/ml) (p < 0.01) and the commercial olive leaf (25.6 lg/ml) (p < 0.05) and grape extract

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(18.4 lg/ml) (p < 0.05) in all tested concentrations from 5 to 80 lg/ ml, indicating the potent radical scavenging effect of POE. At concentrations equal to the IC50 the TEAC value for POE was

120.4 mmol Trolox/g ext. (Table 1), corresponding to 512.3 mmol Trolox/g polyphenol when the concentration is reported as lg of total polyphenols/ml, which is almost equipotent with pure

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Table 2 Major phenolic compounds and non-phenolic secoiridoids present in purified OMWW extract (POE) (dry weight basis) (the content of compounds reported represent an average of triplicate analysis with the percentage standard deviation ranging within 2–5%). Compound

Peak

Retention time (min)

kmax (nm)

Content (mg/g)

Gallic acid Hydroxytyrosol glucoside Hydroxytyrosol 3,4-Dihydroxyphenyl acetic acid DEDA Oleoside Tyrosol 5-Caffeoylquinic acid Caffeic acid Cinnamic acid derivatives Verbascoside p-Coumaric acid Rutin Isoquercitrin Luteolin 7-O-glucoside Elenolic acid Caffeic acid derivative Cinnamic acid derivative Hy-DEDA Oleosidic derivative Oleosidic derivative Luteolin

1 2 3 4 5 6 7 8 9 c1,2 10 11 12 13 14 15 c3 c4 16 e1 e2 17

7.53 8.83 9.95 11.21 11.23 11.88 13.36 14.87 16.85 18.5 21.37 22.21 22.42 23.24 23.52 25.25 25.50 28.43 28.68 30.84 32.97 33.69

273 278 280 280 228 230 278 255, 285, 282, 328 332, 351 345 348 235 328, 312 280, 228 226 350

1.30 23.7 30.8 2.51 268 8.42 6.21 2.51 2.70 4.12 8.61 13.8 9.32 6.71 7.71 163 10.8 21.3 19.8 7.31 7.12 2.81

hydroxytyrosol (528.5 mmol Trolox/g hydroxytyrosol). This further demonstrated the potency of the POE and also indicated a synergic effect between hydroxytyrosol and other phenolic compounds present in the unfractionated POE. 3.2. Effect of storage time and conditions on POE composition Studies on the degradation of biophenols in the original OMWW matrix during extraction process and upon storage of the resulting crude extracts have revealed their poor stability (50–70% decrease in 30 days) due to the complex and reactive nature of OMWW, where oxidation (enzymatic and/or nonenzymatic), condensation, polymerization, and enzymatic hydrolysis can all potentially take place (Obied, Bedgood, Prenzler, & Robards, 2008; Obied et al., 2005b). It has however been reported that isolated pure hydroxytyrosol was stable for 5 days at ambient temperature, exposed to light, and in a direct continuous air current (Fernandez-Bolanos et al., 2002). Antioxidant biophenols are reactive chemical compounds that can scavenge free radicals and can potentially increase shelf life by retarding the process of oxidation and deterioration of food and pharmaceutical products during processing and storage. The stability and antioxidant capacity of bioactive components in POE during storage were investigated under various conditions. Fig. 5 shows the relative changes in the total phenolic content of aqueous solutions of POE monitored over three months of storage. As expected, air (oxygen) had the most significant effect (p < 0.05) on total phenol content of all samples stored at low to high temperatures when compared to samples treated by vacuum and nitrogen. Elevated temperature (above ambient) and sunlight exposure also had significant effect (p < 0.01) on the samples with or without nitrogen treatments, but no difference (p > 0.05) was observed between the nitrogen-protected samples stored at low temperature (4 °C and 23 °C), where 93–98% of the original amounts of total phenols are still remaining until the end of storage. At elevated temperature, the changes of total phenols content were already very pronounced during the first three-week of storage, especially for air-containing samples, when more than 10% decrease had already occurred, and a total decrease of 24% was observed over the storage period. For nitrogen-protected samples, 11% and 20% total phenols were lost by the end of storage when exposed to sunlight or stored

327 322 328 289

289 224

at 45 °C in the dark, respectively. Overall, the biophenols in the purified olive extract were very stable at low temperature which was further improved when stored in absence of air (oxygen), as demonstrated by the degradation kinetics constants of the POE shown in Table 3. The decrease of total phenols was modelled using first-order kinetics for each sample. The degradation reaction rate constants (k) were determined by calculating the slopes after linear regression from a plot of ln[C] against time, the half-life time (t1/2) is that which corresponds to the time required for a 50% reduction of the initial concentration (Table 3). The good fit for the correlation coefficients calculated from the fitting of kinetics suggested that the degradation of the phenols in aqueous solution was satisfactorily represented by first-order kinetics. The degradation constants at elevated temperature (over ambient) were approximately 6–12 times larger than that at lower temperature for all nitrogen-protected samples, and four to eight times larger for air-containing samples, indicating the influence of temperature. The corresponding t1/2 achieved at low temperature (4 °C and 23 °C) was the longest (approx. 6.3 years) for nitrogen-protected samples, and 3.79–1.88 years (4–23 °C) for air-containing samples, suggesting that at subambient storage conditions, air/oxygen is the determinant factors which influence the stability of POE. As the temperature increased to 45 °C, not much difference (p > 0.05) was observed for t1/2 values between samples with and without nitrogen treatments (183 and 160 days). The t1/2 values did not decrease for nitrogen-protected samples when exposed to sunlight, but significant difference (p < 0.05) was found between the air-containing and nitrogen-protected samples (387 and 159 days), indicating the exposure to sunlight did not seem to have significant effect as the exposure to air did on the stability of POE. 3.3. Effect of storage time and conditions on total antioxidant activity of POE and changes in individual phenolic constituents The antioxidant capacity of POE (Fig. 6) showed a slight decrease (1.7–3.2%) at low temperatures over the storage period with a similar trend observed for the changes in total phenols as shown in Fig. 5 under the same storage conditions. This is in agreement with the generalised statement made in many studies that there is a positive correlation between antioxidant activity and total phenolic content (Bouaziz, Chamkha, & Sayadi, 2004; Je-

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OH

HOOC O O

OH

5 DEDA (Dialdehydic form of decarboxymethyl elenolic acid)

OR

2 Hydroxytyrosol glucoside R=glucoside 3 Hydroxytyrosol R=H

OH COOH

OH O

COOH

RhaO

HOOC O

H

OGlu

HO

R OH

11 p-Coumaric acid R=H 9 Caffeic acid R=OH

H

H

O H O

10 Verbascoside O

6 Oleoside

OH

OH O

HO

OH

H O

COOCH3

OH

HOOC O

O-R OH

OH

O

15 Elenolic acid

12 Rutin R=Rutinose 13 Isoquercitrin R=Glucoside OH HO

OH O

RO

OH

O O

O OH

O

O 16 Hy-DEDA (Dialdehydic form of decarboxymethyl elenolic acid linked to hydroxytyrosol)

17 Leutolin R=H 14 Leutolin 7-O-Glucoside

Fig. 3. Structures of main phenolic and oleosidic compounds in POE.

105

4ºC, Nitrogen 4ºC, Air

100

100

23ºC, Nitrogen 23ºC, Air

80

% Initial concentration

radical scavenging effect (%)

90

70 60 50 40 POE extract

30

grape extract olive leaf extract

20

BHT

45ºC, Nitrogen

95

45ºC, Air Sunlight, Nitrogen

90

Sunlight, Air

85 80 75

10 0

70 0

10

20

30

40

50

60

70

80

90

concentration (µg/ml) Fig. 4. Radical scavenging effect of various extracts (POE, olive leaf extract, grape extract) and BHT.

0

20

40

60

80

100

Time (day) Fig. 5. Relative changes in the concentration of total phenols determined by Folin– Ciocalteu method in the POE samples during storage under different conditions.

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Table 3 Degradation rate constants (k), correlation coefficients (R2), half-life periods (t1/2) for total phenols in aqueous POE samples stored at different conditions (The value k reported represent an average of triplicate analysis with the percentage standard deviation ranging within 2–5%). Parameters

Air-containing

k  103 (days1) R2 t1/2 (days)

Nitrogen-protected

4 °C

23 °C

45 °C

Sunlight

4 °C

23 °C

45 °C

Sunlight

0.50 0.9449 1379

1.0 0.9430 686

4.1 0.9205 160

4.2 0.9647 159

0.30 0.9595 2305

0.30 0.9540 2304

3.8 0.9771 183

1.8 0.9969 387

105

% Initial antioxidant capacity

100 95 90 4ºC, Nitrogen

85

4ºC, Air 23ºC, Nitrogen

80

23ºC, Air 45ºC, Nitrogen 45ºC, Air Sunlight, Nitrogen Sunlight, Air

75 70 0

20

40 60 Time (days)

80

100

Fig. 6. Relative changes in the antioxidant capacity of POE samples during storage.

mai, Bouaziz, & Sayadi, 2009). However, at elevated storage temperatures, a different pattern emerged between the samples with and without nitrogen-treatment. The air-containing samples stored at 45 °C or exposed to sunlight had the biggest decrease (18%) in the antioxidant capacity, concomitant with the observed changes in the total phenol content. However, the samples stored under vacuum or nitrogen treatments still showed a particularly high antioxidant capacity (approx. 95% of the initial values) when stored at 45 °C over the storage period, and only slightly decreased to 93% of the initial antioxidant capacity when exposed to sunlight, the decrease in the antioxidant activity (Fig. 6) not being parallel to that of the total phenols shown in Fig. 5. This result indicated that the phenolic compositions of the POE samples had changed during storage at elevated temperature, as the radical scavenging activity in the extract can be attributed to the level of phenolic compounds and, in particular, the nature of monoor poly-phenols (number and position of hydroxyl substituents) (Chimi, Cillard, Cillard, & Rahmani, 1991; Rice-Evans, Miller, Nicholas, & Paganga, 1996). The POE prepared is made up of various classes of simple and complex phenolic compounds. The changes in composition of the POE samples were monitored by HPLC analysis along the storage period. The most notable changes in the HPLC chromatograms of the samples stored at elevated temperature were the gradual increase in hydroxytyrosol and a significant decrease in the amounts of phenolic secoiridoids and some other hydroxytyrosol-containing compounds. As shown in Fig. 2(B), the Hy-DEDA (peak 16) had almost disappeared completely after six-week storage, when compared with the original sample (Fig. 2(A)). The main changes of some individual compounds during storage of the POE samples are shown in Fig. 7. Chemically, Hy-DEDA (Fig. 3) is the ester of hydroxytyrosol linked to the dialdehydic form of decarboxymethyl elenolic acid. It is assumed by many researchers that Hy-DEDA is formed via

enzymatic degradation of oleuropein during the crushing (and malaxing) of olives (Montedoro et al., 1993), thus constituting one of the main phenolic secoiridoids in olive oil and OMWW (Garcia, Brenes, Garcia, Romero, & Garrido, 2003; Montedoro et al., 1993). Over the experimental storage period, an overall decrease in the concentration of Hy-DEDA was observed in all samples, although not to the same extent. The biggest decrease (over 90%) of Hy-DEDA was found in both samples stored at the highest temperature (45 °C) with and without nitrogen-protection, indicating the thermal instability and possible ester hydrolysis reactions of Hy-DEDA in storage of aqueous POE at elevated temperature. However, the degradations of verbascoside and hydroxytyrosol–glucoside were very slow (4–12%) in comparison with that observed for Hy-DEDA (p < 0.01) (Fig. 7), suggesting good thermal stability of the two glycosidic bond-linked hydroxytyrosol derivatives under the present storage conditions. This is consistent with the good stability of these compounds during the extraction process for the recovery of biophenols from olive mill waste observed by Obied et al. (2005b). Additionally, two main flavonoid glycosides (rutin and leutolin-7-glucoside) also showed a comparable slow degradation rate (20–25%) to that of hydroxytyrosol glucoside when compared to that of Hy-DEDA (p < 0.05) under the same storage conditions. In all cases, the presence of air (oxygen) slightly decreased their concentrations during storage (5–7%) when compared to that of the nitrogenprotected samples. Elenolic acid and dialdehydic form of decarboxymethyl elenolic acid (DEDA) are the main non-phenolic secoiridoids in POE and their concentration gradually decreased to 45% and 50% of the original samples, respectively, at the end of storage at 45 °C (Fig. 7). The degradation of these compounds was found to be temperature dependent with storage. At lower temperatures (23 °C and 4 °C) a much higher stability was observed, especially for the DEDA, remaining at 90–97% of the original amount, while 83–92% of the remaining elenolic acid was still present after the same period of storage (data not shown). The absence of oxygen did not have much effect (p > 0.05) on the degradation of these compounds when stored at elevated temperature (Fig. 7) possibly due to its non-phenolic nature. Hydroxytyrosol is the main phenolic component in ripe olives and in the resulting olive mill waste and is one of the most potent antioxidants among the olive phenolic compounds. Contrary to the decrease of most compounds observed over the storage period, an overall increase in the concentration of hydroxytyrosol was found in all POE samples, although not to the same extent. The biggest increase of hydroxytyrosol (75%) was found in the samples stored at 45 °C and treated with nitrogen, while a 49% increase was observed for the air-containing samples (Fig. 7). Each of the changes in hydroxytyrosol concentration can be explained by oxidation of hydroxytyrosol and replacement by fresh hydroxytyrosol formed from the hydrolysis of hydroxytyrosol-containing compounds, especially the phenolic secoiridoid ester Hy-DEDA, as mentioned above. For samples in the presence of air/oxygen, both factors were working simultaneously, antagonizing each other, which accounts for the lower increase in the free hydroxytyrosol concentration. The observed data suggests that the phenolic component is respon-

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200

Before storage Nitrogen-protected at 45 ºC

180

Air-containing at 45 ºC 160

% Initial concentration

140 120 100 80 60 40 20 0 Hydroxytyrosol

Hy-DEDA

Leutolin glucoside

Rutin

Elenolic acid

DEDA

Hydroxytyrosol glucoside

Verbascoside

Fig. 7. Relative changes in concentration in the main components of POE samples stored at 45 °C.

sible for a higher antioxidant potential in free hydroxytyrosol compared to hydroxytyrosol-containing complex phenols. Given the higher antioxidant potential in free hydroxytyrosol and its production during storage at 45 °C, it could be expected that the nitrogen-protected POE samples would still have a high antioxidant capacity when stored at high temperature, as demonstrated in Fig. 6.

4. Conclusions In summary, the results obtained in the present study clearly show that the stabilities of the main phenolic and non-phenolic compounds in purified olive extract during storage were greatly improved when compared to those in original OMWW matrix and crude extract. The content of total phenols and their radical scavenging activity in the POE samples changed slightly (<8% reduction) after 12-week storage at ambient temperature. Determination of the kinetic degradation constants revealed that air/oxygen is the determinant factors which influence the stability of POE at low temperature storage conditions, while the exposure to sunlight did not have any significant effect on their stability. Although an increase in temperature produce a significant decrease in the total concentration of phenolic compounds (20–24% reduction) and non-phenolic secoiridoid aglycons, a high antioxidant capacity was still observed particularly in the nitrogen-protected POE samples (93–95% of the initial value) during storage. The compositional changes monitored by HPLC analysis revealed that the transformation of hydroxytyrosol-containing complex phenols, especially the secoiridoid ester Hy-DEDA, into free hydroxytyrosol could contribute to the maintenance of superior antioxidant potential of POE during storage at elevated temperature. Thus, the preparation of such a stable and potent antioxidant extract as the POE might be of use in protecting food and pharmaceutical products by retarding the process of oxidation and deterioration during processing and storage. Further studies of other bioactivities associated with the non-phenolic secoiridoids (elenolic acid and its derivatives) such as antimicrobial activity, are under investigation.

Acknowledgements This research was supported by an Industry-sponsored Linkage grant from the Australia Research Council (LP0667954) with Australian Olive Oil Brokerage Pty. Ltd. Mr Paul Evangelista of Greenfield Olives is thanked for his assistance.

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