Food Biophysics (2011) 6:295302 DOI 10.

1007/s11483-010-9195-7

SPECIAL ISSUE ARTICLE

Interfacial Behavior and Antioxidant Efficiency of Olive Phenolic Compounds in O/W Olive oil Emulsions as Affected by Surface Active Agent Type
Carla D. Di Mattia & Giampiero Sacchetti & Paola Pittia

Received: 12 August 2010 / Accepted: 9 December 2010 / Published online: 23 December 2010 # Springer Science+Business Media, LLC 2010

Abstract The aim of this study was to evaluate the interfacial behavior of syringic acid, tyrosol and oleuropein, phenolic antioxidant compounds naturally present in olives and olive oils, and their ability to influence the stability to lipid oxidation of olive oil O/W emulsions. To test also the interactions of these molecules with other components and the effects on their activity, two different surfactants were used to prepare the olive oil emulsions, Tween 20, and a whey protein concentrate (WPC). All the antioxidants affected the olive oil/water interfacial tension; among them, oleuropein showed the highest interfacial activity and, thus, is supposed to locate at the interface. In emulsified state, the presence of the phenolic compounds in WPC emulsions did not cause any significant effect on the dispersion degree if compared to the control whilst a general improvement was observed in Tween 20-emulsions, in particular when oleuropein was added systems. The antioxidants were thus proven not to impair the dispersed structure but rather to improve it. As regards the oxidative stability, the antioxidants under investigation caused the occurrence of similar induction phases in the hydroperoxides production not observed in the control emulsions. In the case of secondary oxidation products, the highest inhibition was achieved in both the emulsified systems by oleuropein. In general, however, a lower amount of both primary and secondary oxidation products were observed in WPC emulsions than in Tween 20-emulsions likely due to the antioxidative effect of whey proteins.

Keywords Syringic acid . Tyrosol . Oleuropein . Tween 20 . Whey protein concentrates . Olive oil emulsions . Oxidative stability

Introduction Many natural and processed foods consist of emulsions or have been in an emulsified state at some time during their production.1 Mayonnaise, milk, ice cream, cake batters, butter, and margarine are some examples of emulsified foods in which a lipid (or water) phase is dispersed in form of small droplets in the water (or oil) phase. Investigations undertaken in this field comprises studies on simple to complex and real model systems made by different types of surfactants, solutes, and lipids, depending on the desired nutritional, sensory, and structural properties as well as stability upon storage of the final products. Olive oil represents one of the most important food lipid and ingredient in the European countries; largely used as seasoning, it is also getting more interest in the preparation of formulated and processed foods, like dressings and meat products,24 thanks to its peculiar sensory, nutritional, and functional properties.5,6 Such properties are mostly related to the particular fatty acid composition and to the presence of minor compounds like polar phenolic antioxidants and tocopherols.7 The presence, however, of an aqueous phase in a multiphasic and multicomponent system as in the case of emulsified matrices can arise several problematic aspects regarding both the dispersion state and the physical and chemical stability of the lipidic phase. Besides physical instability, dispersed lipid phases are usually more prone to auto-oxidation than bulk ones, even when characterized by a high content of antioxidant compounds, leading the

C. D. Di Mattia (*) : G. Sacchetti : P. Pittia Department of Food Science, University of Teramo, Via Carlo R. Lerici 1, Mosciano S. Angelo, 64023, Teramo, Italy e-mail: cdimattia@unite.it

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systems to the generation of undesirable off-flavors and offodors as well as potentially toxic reaction compounds. If a wide literature is available about bulk olive oil oxidation as well as the relative contribution of phenolic and tocopherolic compounds to the overall antioxidant activity,813 very few and scarcely coordinated are the information about its oxidative stability when present as dispersed phase in complex food formulations.1417 Due to the high complexity, lipid oxidation in multicomponent and multiphasic systems can be considered an interfacial phenomenon affected by the presence of antioxidant and pro-oxidant compounds and by the interactions between the various ingredients of the system.18 The presence of the aqueous phase, moreover, can often decrease the activity of antioxidants since their hydrogen donation properties result to be weakened by the formation of hydrogen-bonded complexes with water.19 As regards O/W emulsions, lipid oxidation chemistry is very dependent on the physical properties of the waterlipid interface which thus results to play a key role in the general stability of dispersed systems. Several amphyphilic compounds take part to the composition of the interface and to the determination of its properties; these compounds could be naturally present in the lipid phase, such as free fatty acids, phenolic, and tocopherolic antioxidant molecules and some degradation products formed by lipid auto-oxidation (hydroperoxides, aldehydes, ketons, and epoxides). Other amphyphilic ingredients may be intentionally added during formulation to the aim of forming the emulsified structure and confering stability over storage. It is thus of outstanding importance to take into account any possible interaction that may occurr between the various components. According to recent results, for example, it seems that antioxidant molecules like phenolic compounds can interact with proteins forming covalent complexes that can locate at the interface increasing the resistance to oxidation and influencing the antioxidant properties of the system.17,20,21 Oxidation in multiphasic systems has been the aim of many studies for the last two decades; nevertheless, at present, scientific literature is lacking of a comprehensive work that takes into consideration the role of minor compounds, and ingredients in general, in the overall stability of olive oil-based systems. To this regard, recent studies22,23 investigated the effect of the addition of phenolic antioxidant compounds characterized by different polarity (catechin, gallic acid, quercetin) on the colloidal properties and the oxidative stability of olive oil oil-in-water emulsions made with Tween 20 and/or -lactoglobulin. The aim of this work was to investigate the role of some of the phenolic compounds naturally present in olives and olive oil to inhibit lipid oxidation in olive oil-in-water

emulsions; to this aim syringic acid, tyrosol, and oleuropein were thus selected (Figure 1). To study also the role of ingredients on the activity of these molecules, different emulsifiers were used to the stabilization of the olive oilbased emulsions: Tween 20 and a whey protein concentrate.

Materials and Methods Materials Refined, bleached, and deodorized olive oil (Adriaoli, Mosciano S. Angelo (TE), Italy) was used without further purification. Experiments were carried out using oil from a single batch stored under controlled conditions (dark, 15 C) to avoid oxidation. Tween 20, tyrosol, and syringic acid were purchased from SigmaAldrich (Steinheim, Germany) while oleuropein from Extrasynthese (Lyon, France). The whey protein concentrate (WPC) was obtained by Protarmor (Saint Brice en Cogles, France) and was characterized by the following average composition: 62% -lactoglobulin, 12% glycoproteins, 5% -lactoalbumin, 5% humidity, fats<0.8%. Double-distilled and -sterilized water was used throughout the experiments. All the reagents were of analytical grade. Methods Surface and Interfacial Tension Airwater surface tension and olive oil/water interfacial tension measurements were performed at 25 C using a Du Nouy ring tensiometer (Kruss, Hamburg, Germany). Surface and interfacial tension measurements were carried out

Fig. 1 Structures of syringic acid (a), tyrosol (b), and oleuropein (c)

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on buffered solutions of antioxidants in a concentration range from 106 to 103 M. The equilibration time was of 15 mins selected on the basis of asymtosis in value noted in background studies traversing dilute and concentrated regimes. The interfacial tensions results were not corrected as they were reported as the difference between the interfacial tensions of the buffer-olive oil interface and the interfacial tension of the buffered antioxidant solutions olive oil interfacial tensions (=O/W - AOM). Preparation of the O/W emulsions The 20% (w/w) oil-inwater emulsions were prepared by homogenizing refined olive oil and phosphate buffer (50 mM, pH 7) containing Tween 20 or WPC at the concentrations of 0.5% and 2.5%, respectively. Due to the different surface activity of these surfactants, these concentrations were previously selected according to preliminary experiments and chosen in order to achieve a not significant difference (p<0.05) in the initial dispersion degree when O/W olive oil emulsions made only with Tween 20 or whey proteins are taken into account (data not shown). Each antioxidant was individually solubilised in the Tween 20/WPCphosphate buffer aqueous phase prior to homogenization. To test concentration effect, antioxidants were added in four different quantities ranging from 1 105 M to 1 103 M. Emulsions prepared without antioxidant addition were used as control. Homogenization was carried out under nitrogen flux by means of a laboratory homogeniser (YellowLine DI 25 Basic, IKA Werke GmbH & Co, Germany) set to a speed of 20,500 rpm for 180 s on batches of 10-ml total volume. For the shelf-life testing, the emulsions were put and hermetically closed in vials of 20-mL capacity, stored in the dark and allowed to oxidize for 24 days at 35 C in a temperature controlled chamber (Termaks, Bergen, Norway). Samples were collected after 0, 3, 5, 7, 11, 14, 17, and 24 days of storage and submitted to analytical determinations. Dispersion Degree and Interfacial Area Evaluation The dispersion degree of the emulsions was evaluated according to the turbidimetric method of Pearce and Kinsella.24 The absorbance of 1:1000 (A1000) diluted emulsions was measured at =500 nm by means of a Lambda Bio 20 spectrophotometer (Perkin Elmer, Boston, MA). Absorbance data were used to compute the interfacial area of the dispersed oil phase according to Cameron et al.25 and expressed in square meter per milliliter of emulsion. Lipid Hydroperoxides Lipid hydroperoxides were measured according to Shantha and Decker,26 by mixing the emulsion (0.3 mL) with 1.5 mL of isooctane/2-propanol (2:1, v/v), vortexing three times for 30 s and centrifuging

for 2 min at 1000g. Two hundred microliters of the supernatant were taken and 2.8 mL of a methanol:1buthanol solution (3:1, v/v) were added, followed by 15 L of 3.94 M ammonium thiocyanate and 15 L of ferrous iron solution (prepared by adding equal amounts of 0.132 M BaCl2 and 0.144 M FeSO4). After 20 min, the absorbance of the solutions was measured at 510 nm using a Lambda Bio 20 spectrophotometer (Perkin Elmer, Boston, MA). Hydroperoxides concentration was determined using a standard calibration curve prepared with hydrogen peroxide. Thiobarbituric Acid-Reactive Substances Thiobarbituric acid-reactive substances (TBARs)27 were determined by mixing between 0.1 and 1.0 mL (final volume adjusted to 1.0 mL with double-distilled water) of emulsion with 2.0 mL of TBA reagent (15% w/v tricholoracetic acid and 0.375% w/v thiobarbituric acid in 0.25 M HCl) in test tubes and placed in a boiling water bath for 15 min. The tubes were cooled to room temperature for 10 min and then centrifuged (1000g) for 15 min. After 10 min, the absorbance was measured at 532 nm. TBARs concentrations were determined by a standard curve prepared using 1,1,3,3-tetraethoxypropane. Statistical Analysis Data on solutions surface properties were averaged from at least nine measurements while data on systems stability over storage time were averaged from at least six repeats coming from two different batches of samples. All the data are reported as means and standard deviation. The statistical difference of data was determined by a t test between means. Data were processed using the Statistica for Windows (Statsoft, Tulsa, OK) package.

Results and Discussion Interfacial Behavior and Dispersion Degree Syringic acid, tyrosol, and oleuropein were firstly evaluated in terms of their specific surface properties and the results of the air/water surface tension evaluation are shown in Table 1. The addition of syringic acid and tyrosol in the range from 106 to 103 M caused a significant increase of the surface tension of water but no concentration dependency occurred. This represents a quite peculiar result since olive oil phenolic compounds are amphiphilic compounds and are generally reported to be surface active;28 in this case, tyrosol and syringic acid behaved like inorganic compounds such as KOH and NaNO329 or sucrose and glucose30 and contributed to increase the intermolecular attractions between water molecules.

298 Table 1 Air/water surface tensions of tyrosol, syringic acid, and oleuropein, tested in a concentration range of 1106 1103 M Concentrations [M] 1106 1105 5105 1104 5104 1103 Tyrosol (mNm1) 73.30.5 74.20.7 74.30.2 73.50.3 74.60.3 74.70.1

Food Biophysics (2011) 6:295302 Syringic Acid (mNm1) 69.82.3 76.30.1 75.90.1 74.11.0 76.40.1 76.20.3 Oleuropein (mNm1) 61.70.2 61.82.5 60.80.3 62.30.5 53.80.5 56.70.3

A different behavior was shown in the case of oleuropein where a slight and dose-dependent decrease of surface tension values occurred with the increase of its concentration in the water phase. Besides surface tension, interfacial tensions measurements were carried out to determine the affinity of these molecules toward the olive oilwater interfaces and the results, expressed as interfacial pressure =0 , where 0 is the interfacial tension value of the olive oil/buffer system (23.11.1 mNm1) are shown in Figure 2. In general, it can be stated that all the phenolics had an effect at interfacial level since 0 but a different trend in the interfacial tension at the increasing of their concentration in the water phase was observed depending on the molecule. The tyrosol data were characterized by a rather high variability, as shown by the high error bars obtained; nonetheless, it can be affirmed that such compound presented an increasing trend of the surface pressure upon concentrations up to 5104 M. Among the phenolic compounds tested, syringic acid showed the lowest surface pressure with quite similar values at increasing its amount denoting a limited effect on and a scarce dependence upon concentration. A different behavior was observed in the case of oleuropein which caused an increase of surface pressure at increasing concentration, result that could be related to a preferential

Fig. 2 Surface pressure of antioxidant-buffered solutions containing different concentrations of oleuropein, syringic acid, and tyrosol and refined olive oil

tendency of this compound to locate at the olive oil/ water interface and to lower the interfacial tension as a function of its addition to the system. The interfacial properties of oleuropein have already been investigated in literature and this molecule was proven to interact with biomembranes.31,32 By comparing these results with the surface tensions of the same compounds (Table 1), it can be stated that the presence of olive oil, a phase more polar than air, affected their interfacial behavior, in particular in the case of syringic acid and tyrosol. By taking into account the chemical structures of these compounds (Figure 1), the most polar one is oleuropein, followed by syringic acid and then tyrosol. Despite being the most polar, oleuropein was shown to localize on the olive oil/water interface and to affect the interfacial tension upon concentration; this behavior may be ascribed to the fact that this compound presents also some nonpolar features which balance the polarity owing to the sugar moiety and confer amphiphilicity. To test the effect of syringic acid, tyrosol, and oleuropein on the dispersion state, they were individually added in a concentration range of 1105 M1103 M in the control model systems made with 0.5% Tween 20 and 2.5% WPC (w/v), respectively, that based on preliminary experiments are characterized by a comparable dispersion degree. The results, expressed as interfacial area, are shown in Figure 3 (a, b). Model systems with no antioxidant compounds added were used as control. It can be seen that, in general, in the model systems made with Tween 20 (a), the olive phenolic compounds improved the dispersion degree when compared to the control sample, especially when syringic acid and oleuropein were added. The presence of the phenolic antioxidants seems therefore to improve the emulsifying capacity of the systems containing Tween 20 and this effect is likely ascribed both to their amphiphilic nature and to the presence of the surfactant that might have promoted the transposition of all the compounds at the oil/water interface. This result is in agreement with those previously achieved in catechin added Tween 20-olive O/W emulsions,22 even though of different entity, and confirms the importance of the amphyphylic properties of phenolic compounds in affecting the dispersion degree of O/W emulsions.

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effect of the antioxidant addition on the dispersion degree is strongly dependent on the emulsifier used for the stabilization of the dispersed phase and their chemical and polarity properties. In particular, Tween 20 is a low-molecular weight nonionic surfactant which is not charged; the whey proteins, on the contrary, at neutral pH, are mostly negatively charged.33 It can be thus hypothesized that the results obtained in the case of WPC-stabilized emulsions can be ascribed to electrostatic interactions between the interfacial protein layer and the antioxidant compounds which at pH 7 are negatively charged in the case of syringic acid or not charged in the case of oleuropein and tyrosol compounds. Although pH is an important variable affecting the chemical and functional behavior of the compounds under investigation, only experiments at pH 7 were carried out because the interfacial behavior of the emulsifying agents (whey proteins and Tween 20) is well studied and documented at this pH value. Oxidative Stability On the basis of the results previously obtained, the concentration of antioxidant to be added in the O/W emulsions to study the effect on the oxidative stability was chosen equal to 5104 M; this concentration was selected to ensure an evident effect on system oxidation. Previous studies carried out on olive oil emulsions added with phenolic compounds23 showed that the antioxidant addition in low concentration is not able to guarantee, during the auto-oxidation, the presence of an induction phase, index that is usually used to discriminate the antioxidative efficiency of antioxidant compounds. The stability of the emulsified model systems added with antioxidants was carried out by subjecting the emulsions to accelerated oxidation at 35 C for 24 days. The oxidative stability was monitored by means of the changes of lipid hydroperoxides and TBARs, chosen as primary and secondary oxidation indexes, respectively. The evolution of the formation of lipid hydroperoxides over storage time is shown in Figure 4a and b for the systems stabilized by Tween 20 and WPC, respectively. In the case of the Tween 20 emulsions, the control samples showed a gradual increase in the hydroperoxides until the end of the storage while, on the contrary, in all the systems added with the phenolic compounds during the first 10 days of storage a lag phase, where the production of hydroperoxides was quite limited and constant over time, could be clearly noticed. No significant differences were found in the production of primary oxidation products among syringic acid, tyrosol, and oleuropein and thus a similar protective effect was reached. At increasing storage time above 10 days, some differences arose in the oxidation rate of the emulsions among the molecules and the following

Fig. 3 Effect of the addition of different concentrations of oleuropein (filled diamond), syringic acid (empty square), and tyrosol (empty triangle) in comparison to the control (filled circle), in the model systems stabilized by Tween 20 (a) and WPC (b)

The most significant effect was observed as due to the addition of oleuropein (Figure 3a), and this result is in agreement with those obtained from the interfacial tension measurements and it can be supposed that antioxidantsurfactant might have interacted to form complexes characterized by higher interfacial activity. This effect seems to increase up to 1104 M; at higher added concentrations, despite the highest surface pressure exerted from the molecule on the oil/water interface (Figure 2), however, a decrease of the emulsifying effect seems to occur. It is likely that, upon higher oleuropein concentrations, a competitive rather than interactive mechanism between the two molecules might have occurred and this phenomenon could have impaired the emulsifying activity. In the case of the systems stabilized by 2.5% WPC (Figure 3b), the addition of the antioxidant compounds did not cause any pronounced effect on the dispersion state of the droplets since the values, with the exception of tyrosol 1104 M, are generally included in the variability of the control which is higher than that determined in the Tween 20 systems. By comparison of the results at similar concentrations of phenolic compound added in system, it is evident that the

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In general, the evolution of the oxidation reaction in the emulsions stabilized by WPC was quite limited and always lower than that noticed in the systems stabilized by Tween 20 individually added with the same antioxidants. The lower formation of primary oxidation products can be attributed to the presence of whey proteins which, in emulsions, are reported to possess antiradical activity and also to play a synergistic effect with phenolic antioxidant compounds.34,35 It must also take into consideration that protein-stabilized interfaces are usually characterized by higher elasticity modulus and surface viscosity36 and it is thus likely that a more rigid membrane could have hindered the hydroperoxides migration from the interior of the lipid droplet to the aqueous phase. Such a result is in agreement with what was previously reported by other authors35 even though controversial results are also reported.37 The formation of the secondary oxidation products (TBARs) in the Tween 20- and WPC-stabilized systems as a function of storage time is shown in Figure 5a. When the emulsifier was Tween 20, no lag phase was observed in both the control and, with an opposite behavior than in the

Fig. 4 Hydroperoxides production over storage time of the emulsified samples added with oleuropein (filled diamond), syringic acid (empty square), and tyrosol (empty triangle) in comparison to the control (filled circle) in the model systems stabilized by Tween 20 (a) and WPC (b)

ranking in the ability to reduce hydroperoxide formation could be determined: oleuropeinsyringic acid>tyrosol. Furthermore, for what regards tyrosol, it can be seen that from day 14 its behavior was similar to that of the control sample. Figure 4b reports the changes of hydroperoxides occurred in the emulsions stabilized by WPC and individually added with 5104 M of syringic acid, oleuropein, and tyrosol. By comparison of the trend of the primary oxidative products of the control emulsions made only of whey proteins with those observed in the corresponding samples prepared with Tween 20 (Figure 4a), it appears evident that in general the production of hydroperoxides in the former systems is significantly lower and a lag phase in the first 5 days of storage could be also noticed. A longer lag phase with no significant (p<0.05) formation of hydroperoxides was observed in the emulsions added with antioxidants with no significant differences among the phenolic molecules neither in the induction period nor in the following storage time and it can be asserted that comparable protective effects were obtained.

Fig. 5 Evolution of the secondary oxidation products measured as TBARs of the emulsions added with oleuropein (filled diamond), syringic acid (empty square), and tyrosol (empty triangle) in comparison to the control (filled circle) in the model systems stabilized by Tween 20 (a) and WPC (b)

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case of hydroperoxides, in the emulsions added with antioxidants. It can be observed that among the systems tested, the ones added with oleuropein showed the lowest formation of TBARs result that is in agreement with the results of the protective effects of the formation of hydroperoxides shown in Figure 4a. In the WPC-stabilized emulsions, as already evidenced in the formation of hydroperoxides, the production of secondary oxidation products was sensitively lower than in the Tween 20-stabilized systems. The systems added with oleuropein, syringic acid, and tyrosol showed the presence of a lag phase and lower TBARs products in comparison to the control; the induction time resulted to be extended in the case of oleuropein and syringic acid-added emulsions where it lasted until day 11 of storage. In the olive oil emulsions added with tyrosol, the TBARs production was higher than in the other systems. The ranking of the protective activity of the phenolic compounds added in the WPC emulsions in the development of the oxidative reaction and formation of the secondary products is thus, the following: oleuropein>syringic acid>tyrosol. The results reported in Figure 5 highlight that in the case of secondary oxidation, the same behavior and protective ranking could be observed in both the series of system (Tween 20 and WPC stabilized emulsions). The three antioxidants were proven to exert a protective effect in emulsion and were not affected by the type of emulsifier used for the formation and stabilization of the systems. If the TEAC values of these molecules are considered,38,39 the ranking according to their antiradical activity both in ethanol and in refined olive oil was: syringic acid> oleuropein > tyrosol, a ranking which is not strictly respected when we evaluate the evolution of the lipid oxidation in the emulsified state. Oleuropein, even though it is not the most powerful in terms of radical scavenging activity, resulted to be the most protective molecule in terms of oxidative deterioration independently on the emulsifier used in the system. This behavior can be associated to the results obtained from the interfacial tension and emulsifying capacity evaluations that seem to confirm the tendency of this phenolic compound to locate at the oilwater interface where oxidation is more likely to occur. It is probable that syringic acid, despite being the most efficient molecule in terms of antiradical activity, could not exert its potential protective effect due to its polarity and charge which did not allow this compound to stay nearby the interface where oxidative phenomena were occurring. Tyrosol showed the most limited effect from an antioxidative viewpoint, in agreement with its low antiradical activity. The results of these experiments show that the behavior of such olive oil phenolic compounds in dispersed systems

can be explained by their interfacial properties as well as their radical scavenging activity; this latter aspect is also in agreement with the studies carried out by Paiva-Martins and Gordon and Paiva-Martins et al.14,16 on O/W emulsions containing olive oil phenolic compounds. On the basis of these results, it can be asserted that besides their healthy properties, olives and olive oil minor compounds can play an important role in the oxidative stabilization of olive oil-based emulsions and thus this work may provide new practical information that may increase the potentiality of utilization of olive oil or recovered olive oil phenolic compounds in formulated foods.

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