Antioxidant Behavior of Olive Phenolics in Oil-in-Water Emulsions

Article pubs.acs.org/JAFC

Antioxidant Behavior of Olive Phenolics in Oil-in-Water Emulsions Vito Michele Paradiso,*,† Carla Di Mattia,*,‡ Mariagrazia Giarnetti,† Marco Chiarini,‡ Lucia Andrich,‡ and Francesco Caponio† †

Department of Soil, Plant and Food Sciences, University of Bari, Via Amendola 165/a, I-70126 Bari, Italy Faculty of Bioscience and Technology for Agriculture, Food and Environment, University of Teramo, Via Balzarini 1, Campus Coste S. Agostino, 64100 Teramo, Italy

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‡

ABSTRACT: The effect of the surrounding molecular environment (β-lactoglobulin as an emulsion stabilizer and maltodextrin as a viscosity modifier) on the antioxidant activity of three olive oil phenolic compounds (PCs) in olive oil-in-water emulsions was investigated. Oxidation potential, phenolic partitioning, and radical quenching capacity were assessed in solution and in emulsion for oleuropein, hydroxytyrosol, and tyrosol; the influence of β-lactoglobulin and maltodextrin concentration was also evaluated. Finally, the observed properties were related to the oxidative stability of the emulsions containing the PCs to explain their behavior. The order hydroxytyrosol > oleuropein > tyrosol was observed among the antioxidants for both oxidation potential and radical quenching activity. Radical quenching capacity in emulsion and anodic potential were complementary indices of antioxidant effectiveness. As the intrinsic susceptibility of an antioxidant to oxidation expressed by its anodic potential decreased, the environmental conditions (molecular interactions and changes in continuous phase viscosity) played a major role in the antioxidant effectiveness in preventing hydroperoxide decomposition. KEYWORDS: o/w emulsions, olive phenolic antioxidants, cyclic voltammetry, antiradical activity, oxidative stability

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INTRODUCTION Control of lipid oxidation is a main goal of improving nutritional properties and extending the shelf life of foods1 and can be achieved as long as oxidation and antioxidation mechanisms are well understood. Research has shown, in the past several decades, that lipid oxidation is essentially an interfacial phenomenon. Even in bulk oils, oxidative processes have an interfacial nature and take place in physical microenvironments.2,3 With regard to multiphase systems, such as emulsions, the molecular environment plays a fortiori a major role; therefore, location is as important as reactivity for all the molecules involved in oxidation.4 This has also been proven for antioxidants, whose effectiveness strictly depends on both their molecular properties and the environment surrounding them. Several theories have been formulated to explain antioxidant behavior, from the polar paradox formulated by Porter to describe differences between polar and nonpolar antioxidants5 to the cut-off effect applied by Laguerre to elucidate the nonlinear behavior of a homologous series of chlorogenate esters,6 showing more and more complex interactions underlying antioxidant effectiveness and showing that intrinsic molecular properties and environmental conditions are strictly interrelated and influence each other. Therefore, a continuous effort to illuminate mechanisms behind the observed effect of simply adding a molecule to a complex system is fundamental for improving our focus on real systems.7 Olive oil phenolic compounds (PCs) have gained an increasing amount of attention as antioxidant additives in food emulsions. They have proven to be effective in slowing oxidation and, additionally, in affecting the dispersion properties of emulsions.8,9 They can largely differ in their antioxidant efficacies, on the basis of their molecular structure;10 their © 2016 American Chemical Society

antioxidant efficacy did not depend on only molecule polarity, so that other parameters, such as the capacity and rapidity in donating hydrogen, should be considered.11 Some interesting interactions with the molecular environment were also observed. A synergistic activity with surface-active proteins was reported,12 as well as a pH-dependent radical scavenging activity.13 In a recent paper,14 we observed the effectiveness of selected olive oil PCs in slowing oxidative processes in olive oilin-water (O/W) emulsions and found that different concentrations of maltodextrin in the aqueous phase affected these mechanisms. The aim of this research was to investigate the effect that the surrounding molecular environment exerts on the properties and behavior of three differing olive oil PCs (the secoiridoid oleuropein and two phenyl-ethyl alcohols, namely, tyrosol and hydroxytyrosol, differing in the absence or presence of the odiphenol structure) and on their effectiveness in controlling oxidation in olive oil-in-water emulsions. The selected PCs were characterized in solution and in the emulsions by assessing oxidation potential, phase partitioning, and radical quenching activity. The influence of β-lactoglobulin (commonly used as a surface-active protein in the emulsions) and the concentration of maltodextrin (as an emulsion viscosity modulator) on these parameters was evaluated. Finally, the observed properties and behavior were related to the oxidative stability of the emulsions with the PCs added. Received: Revised: Accepted: Published: 5877

April 29, 2016 June 23, 2016 July 5, 2016 July 5, 2016 DOI: 10.1021/acs.jafc.6b01963 J. Agric. Food Chem. 2016, 64, 5877−5886

Article

Journal of Agricultural and Food Chemistry

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Cyclic Voltammetry. The electrochemical behavior of phenolic compounds was studied using a glassy carbon electrode and a computer-controlled potentiostat (Autolab PGSTAT12, Eco Chemie, Utrecht, The Netherlands), monitored by the General Purpose Electrochemical System (GPES3, version 4.9) software package. The instrument included a Ag/AgCl reference electrode and a platinum counter electrode. The working electrode was a glassy carbon electrode (diameter of 3 mm). Measurements were taken in phosphate buffer (50 mM, pH 7) containing 50 mM KCl, as described by Campo Dall’Orto et al.17 The experimental conditions were as follows: scan rate of 0.2 V/s, start potential of −0.3 V, first vertex potential of 0.8 V, second vertex potential of −0.3 V, and step potential of 0.00198 V for oleuropein and hydroxytyrosol; scan rate of 0.2 V/s, start potential of −0.3 V, first vertex potential of 1.0 V, second vertex potential of −0.3 V, and step potential of 0.00198 V for tyrosol. Measurements were first taken on 10−4 M buffered solutions of oleuropein, tyrosol, and hydroxytyrosol, on binary solutions made of antioxidants with 10−5 M β-lactoglobulin, and on ternary solutions made of antioxidants, 10−5 M β-lactoglobulin, and maltodextrins tested at two concentrations [1 and 15% (w/w)]. A blank consisting of systems with no antioxidants was used before each measurement. Electron Spin Resonance Measurements. The antiradical activities of the emulsions containing oleuropein, tyrosol, and hydroxytyrosol were measured by the ability of the continuous phase to quench Fremy’s radical (potassium nitrosidsulfonate). Emulsions were analyzed as they were, and aliquots of each sample were added to an equal volume of Fremy’s salt (1 mM). The same procedure was applied to evaluate the antiradical activity also on the aqueous solutions before homogenization. The electron paramagnetic resonance (EPR) spectra were recorded after 20 min at 23 °C (average temperature) on a Bruker (Karlsruhe, Germany) EMX X-band microwave bridge spectrometer, equipped with an EMX highsensitivity cavity. The microwave power and modulation amplitude were set at 0.2 mW and 0.1 mT, respectively. The other instrumental parameters were as follows: sweep width, 5 mT; sweep time, 10 s; receiver mode, first; time constant, 10 ms; modulation frequency, 100 kHz. All measurements were taken in quartz cells with an inner diameter of 0.90 mm and an outer diameter of 1.10 mm (Wilmad Glass). Spectra were recorded and analyzed using WinEPR software (Bruker). Spectra of the unreacted radical (aliquots of control emulsions or the aqueous phase prior to homogenization with no antioxidant added) were used for the computation of the percentage of radical quenching. Accelerated Oxidation of O/W Emulsions. In our previous study,14 we performed an accelerated oxidation test with the same systems that are being examined here. 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) was selected as a radical initiator of oxidation conducted in the dark at 25 °C.18 Lipid hydroperoxides were measured according to the method of Shantha and Decker19 to monitor primary oxidation, while hexanal, which arises from the oxidation of linoleic acid, and nonanal, which arises from the oxidation of oleic acid,20 were measured by HS-SPME/GC/MS as volatile secondary oxidation products. In particular, the extraction was performed with a 75 μm Carboxen/polydimethylsiloxane (CAR/ PDMS) fiber (Supelco, Bellefonte, PA) exposed in the headspace of the sample at 35 °C for 15 min. Gas chromatographic analysis of volatile compounds was conducted in splitless mode on a HP-Innowax (20 m × 0.18 mm, 0.18 μm film thickness) polar capillary column (Agilent Technologies, Santa Clara, CA). The GC peak areas were converted into amounts of each volatile compound (micrograms per milliliter) according to calibration curves. More details about the methods are reported in our previous paper.14 Curves of the oxidation products during the 28 day oxidation test were reported. In the work presented here, the areas under the curve (AUC) of hydroperoxides after oxidation for 7, 16, and 28 days and of hexanal and nonanal after oxidation for 16 and 28 days (because after 7 days the AUC could be neglected) were considered for a correlation study with the assessed antioxidant properties. Statistical Analysis. Analysis of variance (ANOVA), post hoc Fisher’s LSD test, correlation analysis, linear regression, and surface

MATERIALS AND METHODS

Materials. Crystallized and lyophilized β-lactoglobulin (BLG) from bovine milk (90% polyacrylamide gel electrophoresis purity, lot no. 030M7025V) was purchased from Sigma-Aldrich Ltd. (Steinheim, Germany). Highly refined olive oil (CAS Registry Number 8001-25-0; tested according to United States Pharmacopeia and European Pharmacopoeia; Fluka Chemie AG, Buchs, Switzerland) was used without further purification; before its use, the oil was preliminarily characterized by high-performance liquid chromatography (HPLC) analysis, and its interfacial properties were determined to exclude the presence of both phenolic and amphiphilic compounds. Experiments were performed by using oil from a single batch stored under controlled conditions (dark, 15 °C) to avoid oxidation. Three olive phenolic compounds [oleuropein (Oleu), HPLC purity of ≥90%, CAS Registry Number 32619-42-4; tyrosol (Tyr), HPLC purity of ≥99%, CAS Registry Number 501-94-0; hydroxytyrosol (OH-Tyr), HPLC purity of ≥98%, CAS Registry Number 10597-60-1] were purchased from Extrasynthese (Lyon, France). Maltodextrins (MD) from native corn starch, characterized by DE 7.5−9 and M = 30000, were obtained from Cargill (Milan, Italy). Ultrapure water from the USFELGA water purifier system from Purelab Plus (Ransbach-Baumbach, Germany) was used throughout the experiments. Phosphate buffer, sodium azide, sucrose, and Fremy’s radical were purchased from Sigma-Aldrich Ltd. All the reagents were of analytical grade. Preparation of Emulsions. Olive oil-in-water emulsions [20% (w/v)] were prepared by using a 0.5% (w/v) BLG solution in 50 mM phosphate buffer (pH 7) as continuous phase supplemented with 0.03% (w/v) sodium azide (NaN3) prior to emulsification as an antibacterial agent. Model emulsified systems were prepared either without maltodextrins (MD0) or with 1% (w/v) MD1 or 15% MD15 maltodextrin as a thickening agent. To these systems were added olive oil polyphenols oleuropein (Oleu), tyrosol (Tyr), and hydroxytyrosol (OH-Tyr) at concentrations of 10−4 M by their solubilization in the aqueous phase prior to homogenization. Maltodextrins were preliminarily dispersed in pure water at a 50% (w/v) concentration, and then the mixture was stirred with a magnetic stirrer for 45 min and slighlty heated (≈40 °C) to allow complete solubilization. The emulsions with maltodextrins were prepared by mixing an appropriate volume of a concentrated emulsion and MD aqueous solutions to obtain the desired phase volume and MD concentration in the final emulsion. Emulsions were prepared in two successive steps. A pre-emulsion was obtained with a high-shear mixing device (Ultra-Turrax yellow line DI25 basic, Ika-Werke GmbH & Co.) at 20000 rpm for 1 min. Highpressure homogenization of the pre-emulsion was then performed using a Panda Plus 2000 homogenizer (GEA Niro Soavi, Parma, Italy) by applying five homogenization cycles at 150 bar. Phenolic Partitioning. Phenolic partitioning in the aqueous phases was assessed by HPLC measurements according to the method described by Pirisi et al.15 and calculated as the mass balance between the concentrations in the aqueous phase before and after homogenization. After emulsification, the aqueous phases were separated with a method adapted from ref 16. A volume of 1 mL of fresh emulsion was diluted in 1 mL of a sucrose solution [500 g L−1 in 50 mM phosphate buffer (pH 7)]. This mix was centrifuged (5415 D centrifuge, Eppendorf) twice at 6000 rpm for 2 h. After centrifugation, two phases were observed: the creamed oil droplets at the top of the centrifuge tube and the aqueous phase of the emulsion at the bottom. The tubes were immediately frozen at −20 °C and then cut just beneath the cream phase, to separate the phases. The bottom aqueous phase was carefully extracted with the aid of a micropipette. The determination of antioxidants in the continuous and separated aqueous phases was performed with a 225 nm UV−vis detection HPLC method (Series 200, UV−vis detector, Perkin-Elmer, Norwalk, CT) according to the procedure described by Pirisi et al.;15 the percentage of partitioning in the aqueous phase was calculated by considering the initial concentration in the continuous phase. 5878

DOI: 10.1021/acs.jafc.6b01963 J. Agric. Food Chem. 2016, 64, 5877−5886

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Figure 1. Cyclic voltammograms (CVs) recorded in the solutions containing the antioxidants, the antioxidants with β-lactoglobulin (BLG), and the antioxidants with BLG and maltodextrins (MD) at different concentrations.

Table 1. Cyclic Voltammetry Parameters Measured at a 3 mm Glassy Carbon Electrode for 10−4 M Solutions of Pure Olive Phenolic Compounds, Along with Their Binary and Ternary Solutions in the Presence of β-Lactoglobulin (BLG) and Maltodextrins (MD)a Ep,a (mV) antioxidant oleuropein

tyrosol

hydroxytyrosol

BLG

MD (%)

− ● ● ● − ● ● ● − ● ● ●

0 0 1 15 0 0 1 15 0 0 1 15

Ip,a (μA)

mean

LSD testb

± ± ± ± ± ± ± ± ± ± ± ±

B,b B,ab B,b B,a A,b A,b A,a A,a B,b C,b B,b B,a

92 106 90 118 446 476 520 530 78 74 83 131

14 8 15 4 25 32 18 9 16 3 9 4

mean

LSD test

ΔE (mV)

Ip,a/Ip,c

± ± ± ± ± ± ± ± ± ± ± ±

B,a B,a A,b A,c B,a C,b B,c AB,c A,b A,a A,c B,d

46 34 44 54

1.23 1.34 1.68 1.69

39 25 48 53

1.07 1.13 1.04 0.91

6.21 6.42 5.06 3.85 6.25 5.30 3.75 3.58 7.28 8.73 5.59 3.22

1.04 0.50 0.71 0.38 0.45 0.45 0.20 0.08 0.26 0.24 0.18 0.12

a Legend: Ep,a, anodic potential; Ip,a, anodic current intensity; Ip,c, cathodic peak current; ΔE, difference between anodic and cathodic peak potentials; −, no added β-lactoglobulin; ●, with 10−5 M β-lactoglobulin; MD, maltodextrin. bFisher’s LSD test for multiple comparisons (p = 0.05). Uppercase letters are used to compare parameters of different antioxidants in the same molecular environment; lowercase letters are used to compare parameters of the same antioxidant in different molecular environments.

antioxidant effectiveness.10 The o-diphenolic antioxidants (hydroxytyrosol and oleuropein) showed, as expected, Ep,a values much lower than that of the monophenolic compound tyrosol.23 Because a strong correlation has been observed between the anodic potential of the compounds and their antioxidant properties, i.e., the lower the potential, the stronger the ability of the compound to exert its antioxidant capacity,24−26 the highest antioxidant power was thus shown by hydroxytyrsol, followed by oleuropein and then tyrosol. For all the compounds, it was possible to observe that the addition of BLG and MD generally caused a shift in the anodic peaks to higher potentials, variations that were statistically significant when MD was added at the highest concentration. The only exception was observed for hydroxytyrosol in the presence of BLG: as a consequence, hydroxytyrosol showed a potential lower than that of oleuropein in the presence of BLG. On the other hand, significant differences were found in anodic current Ip,a, where lower currents were detected as the complexity of the systems increased, especially in the presence of MD. Because the anodic current indicates the amount of compound that is oxidized at the electrode, it is mainly a diffusioncontrolled response and could be also influenced by interactions leading to antioxidant regeneration. Also, in this

response regression were performed using XLStat software (Addinsoft SARL) and Minitab 17 (Minitab Inc.).

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RESULTS AND DISCUSSION Cyclic Voltammetry of Phenolic Mixed Solutions. It is well-known from the literature that phenolic compounds can be easily detected at carbon-based electrodes and that the electrochemical response can be used to characterize the phenolic content of a sample in terms of both total amount and functional properties.17,21,22 On the other hand, when performed on equimolar solutions, electrochemical analysis can be used to evaluate and compare the intrinsic antioxidant capacity of molecules.10 Cyclic voltammograms (CVs) recorded in the solutions containing the antioxidants, the antioxidants with BLG, and the antioxidants with BLG and MD at different concentrations are illustrated in Figure 1, and the voltammetric parameters are reported in Table 1. On the scan from −300 to 1000 mV, all the compounds showed an anodic peak that occurred at 92 ± 14, 446 ± 25, and 78 ± 16 mV for oleuropein, tyrosol, and hydroxytyrosol, respectively, confirming that all three molecules can act as radical scavengers, though with quite differing energy requirements for oxidation and, therefore, differing levels of 5879


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separation, also considering the fact that both the radical initiator AAPH and Fremy’s radical for EPR measurements were water-soluble. This approach, though providing partial information, has been frequently adopted in the literature and can contribute to the interpretation of antioxidant behavior.11,38−40 Moreover, the pseudophase model, despite being widely used in surfactant-stabilized model emulsions, needs to be further validated in more complex systems such as proteinstabilized emulsions.41 The partitioning of the olive phenolic antioxidants in the continuous phase as affected by emulsion formulation is reported in Figure 2. Among the compounds,

case, the hydroxytyrosol/BLG system was an exception, because the current intensity increased with respect to that with the sole antioxidant. BLG, therefore, either enhanced or weakened the antioxidant properties of the molecules under examination. This protein has previously been reported to form noncovalent complexes, based on hydrogen bonds and van der Waals forces with phenolic antioxidants.27−30 With regard to the effect of such interactions, Ozdal et al.28 reported, in a comprehensive review, contrasting results of either enhanced or lowered antioxidant activity of protein−phenolic complexes. Dubeau et al.,31 who examined the interactions between milk proteins and tea polyphenols also by means of voltammetry, pointed out a general decrease in the antioxidant activity of phenolic antioxidants, but also that the redox capacities of tea antioxidants were not equally affected by milk proteins. Most of the available data, however, regard polyphenols as single or mixed as extracted, and only one work from the literature investigated the interaction of phenolic compounds from olive oil with different food proteins, including BLG;32 however, the affinity of olive oil phenolics for the different food proteins was found to be relatively weak compared with that of tannic acid. Amorati and Valgimigli33 reported that noncovalent bonding, involving both reactive and nonreactive functional groups, could lead to either an increase or a decrease in antioxidant activity in phenolic compounds. Another possible mechanism that may explain the observed behavior is related to the interaction of quinones, formed by oxidation of diphenols, with an array of functional groups, including amino and sulfhydryl groups, to form adducts with a resulting reduction of the molecule and regeneration of its antioxidant capacity.34,35 This would explain why the peak current was affected by the interaction with BLG more than the peak potential was. When maltodextrins were added to the solutions containing antioxidants and BLG, the differences in Ep,a values among antioxidants were confirmed, while increases (significant with 15% MD) were observed as the MD content increased, with the exception of that of tyrosol, which showed a significant increase even with 1% MD. With regard to the peak current, Ip,a of oleuropein was higher than that of tyrosol, and no longer comparable as in solutions without MD. Increasing amounts of MD determined decreased peak currents. Also, hydroxytyrosol suffered the greatest reduction in peak current, which was comparable to that of the other antioxidants in 15% MD solutions. The lower anodic current registered in the solutions supplemented with MD may be due to the slower diffusion of the species to the electrode caused by steric hindrance phenomena and differences in viscosity. Nevertheless, some other interaction should be hypothesized, because the peak potential of the antioxidants was also affected by maltodextrin. After the scan direction had been reversed from 1000 to −300 mV, a cathodic peak appeared for oleuropein and hydroxytyrosol, showing the reversibility for the oxidation reactions. The cathodic peak corresponds to the reversible reduction of the catechol moiety oxidation product, o-quinone, to the o-phenol moiety.36 If the ratio Ip,a/Ip,c is considered, it can be affirmed that a quasi-reversible reaction and a reversible reaction occurred for oleuropein and hydroxytyrosol, respectively. Phenolic Partitioning and Radical Quenching in the Aqueous Phase. Even though existing methods for evaluating antioxidant partitioning in intact emulsions that refer to the pseudophase model are available,37 in this work the partitioning was conducted in the continuous phase after emulsion

Figure 2. Partitioning of the olive phenolic antioxidants (%, w/w in aqueous phase) as affected by emulsion formulation: MD0, no added maltodextrins; MD1, with 1% maltodextrins; MD15, with 15% maltodextrins. Fisher’s LSD test was applied for multiple comparisons (p = 0.05). Uppercase letters are used to compare partitioning of different antioxidants in the same molecular environment; lowercase letters are used to compare partitioning of the same antioxidant in different molecular environments.

tyrosol showed the greatest partitioning in the aqueous phase of the MD0 emulsions, while oleuropein and hydroxytyrosol partitioned poorly with similar percentages (23−27%). The degree of partitioning decreased in the following order: tyrosol > hydroxytyrosol > oleuropein. This order is in accord with what was previously observed in the literature for oleuropein and hydroxytyrosol by using the 1-octanol/water method.42 As expected, the lowest partitioning was observed with oleuropein, in accord with its amphiphilic structure and interfacial properties that favored its location near the oil−water interface.9,14,43 The addition of MD caused a partitioning of the compounds in the aqueous phase that was significantly higher than that observed in MD0 systems, especially for oleuropein and hydroxytyrosol, with concentration-dependent behavior. The higher partitioning of oleuropein and hydroxytyrosol as a consequence of MD addition may be thus ascribed to the increase in the hydrophobicity of the aqueous phase; maltodextrins are indeed reported to vary the physicochemical properties of solutions by regrouping themselves in aqueous media and creating a local hydrophobic medium.44 The antiradical activity of the aqueous phase of the emulsions is reported in Figure 3-I. The hydrophilic Fremy’s stable radical has been used with ESR detection to allow measurements of emulsions that are not optically transparent. The aqueous radical solution was mixed with an equal volume of emulsion, and the change in the intensity of the Fremy’s radical EPR spectrum as a result of its interaction with the H-donating species partitioned in the 5880


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Figure 3. Antiradical activity (% of radical quenched) of (I) the aqueous phase of the emulsions and (II) the same aqueous solutions that were used for the cyclic voltammetry measurements: MD0, no added maltodextrins; MD1, with 1% maltodextrins; MD15, with 15% maltodextrins. Fisher’s LSD test was applied for multiple comparisons (p = 0.05). Uppercase letters are used to compare antiradical activity of different antioxidants in the same molecular environment; lowercase letters are used to compare antiradical activity of the same antioxidant in different molecular environments.

Table 2. Pearson Correlation Matrix of the Parameters Measured for Each Emulsion Type variablea

LOH-7

LOH-16

LOH-28

C6−16

C6−28

C9−16

C9−28

Ep,a Ip,a partitioning quenching (solution) quenching (emulsion) MD %

0.805 −0.163 0.580 -0.692 -0.828 −0.372

0.898 −0.438 0.851 -0.845 -0.741 0.094

0.840 −0.471 0.841 -0.814 −0.643 0.186

0.477 0.059 0.345 −0.566 −0.244 −0.621

0.809 −0.148 0.633 -0.805 -0.716 −0.239

0.332 0.123 0.225 −0.375 −0.316 -0.739

0.476 0.001 0.376 −0.484 −0.438 −0.658

a

Legend: LOH-7, LOH-16, and LOH-28, areas under the curve of hydroperoxides after accelerated oxidation for 7, 16, and 28 days, respectively; C6−16 and C6−28, areas under the curve of hexanal after accelerated oxidation for 16 and 28 days, respectively; C9−16 and C9−28, areas under the curve of nonanal after accelerated oxidation for 16 and 28 days, respectively; Ep,a, anodic potential; Ip,a, anodic current intensity; MD, maltodextrins.

discrepancy has already been evidenced in the literature, and the differences were ascribed to the fact that different methods were used for the measurement of the antiradical activity; however, further investigation may be needed to elucidate the different response.46,47 A Correlation Study To Assess the Antioxidant Behavior. To assess how the properties of the phenolic molecules described above and the surrounding environment can influence their antioxidant properties, we considered the results of our previous study of the oxidative stability of similar emulsions containing the antioxidants under examination.14 In that study, emulsions were submitted to accelerated oxidation tests and hydroperoxides and volatile oxidation compounds were determined to evaluate antioxidant activities. Hydroxytyrosol was the most effective compound in slowing oxidation, followed by oleuropein, while the effectiveness of tyrosol was limited. On the other hand, the addition of MD resulted in both the decomposition of hydroperoxides and the effectiveness of phenolic antioxidants, improving also tyrosol activity for the prevention of hydroperoxide decomposition. In the work presented here, the areas under the curve (AUC) of hydroperoxides after oxidation for 7, 16, and 28 days and of hexanal and nonanal after oxidation for 16 and 28 days (because after 7 days the AUC could be neglected) were considered as indices of primary and secondary oxidation, respectively. Correlation and regression analyses were used to relate the intrinsic and environmentally dependent properties of the antioxidant molecules with the oxidative stability of the emulsions, where the relation between the intrinsic properties of antioxidants and their effectiveness is mediated by environmental conditions and interactions. The antioxidant

continuous phase was measured. Despite it being almost completely partitioned in the aqueous phase, tyrosol was less effective in reducing the stable radical; the addition of MD caused some significant changes in the antiradical activity even though the percentage of quenching was oleuropein > tyrosol. The trend of the radical quenching generally reflected the partitioning of the compounds in the aqueous phase with the exception of the systems containing the highest percentage of MD. Such a result may be related to the increase in the viscosity caused by the high concentration of maltodextrins (η = 2.38, 2.50, and 7.34 mPa s, cv hydroxytyrosol > tyrosol. The addition of MD to the solutions led to comparable antiradical activity for oleuropein and hydroxytyrosol. This did not correspond completely to the behavior previously observed in the emulsified systems and also in the literature when both ABTS and DPPH antioxidant methods were used. 10,45 This 5881


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Journal of Agricultural and Food Chemistry effect of a phenolic compound in a colloidal system can be considered to be related to its intrinsic antioxidant properties as well as to its localization in the different pseudophaseson the assumption that water, oil, and the interface can be regarded as discrete reaction regions where partition equilibria, reaction rate, and local concentrations may be consideredto interactions with other molecules of the molecular environment, and to factors affecting diffusion of reactants and products. Table 2 reports the Pearson correlation matrix of the parameters measured for each emulsion type with their indices of oxidative stability. With regard to primary oxidation, the Ep,a potential showed a good correlation with the hydroperoxide levels (p < 0.01 in all cases). Nevertheless, the correlation suffered the high leverage of tyrosol, which showed a potential largely higher than those of the other antioxidants, as can be seen in Figure 4. On the other hand, for smaller variations in

Figure 5. Antioxidant partitioning in the aqueous phase of emulsions containing the olive phenolic antioxidants and maltodextrins (MD) at different concentrations plotted vs the oxidative stability of the corresponding emulsions, expressed as the area under the curve (AUC) of hydroperoxides after 7 (LOH-7, red), 16 (LOH-16, green), and 28 (LOH-28, blue) days under accelerated oxidation conditions. Fitted linear regressions are reported for significant correlations (see Table 2): 0, no added maltodextrins; 1, 1% maltodextrins added; 15, 15% maltodextrins added. See ref 13 for the accelerated oxidation tests.

Therefore, in spite of great variations in partitioning, hydroxytyrosol showed the same effectiveness in preventing hydroperoxide formation. This would indicate that hydroxytyrosol was an effective scavenger toward the radicals formed by the initiator in the aqueous phase as well as toward lipid radicals at interfaces. With regard to the quenching ability of antioxidants, when it was measured in solution, it showed a good correlation with hydroperoxide formation over long times; on the contrary, the measurement of quenching ability directly in an emulsion gave values that correlated well with early stage hydroperoxide formation, as shown in the scatter plots in Figure 6. In the initial stages of oxidation in such multiphase systems, antioxidant activity is specially affected by interaction of the reactant with the molecular environment. Directly measuring the quenching behavior of antioxidants in real systems allowed a better focus on both of these aspects, taking into account also changes in the surrounding environment, such as those induced by different amounts of maltodextrin. On the other hand, the measurement of antiradical activity in solution showed better relation with advanced stages of oxidation, when the radical species are abundant and interactions are less limiting toward radical reactions. Moreover, the plot shows a grouping of data points by antioxidant molecules, revealing that this parameter is suited to pointing out the molecule properties more than their response in complex systems. With regard to hexanal, as the major volatile secondary oxidation product, the AUC over 16 days of oxidation showed no significant correlation. The only correlation that was close to the threshold p value was that with the maltodextrin content (p = 0.07). The AUC at 28 days was, instead, significantly related with oxidation potential Ep,a, quenching in solution, and quenching in emulsion. Moreover, it was significantly correlated with the hydroperoxide AUC after 7 days (p = 0.01), showing that overall headspace hexanal directly depended on the

Figure 4. Anodic peak potential (Ep,a) of the solutions containing the olive phenolic antioxidants with β-lactoglobulin and maltodextrins (MD) at different concentrations plotted vs the oxidative stability of the corresponding emulsions, expressed as the area under the curve (AUC) of hydroperoxides after 7 (LOH-7, red), 16 (LOH-16, green), and 28 (LOH-28, blue) days under accelerated oxidation conditions. Fitted linear regressions are reported for significant correlations (see Table 2): 0, no added maltodextrins; 1, 1% maltodextrins added; 15, 15% maltodextrins added. See ref 13 for the accelerated oxidation tests.

Ep,a, the relation with hydroperoxide formation was not evident, as can be seen in the left part of the scatter plot in Figure 4. Therefore, Ep,a could be considered a preliminary test for screening bad and good antioxidants. Antioxidant partitioning in water was positively correlated with hydroperoxide formation over 16 and 28 days and not with hydroperoxides after 7 days (Figure 5). Considering that the oxidation initiator was in the aqueous phase, this would mean that antioxidant localization near the oxidation substrate (i.e., oil droplets) would be a key aspect to be considered for hydroperoxide formation over prolonged oxidation, while antioxidant molecules in the aqueous phase would not be insignificant during initiation and the beginning of propagation. However, hydroxytyrosol behaved differently with respect to the other antioxidants, because its partitioning seemed not to affect the overall hydroperoxide formation, which was kept almost unchanged irrespective of changes in partitioning. 5882


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Figure 6. Antiradical activity of the aqueous phase of (I) the emulsions and (II) the solutions containing the olive phenolic antioxidants and maltodextrins (MD) at different concentrations plotted vs the oxidative stability of the corresponding emulsions, expressed as the area under the curve (AUC) of hydroperoxides after 7 (LOH-7, red), 16 (LOH-16, green), and 28 (LOH-28, blue) days under accelerated oxidation conditions. Fitted linear regressions are reported for significant correlations (see Table 2): 0, no added maltodextrins; 1, 1% maltodextrins added; 15, 15% maltodextrins added. See ref 13 for the accelerated oxidation tests.

Figure 7. (I) Antiradical activity of the separated aqueous phase of the emulsions, (II) antiradical activity of the solutions, and (III) anodic peak potential (Ep,a) of the solutions containing the olive phenolic antioxidants with β-lactoglobulin and maltodextrins (MD) at different concentrations plotted vs the oxidative stability of the corresponding emulsions, expressed as the area under the curve (AUC) of hexanal after 28 days under accelerated oxidation conditions. Fitted linear regressions are reported for significant correlations (see Table 2): 0, no added maltodextrins; 1, 1% maltodextrins added; 15, 15% maltodextrins added. See ref 13 for the accelerated oxidation tests.

Figure 8. Results of the regression analysis of the oxidative stability (expressed as the area under the curve, AUC, of hexanal after 28 days under accelerated oxidation conditions) of emulsions containing the olive phenolic antioxidants and maltodextrins (MD) at different concentrations, as a function of the antiradical activity of their aqueous phase and of the anodic peak potential (Ep,a) of the corresponding solutions. (I) Plot of observed values vs calculated values and (II) contour plot of the AUC of hexanal after 28 days as a function of the anodic peak potential of the antioxidant and of antiradical activity of the aqueous phase of emulsions: 0, no added maltodextrins; 1, 1% maltodextrins added; 15, 15% maltodextrins added. See ref 13 for the accelerated oxidation tests.

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Journal of Agricultural and Food Chemistry amount of hydroperoxides formed in the initial stages of oxidation. Figure 7 plots the hexanal AUC after 28 days against the analytical parameters correlated. The plots showed some limitations of the observed correlations. The correlation of hexanal with the oxidation potential was again leveraged, as observed above, by the outstanding tyrosol, while hydroxytyrosol and oleuropein did not show a clear relation. The antiradical activity measured in solution described well the ability of each antioxidant to prevent hexanal formation, yet it was not able to describe changes in behavior as the molecular environment changed, as shown by the grouping of data points by antioxidant molecule. Finally, also the antiradical activity measured in the emulsion did not show a satisfactory relation, as evidenced by the scarce alignment of data points for hydroxytyrosol and oleuropein. A significant improvement in linearity was obtained by evaluating a regression of the AUC of hexanal as a function of antiradical activity in the emulsion and Ep,a, likely due to the fact that these two parameters, when coupled, can provide indications of the two main reaction mechanisms by which the antioxidant compounds can exert their antioxidant activity, which are H-donor and electron transfer activities, respectively. Another possible explanation for the good complementarity of these indices is that they would describe different kinds of interactions of antioxidants with the molecular environment, which can be ascribed in different degrees to electron mobility and diffusion of molecules. Figure 8 plots the observed data versus the data calculated through the obtained regression (adjusted R2 = 0.901; p values, 0.002 for regression, 0.026 for antiradical activity, 0.002 for Ep,a, and 0.005 for antiradical activity *Ep,a) and reports the obtained contour plot. The contour plot is the graphical representation of the second-order regression equation and reports the variation of the AUC of hexanal after 28 days as a function of antioxidant Ep,a and antiradical activity in emulsion. The significant interaction between the factors points out that as the intrinsic susceptibility to oxidation of an antioxidant (expressed by its anodic potential) decreases, molecular diffusivity in the environment plays a major role in its overall effectiveness in preventing hydroperoxide decomposition. Finally, the AUC of nonanal showed a correlation with only the maltodextrin content (p = 0.023 and 0.054 after 16 and 28 days, respectively). This was due to the low values of the nonanal AUC in systems with 15% maltodextrin (Figure 9). Because nonanal characterizes advanced oxidation stages in olive oil,48 its formation was presumably influenced by radical mobility more than by antioxidant performance. In our previous work, we suggested that the effect of MD on oxidative stability was related not only to the increased viscosity in the systems but also to interactions involving the emulsifier, phenolic compounds, and maltodextrins.14 The work presented here confirms this view, by combining different analytical approaches to describe antioxidant behavior. During the oxidation of lipids in heterogeneous systems, the formation of oxidation products may be affected by emulsion structure because it determines the local concentration of both reagents and products of the oxidation reactions. Lipid hydroperoxides are surface-active compounds and are thus able to accumulate at the lipid−water interface of emulsion droplets. The existence of pro-oxidants in the aqueous phase and lipid hydroperoxides at the emulsion droplet surface suggests that lipid oxidation in oil-in-water emulsions primarily occurs at the emulsion droplet interface.49 At the same time,

Figure 9. Antioxidant partitioning in the aqueous phase of emulsions containing the olive phenolic antioxidants and maltodextrins (MD) at different concentrations plotted vs the oxidative stability of the corresponding emulsions, expressed as the area under the curve (AUC) of nonanal after 16 (C9−16, green) and 28 (C9−28, blue) days under accelerated oxidation conditions. Fitted linear regressions are reported for significant correlations (see Table 2): 0, no added maltodextrins; 1, 1% maltodextrins added; 15, 15% maltodextrins added. See ref 13 for the accelerated oxidation tests.

closer interactions, of electrostatic or covalent nature, may involve not only the compounds directly involved in oxidation but also those composing the surrounding molecular environment. When all these things are considered, the thorough comprehension of the factors affecting lipid oxidation in heterogeneous systems such as emulsions may lead to the creation of tools for the protection of complex foods from deterioration. In our previous study,14 we reported that phenolic antioxidants delayed hydroperoxide decomposition with effectiveness decreasing in the following order: hydroxytyrosol > oleuropein > tyrosol. We also reported that altodextrin affected antioxidant effectiveness. The study presented here points out a significant aspect of antioxidant behavior and interactions with the molecular environment, underlying their different responses in emulsions. Both a surfactant (βlactoglobulin) and a viscosity modifier (maltodextrin) were shown to be involved in interactions with antioxidant molecules, affecting therefore their activity and the physical properties of the emulsions. Cyclic voltammetry performed on the antioxidant and electron spin resonance measured directly on the emulsion proved to give rapid and complementary indices correlated with the real behavior of the molecule in a specific molecular environment.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +39 (0)80 544 2272. *E-mail: [email protected]. Phone: +39 (0)861 266912. Funding

This research was funded within the national research project FIRB 2010, “Futuro in Ricerca”, Ministero dell’Istruzione, dell’Università e della Ricerca (code RBFR10876O, D.M. 21 September 2011, prot. n.556, Ric.21). Notes

The authors declare no competing financial interest. 5884


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