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

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The 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 J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01963 • Publication Date (Web): 05 Jul 2016 Downloaded from http://pubs.acs.org on July 10, 2016

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Journal of Agricultural and Food Chemistry

The antioxidant behavior of olive phenolics in oil-in-water emulsions

1 2 3 4 5

Vito Michele Paradiso1*, Carla Di Mattia2*, Mariagrazia Giarnetti1, Marco Chiarini2, Lucia Andrich2, Francesco Caponio1

6 7 8 9 10

1

Department of Soil, Plant and Food Sciences, University of Bari, Via Amendola 165/a, I-70126, Bari (Italy); 2Faculty of Bioscience and Technology for Agriculture, Food and Environment, University of Teramo, Via Balzarini 1, Campus Coste S. Agostino, 64100 Teramo (Italy)

11 12 13

* corresponding authors: [email protected], +39 (0)80 544 2272 (V M Paradiso); [email protected], +39 (0)861 266912 (C Di Mattia)

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Abstract

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The effect of the surrounding molecular environment (β-lactoglobulin as emulsion stabilizer,

17

maltodextrin as viscosity modifier) on the antioxidant activity of three olive oil phenolic

18

compounds (PCs) in olive oil-in-water emulsions was investigated. Oxidation potential, phenolic

19

partitioning and radical quenching capacity were assessed in solution and in emulsion for

20

oleuropein, hydroxytyrosol and tyrosol; the influence by β-lactoglobulin and maltodextrin

21

concentration was also evaluated. Finally, the observed properties were related to the oxidative

22

stability of the emulsions containing the PCs to explain their behavior. The order

23

hydroxytyrosol>oleuropein>tyrosol was observed among the antioxidants for both oxidation

24

potential and radical quenching activity. Radical quenching capacity in emulsion and anodic

25

potential resulted to be complementary indices of antioxidant effectiveness. As the antioxidant

26

intrinsic susceptibility to oxidation expressed by its anodic potential decreased, the environmental

27

conditions (molecular interactions, changes in continuous phase viscosity) played a major role in

28

the antioxidant effectiveness in preventing hydroperoxide decomposition.

29

30

Keywords

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o/w emulsions; olive phenolic antioxidants; cyclic voltammetry; antiradical activity; oxidative stability

32 33


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Introduction

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Control of lipid oxidation is a main aim in order to improve nutritional properties and extend shelf

36

life of foods,1 and can be achieved as far as oxidation and anti-oxidation mechanisms are well

37

understood.

38

Research has shown, in last decades, that lipid oxidation is essentially an interfacial phenomenon.

39

Even in bulk oils, oxidative processes have interfacial nature and take place in physical

40

microenviroments.2,3 As regards multiphase systems, such as emulsions, the molecular

41

environment plays a fortiori a major role: therefore, location is as important as reactivity for all the

42

molecules involved in oxidation.4 This has been proved also for antioxidants, whose effectiveness

43

strictly depends on both their molecular properties and the environment surrounding them.

44

Several theories have been formulated to explain antioxidant behavior – from the Polar paradox

45

formulated by Porter to describe differences between polar and non-polar antioxidants,5 to the

46

Cut-off effect applied by Laguerre to elucidate the nonlinear behavior of a homologous series of

47

chlorogenate esters6 – showing more and more complex interactions underlying antioxidant

48

effectiveness and pointing out that intrinsic molecular properties and environmental conditions

49

are strictly interrelated and influence each other. Therefore, a continuous effort to illuminate

50

mechanisms behind the observed effect of simply adding a molecule to a complex system is

51

fundamental to better focus on real systems.7

52

Olive oil phenolic compounds (PCs) have gained increasing attention as antioxidant additives in

53

food emulsions. They have been proved to be effective in slowing down oxidation, and

54

additionally, to affect dispersion properties of emulsions.8,9 They can largely differ for their

55

antioxidant efficacy, on the basis of their molecular structure:10 their antioxidant efficacy resulted

56

not to depend only on molecule polarity, so that other parameters, such as the capacity and

57

rapidity in donating hydrogen, should be considered.11 Some interesting interactions with the 3 ACS Paragon Plus Environment


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molecular environment were also observed. A synergistic activity with surface active proteins was

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reported,12 as well as a pH-dependent radical scavenging activity.13 In a recent paper14 we

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observed the effectiveness of selected olive oil PCs in slowing down oxidative processes in olive

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oil/water emulsions and that different concentrations of maltodextrin in the acqueous phase

62

affected these mechanisms.

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The aim of the present research was to investigate on the effect that the surrounding molecular

64

environment exerts on the properties and behavior of three differing olive oil PCs (the secoiridoid

65

oleuropein, and two phenyl-ethyl alcohols, namely tyrosol and hydroxytyrosol, differing for

66

absence or presence of o-diphenol structure) and on their effectiveness in controlling oxidation in

67

olive oil/water emulsions. The selected PCs were characterized in solution and in emulsion by

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assessing oxidation potential, phase partitioning and radical quenching activity; the influence on

69

these parameters by β-lactoglobulin (commonly used as surface-active protein in the emulsions)

70

and concentration of maltodextrin (as emulsion viscosity modulator) was evaluated; finally, the

71

observed properties and behavior were related with the oxidative stability of the emulsions added

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with the PCs.

73 74

Materials & Methods

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Materials

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Crystallised and lyophilised β-lactoglobulin (BLG) from bovine milk (90% PAGE purity, lot n.

77

030M7025V) was purchased from Sigma-Aldrich Ltd. (Steinheim, Germany). Highly refined olive oil

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(CAS number: 8001-25-0; tested according to United States Pharmacopeia and European

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Pharmacopoeia; Fluka Chemie AG, Buchs, Switzerland) was used without further purification;

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before its use, the oil was preliminary characterised by HPLC analysis and interfacial properties

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determination to exclude the presence of both phenolic and amphiphilic compounds. Experiments 4 ACS Paragon Plus Environment

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were carried out by using oil from a single batch stored under controlled conditions (dark, 15°C) to

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avoid oxidation. Three olive phenolic compounds: oleuropein (Oleu), HPLC purity ≥ 90%, CAS

84

number 32619-42-4, tyrosol (Tyr), HPLC purity ≥ 99%, CAS number 501-94-0, hydroxytyrosol (OH-

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Tyr), HPLC purity ≥ 98%, CAS number 10597-60-1, were purchased from Extrasynthese (Lyon,

86

France). Maltodextrins (MD) from native corn starch, characterised by DE 7.5-9 and M= 30.000,

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were obtained from Cargill (Milan, Italy). Ultrapure water was used throughout the experiments

88

and was from the water purifier system USFELGA from Purelab Plus (Ransbach-Baumbach,

89

Germany). Phosphate buffer, sodium azide, sucrose, Fremy’s radical were purchase from Sigma-

90

Aldrich Ltd. (Steinheim, Germany). All the reagents were of analytical grade.

91 92

Emulsions preparation

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Olive oil-in-water emulsions (20% w/v) were prepared by using as aqueous phase a 0.5% (w/v) BLG

94

solution in 50 mM phosphate buffer (pH 7) and added with 0.03% (w/v) Sodium Azide (NaN3) prior

95

to emulsification as antibacterial agent.

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Model emulsified systems were prepared either without maltodextrins (MD0) or added with 1%

97

(w/v) (MD1) or 15% (MD15) maltodextrins, respectively, as thickening agent. To these systems

98

olive oil polyphenols oleuropein (Oleu), tyrosol (Tyr) and hydroxytyrosol (OH-Tyr) at a 10-4 M

99

concentration were added by solubilising them in the aqueous phase prior to homogenisation.

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Maltodextrins were preliminarily dispersed in pure water at a 50 % (w/v) concentration and then

101

the mixture was stirred by magnetic stirrer for 45 minutes and slighlty heated (≈40 °C) to allow

102

complete solubilisation. The emulsions added with maltodextrins were prepared by mixing

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appropriate volume of concentrated emulsion and MD aqueous solutions in order to get the

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desired phase volume and MD concentration in the final emulsion.



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Emulsions were prepared by two successive steps: a pre-emulsion was obtained with a high shear

106

mixing device (Ultra-Turrax yellow line DI25 basic, Ika-Werke GmbH & Co, Germany) at 20000 rpm

107

for 1 minute. High pressure homogenization of the pre-emulsion was then carried out using a

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Panda Plus 2000 homogenizer (GEA Niro Soavi, Parma, Italy) by applying five homogenization

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cycles at 150 bar.

110 111

Phenolic partitioning

112

Phenolic partitioning in the aqueous phases was determined by means of HPLC measurements

113

according to the method described by Pirisi et al.15 and calculated as mass balance between the

114

concentrations in the aqueous phase before and after homogenization.

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After emulsification, the aqueous phases were separated with a method adapted from Patton and

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Huston.16 A volume of 1 ml of fresh emulsion was diluted in 1 ml of sucrose solution (500 gL-1 in 50

117

mM pH 7 phosphate buffer). This mix was centrifuged (5415 D centrifuge, Eppendorf) twice at

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6000 rpm for 2 h. After centrifugation, two phases were observed: the creamed oil droplets at the

119

top of the centrifuge tube and the aqueous phase of the emulsion at the bottom. The tubes were

120

immediately frozen at ˗20°C, and then cut just beneath the cream phase, so as to separate the

121

phases. The bottom aqueous phase was carefully extracted with the aid of a micropipette. The

122

determination of antioxidants in the continuous and separated aqueous phases was performed

123

with a 225 nm UV-VIS detection HPLC method (Series 200, UV-VIS detector, Perkin Elmer, Norwalk

124

USA) according to the procedure described by Pirisi et al;15 the percentage of partitioning in the

125

aqueous phase was calculated by considering the initial concentration in the continuous phase.

126 127

Cyclic voltammetry


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The electrochemical behavior of phenolic compounds was studied using a glassy carbon electrode

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and a computer-controlled potentiostat Autolab PGSTAT12 (Eco Chemie, Holland), monitored by

130

the General Purpose Electrochemical System (GPES3, version 4.9) software package. The

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instrument included an Ag-AgCl reference electrode and a platinum counter electrode. The

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working electrode was a glassy carbon electrode (diameter: 3 mm). Measurements were carried

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out in phosphate buffer (50mM, pH 7) containing 50mM KCl, as described by Campo Dall’Orto et

134

al.17 The experimental conditions were: scan rate (V/s) = 0.2; start potential (V) = ˗ 0.3; first vertex

135

potential (V) = 0.8; second vertex potential (V) = ˗ 0.3; step potential (V) = 0.00198 for oleuropein

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and hydroxytyrosol; scan rate (V/s) = 0.2; start potential (V) = ˗ 0.3; first vertex potential (V) = 1.0;

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second vertex potential (V) = ˗ 0.3; step potential (V) = 0.00198 for tyrosol. Measurements were

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first carried out on 10-4 M buffered solutions of oleuropein, tyrosol and hydroxytyrosol, on binary

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solutions made of antioxidants plus β-lactoglobulin 10-5 M and ternary solutions made of

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antioxidants plus β-lactoglobulin 10-5 M plus maltodextrins tested at two concentrations (1 and

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15% w/w). A blank consisting of systems with no antioxidants was carried out before each

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measurement.

143 144

Electron Spin Resonance measurements

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The antiradical activity of the emulsions containing oleuropein, tyrosol and hydroxytyrosol were

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measured by the ability of the continuous phase to quench the Fremy’s radical (potassium

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nitrosidsulfonate). Emulsions were analysed as they were and aliquots of each sample were added

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to an equal volume of Fremy’s salt (1mM). The same procedure was applied to evaluate the

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antiradical activity also on the aqueous solutions before homogenisation. The EPR spectra were

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obtained after 20 mins, at 23°C (average temperature) on a Bruker EMX X-band microwave bridge

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spectrometer (Karlsruhe, Germany), equipped with a EMX high sensivity cavity. The microwave 7 ACS Paragon Plus Environment


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power and modulation amplitude were set at 0.2 mW and 0.1 mT, respectively. The other

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instrumental parameters were: sweep width, 5 mT; sweep time, 10 s; receiver mode, 1st; time

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constant,10 ms; modulation frequency, 100 kHz. All measurements were made in quartz cells

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0.90x1.10 mm, inner and outer diameters, respectively (Wilmad Glass, NJ, USA). Spectra were

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acquired and analysed using WinEPR software (Bruker, Germany). Spectra of the unreacted radical

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(aliquots of control emulsions or aqueous phase prior homogenization with no antioxidant added)

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were used for the computation of the percentage of radical quenching.

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Accelerated oxidation of O/W emulsions

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In our previous study, 14 we carried out an accelerated oxidation test with the same systems under

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examination in the present research. 2,2′-Azobis(2-methylpropionamidine) dihydrochloride

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(AAPH) was selected as radical initiator of oxidation carried out in dark at 25 °C.18 Lipid

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hydroperoxides were measured according to Shantha and Decker19 to monitor primary oxidation,

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while hexanal, which arise from the oxidation of linoleic acid, and nonanal, which arise from the

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oxidation of oleic acid,20 were measured by HS-SPME/GC/MS as volatile secondary oxidation

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products. In particular, the extraction was performed by a 75 µm Carboxen/polydimethylsiloxane

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(CAR/PDMS) fiber (Supelco, PA, USA) exposed in the headspace of the sample at 35 °C for 15 min.

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Gas–chromatographic analysis of volatile compounds was carried out in splitless mode on a HP-

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Innowax (20 m × 0.18 mm, 0.18 µm film thickness) polar capillary column (Agilent Technologies,

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CA, United States). The GC peak areas were converted into amounts of for each volatile

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compounds (µg/mL) according to calibration curves. More details about the methods are reported

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in our previous paper.14 Curves of the oxidation products along the 28-days oxidation test were

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reported. In the present work, the areas under the curve (AUC) of hydroperoxides, after 7, 16 and

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28 days of oxidation, and of hexanal and nonanal after 16 and 28 days of oxidation (since after 7 8 ACS Paragon Plus Environment

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days the AUC were neglectable) were considered for a correlation study with the assessed

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antioxidant properties.

178 179

Statistical analysis

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Analysis of variance (ANOVA), post-hoc Fisher’s LSD test, correlation analysis, linear regression and

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surface response regression were performed using XLStat software (Addinsoft SARL, New York,

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USA) and Minitab 17 (Minitab Inc., Pennsylvania, USA).

183 184

Results and Discussion

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Cyclic voltammetry of phenolic mixed solutions

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It is well known from the literature that phenolic compounds are easily detectable at carbon based

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electrodes, and that the electrochemical response can be used to characterize the phenolic

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content of a sample in terms of both total amount and functional properties.17,21,22 On the other

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hand, when carried out on equimolar solutions, electrochemical analysis can be used to evaluate

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and compare intrinsic antioxidant capacity of molecules.10 Cyclic Voltammograms (CVs) recorded

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in the solutions containing the antioxidants, the antioxidants plus BLG, and the antioxidants plus

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BLG plus MD at different concentrations are illustrated in Figure 1 whilst the voltammetric

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parameters are reported in Table 1.

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On the scan from -300 up to 1000mV, all the compounds showed an anodic peak which occurred

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at 92±14, 446±25, 78±16 mV for oleuropein, tyrosol and hydroxytyrosol, respectively, confirming

196

that all of the three molecules can act as radical scavengers, though with quite differing energy

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requirements for oxidation and, therefore, differing antioxidant effectiveness.10 The o-diphenolic

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antioxidants (hydroxytyrosol and oleuropein) showed, as expected, much lower Ep,a values than

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the monophenolic compound tyrosol.23. Since a strong correlation has been observed between the 9 ACS Paragon Plus Environment


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anodic potential of the compounds and their antioxidant properties – i.e. the lower the potential,

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the higher the ability of the compound to exert its antioxidant capacity –24–26 the highest

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antioxidant power was thus shown by hydroxytyrsol, followed by oleuropein and then by tyrosol.

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For all the compounds it was possible to observe that the addition of BLG and MD generally

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caused a shift in the anodic peaks to higher potentials, variations which were statistically

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significant when MD was added at the highest concentration. The only exception was observed for

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hydroxytyrosol in presence of BLG: as a consequence, hydroxytyrosol showed lower potential than

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oleuropein in presence of BLG. On the contrary, significant differences were found in the anodic

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current Ip,a, where lower currents were detected as the complexity of the systems increased,

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especially in the presence of MD. Since anodic current indicates the amount of compounds which

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is oxidised at the electrode, it is mainly a diffusion controlled response and could be also

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influenced by interactions leading to antioxidant regeneration. Also in this case, the system

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hydroxytyrosol-BLG was an exception, since the current intensity increased respect to the sole

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antioxidant. BLG showed, therefore, to either enhance or lower the antioxidant properties of the

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molecules under examination. This protein has been previously reported to form non-covalent

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complexes, based on hydrogen bonds and Van der Waals forces with phenolic antioxidants.27–30 As

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regards the effect of such interactions, Ozdal et al.28 reported, in a comprehensive review,

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contrasting results of either enhanced or lowered antioxidant activity of protein–phenolic

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complexes. Dubeau et al.,31 who examined the interactions between milk proteins and tea

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polyphenols also by means of voltammetry, pointed out a general decrease of the antioxidant

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activity of phenolic antioxidants, but also that the redox capacities of tea antioxidants were not

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equally affected by milk proteins. Most of the available data, however, regards polyphenols, single

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or mixed as extracted and only one work from literature investigated on the interaction of

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phenolic compounds from olive oil with different food proteins including BLG;32 however, the 10 ACS Paragon Plus Environment

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affinity of olive oil phenolics for the different food proteins was found to be relatively weak when

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compared with tannic acid. Amorati and Valgimigli33 reported that non-covalent bonding, both

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involving reactive and non-reactive functional gropus, could lead to either increase or decrease of

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antioxidant activity in phenolic compounds.

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Another possible mechanism that may explain the behavior observed is related to the interaction

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of quinones, formed by oxidation of diphenols, with an array of functional groups, including amino

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and sulphydryl groups, to form adducts with a resulting reduction of the molecule and

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regeneration of its antioxidant capacity.34,35 This would explain why peak current was affected by

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the interaction with BLG more than peak potential. When maltodextrins were added to the

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solutions containing antioxidants and BLG, the differences in Ep,a values among antioxidants were

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confirmed, while increases (significant with 15 % MD) were observed as the MD content

235

increased, with the exception of tyrosol, that showed a significant increase even with 1% MD. As

236

regards the peak current, Ip,a of oleuropein was higher than that of tyrosol, and no longer

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comparable as in solutions without MD. Increasing amounts of MD determined lowering peak

238

currents. Also, hydroxytyrosol suffered the greatest reduction of peak current, which was

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comparable to that of the other antioxidants in 15 % MD solutions. The lower anodic current

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registered in the solutions added with MD may be due to a slower diffusion of the species to the

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electrode caused by steric hindrance phenomena and differences in viscosity. Nevertheless, some

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other interaction should be hypothesized, since also the peak potential of the antioxidants was

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affected by maltodextrin. After reversing the scan direction from 1000mV to -300mV, a cathodic

244

peak appeared for oleuropein and hydroxytyrosol, showing the reversibility for the oxidation

245

reactions. The cathodic peak corresponds to the reversible reduction of the catechol moiety

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oxidation product, ortho-quinone, to the ortho-phenol moiety.36 If the ratio Ip,a/Ip,c is considered, it



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can be affirmed that a quasi-reversible and a reversible reaction occurred for oleuropein and

248

hydroxytyrosol, respectively.

249 250

Phenolic partitioning and radical quenching in the aqueous phase

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Even though existing methods to evaluate antioxidant partitioning in intact emulsions are

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available which refer to the pseudophase model,37 in the present work the partitioning was carried

253

out in the continuous phase after emulsion separation, also in consideration of the fact that both

254

the radical initiator AAPH and Fremy’s radical for EPR measurements were water-soluble. This

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approach, though providing partial information, results to be frequently adopted in literature and

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can contribute to the interpretation of antioxidant behavior.11,38–40 Moreover, the pseudophase

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model, despite being widely used in surfactant-stabilized model emulsions, needs to be further

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validated in more complex systems such as protein-stabilized emulsions.41 The partitioning of the

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olive phenolic antioxidants in the continuous phase as affected by emulsion formulation is

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reported in Figure 2. Amongst the compounds, tyrosol showed the highest partitioning in the

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aqueous phase of the MD0 emulsions, whilst oleuropein and hydroxytyrosol partitioned poorly

262

with

263

tyrosol>hydroxytyrosol>oleuropein, result which is in accordance with what previously observed in

264

literature for oleuropein and hydroxytyrosol by using the 1-octanol/water method.42 As expected,

265

the lowest partitioning was observed with oleuropein, as in accordance with its amphiphilic

266

structure and interfacial properties which favoured its location nearby the oil/water

267

interface.9,14,43 The addition of MD caused a partition of the compounds in the aqueous phase

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significantly higher when compared to MD0 systems, especially for oleuropein and hydroxytyrosol,

269

with a concentration dependent behavior. The higher partitioning of oleuropein and

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hydroxytyrosol as a consequence of MD addition may be thus ascribed to the increase of

similar

percentages

(23-27%).

The

order


of

partitioning

was

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hydrophobicity of the aqueous phase; maltodextrin are indeed reported to vary the

272

physicochemical properties of solutions by regrouping themselves in aqueous media and creating

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a local hydrophobic medium.44

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The antiradical activity of the aqueous phase of the emulsions is reported in Figure 3-I.

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The hydrophylic Fremy's stable radical has been used with ESR detection in order to allow

276

measurements on not optically transparent emulsions. The aqueous radical solution was mixed

277

with an equal volume of emulsion and the change of intensity of the Fremy's radical EPR spectrum

278

as a result of its interaction with the H-donating species partitioned in the continuous phase was

279

measured. Despite almost completely partitioned in the aqueous phase, the less effective in

280

reducing the stable radical was tyrosol; the addition of MD caused some significant changes in the

281

antiradical activity even though the percentage of quenching resulted less than 10%. Amongst the

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olive phenolic compounds, hydroxytyrosol showed the highest antiradical activity both in absence

283

and in presence of MD. In all the systems considered the order of antiradical activity was

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hydroxytyrosol>oleuropein>tyrosol. The trend of the radical quenching generally reflected the

285

partitioning of the compounds in the aqueous phase with the exception of the systems containing

286

the highest percentage of MD. Such a result may be related to the increase of viscosity due to the

287

high concentration of maltodextrins (η=2.38, 2.50 and 7.34 mPa•s, cvhydroxytyrosol>tyrosol, in absence 13 ACS Paragon Plus Environment


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of MD. The addition of MD to the solutions lead to comparable antiradical activity for oleuropein

296

and hydroxytyrosol. This did not correspond completely to the behaviour previously observed in

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the emulsified systems and also in literature when both ABTS and DPPH antioxidant methods were

298

used.10,45 This discrepancy has already been evidenced in literature and the differences in the

299

answers were ascribed to the fact that different methods were used for the measurement of the

300

antiradical activity; however, further investigation may be needed to elucidate the different

301

response.46,47

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A correlation study to assess the antioxidant behavior

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In order to assess how the above described properties of the phenolic molecules and the

304

surrounding environment can influence their antioxidant properties, we considered the results of

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our previous study about oxidative stability on similar emulsions containing the antioxidants under

306

examination.14 In that study emulsions were submitted to accelerated oxidation tests and

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hydroperoxides and volatile oxidation compounds were determined to evaluate antioxidant

308

activities. Hydroxytyrosol resulted to be the most effective compound in slowing down oxidation,

309

followed by oleuropein, while tyrosol effectiveness was limited. On the other hand, the addition of

310

MD resulted to affect both the decomposition of hydroperoxides and the effectiveness of phenolic

311

antioxidants, improving also tyrosol activity in prevention of hydroperoxide decomposition. In the

312

present work, the areas under the curve (AUC) of hydroperoxides, after 7, 16 and 28 days of

313

oxidation, and of hexanal and nonanal after 16 and 28 days of oxidation (since after 7 days the

314

AUC were neglectable) were considered as indices of primary and secondary oxidation

315

respectively. Correlation and regression analyses were used to relate the intrinsic and

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environment-dependent properties of the antioxidant molecules with the oxidative stability of the

317

emulsions, where the relation between the intrinsic properties of antioxidants and their

318

effectiveness is mediated by environmental conditions and interactions. The antioxidant effect of 14 ACS Paragon Plus Environment

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a phenolic compound in a colloidal system can be considered as related to its intrinsic antioxidant

320

properties as well as to its localization in the different pseudophases – on the assumption that

321

water, oil and interface can be regarded as discrete reaction regions where partition equilibria,

322

reaction rate and local concentrations may be considered –, to interactions with other molecules

323

of the molecular environment, and to factors affecting diffusion of reactants and products. Table 2

324

reports the Pearson correlation matrix of the parameters measured for each emulsion type with

325

their indices of oxidative stability. As regards primary oxidation, the Ep,a potential showed a good

326

correlation with the hydroperoxide levels (p < 0.01 in all cases). Nevertheless, the correlation

327

suffered the high leverage of tyrosol, which showed largely higher potential than the other

328

antioxidants, as can be seen in Figure 4. On the other hand, for smaller variations of Ep,a, the

329

relation with hydroperoxide formation was not evident, as can be seen in the left part of the

330

scatter plot in Figure 4. Therefore, Ep,a could be considered a preliminary test for screening bad

331

and good antioxidants. Antioxidant partitioning in water was positively correlated with

332

hydroperoxide formation over 16 and 28 days and not with hydroperoxides after 7 days (Figure 5).

333

Considering that the oxidation initiator was in the aqueous phase, this would mean that

334

antioxidant localization near oxidation substrate (i.e. oil droplets) would be a key aspect to be

335

considered for hydroperoxide formation over prolonged oxidation, while antioxidant molecules in

336

the aqueous phase would not be insignificant during initiation and beginning of propagation.

337

Yet, hydroxytyrosol behaved differently respect to the other antioxidants, since its partitioning

338

seemed not to affect the overall hydroperoxide formation, that was kept almost unchanged

339

irrespective to changes in partitioning. Therefore, in spite of great variations in partitioning,

340

hydroxytyrosol showed the same effectiveness in preventing hydroperoxide formation. This would

341

indicate that hydroxytyrosol was an effective scavenger towards the radicals formed by the

342

initiator in the aqueous phase as well as towards lipid radicals at interfaces. As regards the 15 ACS Paragon Plus Environment


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quenching ability of antioxidants, when it was measured in solution, it showed a good correlation

344

with the hydroperoxide formation over long time; on the contrary, the measurement of quenching

345

ability directly in emulsion gave values well correlated with the early-stage hydroperoxide

346

formation, as shown in the scatter plots in Figure 6. In the initial stages of oxidation in such

347

multiphase systems, antioxidant activity is specially affected by reactant interaction with the

348

molecular environment. Directly measuring the quenching behavior of antioxidants in real systems

349

allowed a better focus on both these aspects, taking into account also changes in the surrounding

350

environment, such as those induced by different amounts of maltodextrin. On the other hand, the

351

measurement of antiradical activity in solution showed better relation with advanced stages of

352

oxidation, when the radical species are abundant and interactions are less limiting towards radical

353

reactions. Moreover, the plot shows a grouping of data points by antioxidant molecules, revealing

354

that this parameter is suited to point out the molecule properties more than their response in

355

complex systems.

356

As regards hexanal, as the major volatile secondary oxidation product, the AUC over 16 days of

357

oxidation showed no significant correlation. The only correlation that was close to the threshold p-

358

value was with the maltodextrin content (p = 0.07). The AUC at 28 days was, instead, significantly

359

related with oxidation potential Ep,a, quenching in solution and quenching in emulsion. Moreover,

360

it was significantly correlated with the hydroperoxides AUC after 7 days (p = 0.01), showing that

361

overall headspace hexanal directly depended on the amount of hydroperoxides formed in the

362

initial stages of oxidation.

363

Figure 7 plots the hexanal AUC after 28 days against the analytical parameters correlated. The

364

plots showed some limitations of the observed correlations. The correlation of hexanal with the

365

oxidation potential was again leveraged, as observed above, by the outstanding tyrosol, whilst

366

hydroxytyrosol and oleuropein did not show a clear relation. The antiradical activity measured in 16 ACS Paragon Plus Environment

Page 16 of 37

Page 17 of 37


367

solution well described the ability of each antioxidant to prevent hexanal formation, yet it was not

368

able to describe changes in behavior as the molecular environment changed, as shown by the

369

grouping of data points by antioxidant molecule. Finally, also the antiradical activity measured in

370

emulsion did not show a satisfactory relation, as evidenced by the scarce alignment of data points

371

for hydroxytyrosol and oleuropein. A significant improvement of linearity was obtained by

372

evaluating a regression of AUC of hexanal as a function of antiradical activity in emulsion and Ep,a,

373

likely due to the fact that these two parameters, when coupled, can provide indications on the

374

two main reaction mechanisms by which the antioxidant compounds can exert their antioxidant

375

activity, which are H-donor and electron transfer activities, respectively. Another possible

376

explanation of the good complementarity of these indices is that they would describe different

377

kinds of interactions of antioxidants with the molecular environment, ascribable in different

378

degrees to electron mobility and diffusion of molecules. Figure 8 plots the observed data versus

379

the data calculated through the obtained regression (adjusted R2 = 0.901; p-values: regression =

380

0.002; antiradical activity = 0.026; Ep,a = 0.002; antiradical activity *Ep,a = 0.005) and reports the

381

obtained contour plot. The contour plot is the graphical representation of the second order

382

regression equation and reports the variation of the AUC of hexanal after 28 days as a function of

383

antioxidant Ep,a and antiradical activity in emulsion. The significant interaction between the factors

384

points out that as the intrinsic susceptibility to oxidation of an antioxidant (expressed by its anodic

385

potential) decreases, molecular diffusivity in the environment plays a major role in its overall

386

effectiveness in preventing hydroperoxide decomposition.

387

Finally, the AUC of nonanal showed correlation only with the maltodextrin content (p = 0.023 and

388

0.054 after 16 and 28 days respectively). This was due to the low values of nonanal AUC in systems

389

with 15% of maltodextrin (Figure 9). Since nonanal characterizes advanced oxidation stages in

390

olive oil,48 its formation was presumably influenced by radical mobility more than by antioxidant 17 ACS Paragon Plus Environment


391

performances. In our previous work, we suggested that the effect of MD on oxidative stability was

392

not only related to the increased viscosity in the systems, but also to interactions involving

393

emulsifier, phenolic compounds and maltodextrins.14 The present paper confirmed this view, by

394

combining different analytical approaches to describe antioxidant behavior.

395

During the oxidation of lipids in heterogeneous systems the formation of oxidation products may

396

be affected by emulsion structure because it determines the local concentration of both reagents

397

and products of the oxidation reactions. Lipid hydroperoxides are surface-active compounds and

398

are thus able to accumulate at the lipid–water interphase of emulsion droplets. The existence of

399

pro-oxidants in the aqueous phase and lipid hydroperoxides at the emulsion droplet surface

400

suggests that lipid oxidation in oil-in-water emulsions primarily occurs at the emulsion droplet

401

interphase.49 At the same time, closer interactions, either of electrostatic or covalent nature, may

402

involve not only the compounds directly involved in oxidation, but also those composing the

403

surrounding molecular environment. All these things considered, the thorough comprehension of

404

the factors affecting lipid oxidation in heterogeneous systems such as emulsions, may lead to the

405

attainment of tools for the protection of complex foods from deterioration.

406

In our previous study14 we reported that phenolic antioxidants delayed hydroperoxide

407

decomposition in the following order of effectiveness: hydroxytyrosol>oleuropein>tyrosol, and

408

that maltodextrin affected antioxidant effectiveness. The present study pointed out significant

409

aspect of antioxidant behavior and interactions with the molecular environment, underlying their

410

different response in emulsions. Both surfactant (β-lactoglobulin) and viscosity modifier

411

(maltodextrin) showed to be involved in interactions with antioxidant molecules affecting

412

therefore their activity besides physical properties of the emulsions. Cyclic voltammetry

413

performed on the antioxidant and electron spin resonance measured directly on the emulsion


Page 18 of 37

Page 19 of 37


414

proved to give rapid and complementary indices correlated with the real behavior of the molecule

415

in a specific molecular environment.

416 417

Acknowledgments

418

This research was funded within the national research project FIRB 2010 - “Futuro in Ricerca”,

419

Ministero dell’Istruzione, dell’Università e della Ricerca (code:RBFR10876O, D.M. 21 September

420

2011, prot. n.556, Ric.21).

421 422

References

423 424 425

(1)

Waraho, T.; Cardenia, V.; Decker, E. A.; McClements, D. Lipid Oxidation in Emulsified Food Products. In Oxidation in foods and beverages and antioxidant applications; Decker, E. A. ., Elias, R. J. ., McClements, D. J., Eds.; Woodhead Publishing, 2010; pp 306–343.

426 427

(2)

Budilarto, E. S.; Kamal-Eldin, A. The Supramolecular Chemistry of Lipid Oxidation and Antioxidation in Bulk Oils. Eur. J. Lipid Sci. Technol. 2015, 117 (8), 1095–1137.

428 429

(3)

Chaiyasit, W.; Elias, R. J.; McClements, D. J.; Decker, E. A. Role of Physical Structures in Bulk Oils on Lipid Oxidation. Crit. Rev. Food Sci. Nutr. 2007, 47, 299–317.

430 431 432

(4)

McClements, D.; Decker, E. Lipid Oxidation in Oil- in- Water Emulsions: Impact of Molecular Environment on Chemical Reactions in Heterogeneous Food Systems. J. Food Sci. 2000, 65 (8), 1270–1282.

433 434 435

(5)

Porter, W. L.; Black, E. D.; Drolet, A. M. Use of Polyamide Oxidative Fluorescence Test on Lipid Emulsions: Contrast in Relative Effectiveness of Antioxidants in Bulk versus Dispersed Systems. J. Agric. Food Chem. 1989, 37 (3), 615–624.

436 437 438 439

(6)

Laguerre, M.; Giraldo, L. J. L.; Lecomte, J.; Figueroa-Espinoza, M.-C.; Baréa, B.; Weiss, J.; Decker, E. A.; Villeneuve, P. Chain Length Affects Antioxidant Properties of Chlorogenate Esters in Emulsion: The Cutoff Theory behind the Polar Paradox. J. Agric. Food Chem. 2009, 57 (23), 11335–11342.

440 441 442

(7)

Laguerre, M.; Bayrasy, C.; Panya, A.; Weiss, J.; McClements, D. J.; Lecomte, J.; Decker, E. A.; Villeneuve, P. What Makes Good Antioxidants in Lipid-Based Systems? The next Theories beyond the Polar Paradox. Crit. Rev. Food Sci. Nutr. 2015, 55 (August 2014), 183–201.

443 444 445

(8)

Di Mattia, C. D.; Sacchetti, G.; Mastrocola, D.; Sarker, D. K.; Pittia, P. Surface Properties of Phenolic Compounds and Their Influence on the Dispersion Degree and Oxidative Stability of Olive Oil O/W Emulsions. Food Hydrocoll. 2010, 24 (6-7), 652–658.

446 447 448

(9)

Di Mattia, C. D.; Sacchetti, G.; Pittia, P. Interfacial Behavior and Antioxidant Efficiency of Olive Phenolic Compounds in O/W Olive Oil Emulsions as Affected by Surface Active Agent Type. Food Biophys. 2011, 6, 295–302. 19 ACS Paragon Plus Environment


449 450 451 452

(10)

Carrasco-Pancorbo, A.; Cerretani, L.; Bendini, A.; Segura-Carretero, A.; Del Carlo, M.; Gallina-Toschi, T.; Lercker, G.; Compagnone, D.; Fernández-Gutiérrez, A. Evaluation of the Antioxidant Capacity of Individual Phenolic Compounds in Virgin Olive Oil. J. Agric. Food Chem. 2005, 53 (23), 8918–8925.

453 454 455

(11)

Di Mattia, C. D.; Sacchetti, G.; Mastrocola, D.; Pittia, P. Effect of Phenolic Antioxidants on the Dispersion State and Chemical Stability of Olive Oil O/W Emulsions. Food Res. Int. 2009, 42 (8), 1163–1170.

456 457 458 459

(12)

Bonoli-Carbognin, M.; Cerretani, L.; Bendini, A.; Almajano, M. P.; Gordon, M. H. Bovine Serum Albumin Produces a Synergistic Increase in the Antioxidant Activity of Virgin Olive Oil Phenolic Compounds in Oil-in-Water Emulsions. J. Agric. Food Chem. 2008, 56 (16), 7076– 7081.

460 461

(13)

Paiva-Martins, F.; Gordon, M. H. Effects of pH and Ferric Ions on the Antioxidant Activity of Olive Polyphenols in Oil-in-Water Emulsions. J. Am. Oil Chem. Soc. 2002, 79 (6), 571–576.

462 463 464

(14)

Di Mattia, C.; Paradiso, V. M.; Andrich, L.; Giarnetti, M.; Caponio, F.; Pittia, P. Effect of Olive Oil Phenolic Compounds and Maltodextrins on the Physical Properties and Oxidative Stability of Olive Oil O/W Emulsions. Food Biophys. 2014, 9 (4), 396–405.

465 466 467

(15)

Pirisi, F. M.; Cabras, P.; Cao, C. F.; Migliorini, M.; Muggelli, M. Phenolic Compounds in Virgin Olive Oil. 2. Reappraisal of the Extraction, HPLC Separation, and Quantification Procedures. J. Agric. Food Chem. 2000, 48 (4), 1191–1196.

468 469

(16)

Patton, S.; Huston, G. E. A Method for Isolation of Milk Fat Globules. Lipids 1986, 21 (2), 170–174.

470 471 472

(17)

Dall’Orto, V. C.; Danilowicz, C.; Rezzano, I.; Carlo, M. Del; Mascini, M. Comparison between Three Amperometric Sensors for Phenol Determination in Olive Oil Samples. Anal. Lett. 1999, 32 (10), 1981–1990.

473 474 475

(18)

Paradiso, V. M.; Giarnetti, M.; Di Mattia, C. D.; Andrich, L.; Cosmai, L.; Summo, C.; Caponio, F. Accelerated Oxidation Tests in Emulsions. A Comparison of Different Oxidation Promoters. Ind. Aliment. 2014, 53, 5–10.

476 477

(19)

Shantha, N. C.; Decker, E. A. Rapid, Sensitive, Iron-Based Spectrophotometric Methods for Determination of Peroxide Values of Food Lipids. J. AOAC Int. 1994, 77, 421–424.

478 479 480

(20)

Paradiso, V. M.; Summo, C.; Pasqualone, A.; Caponio, F. Evaluation of Different Natural Antioxidants as Affecting Volatile Lipid Oxidation Products Related to off-Flavours in Corn Flakes. Food Chem. 2009, 113 (2), 543–549.

481 482

(21)

Liu, W.; Guo, R. Interaction between Flavonoid, Quercetin and Surfactant Aggregates with Different Charges. J. Colloid Interface Sci. 2006, 302 (2), 625–632.

483 484 485 486

(22)

Ávila, M.; Crevillén, A. G.; González, M. C.; Escarpa, A.; Hortigüela, L. V.; de Lorenzo Carretero, C.; Pérez Martín, R. A. Electroanalytical Approach to Evaluate Antioxidant Capacity in Honeys: Proposal of an Antioxidant Index. Electroanalysis 2006, 18 (18), 1821– 1826.

487 488 489

(23)

Del Carlo, M.; Amine, a.; Haddam, M.; della Pelle, F.; Fusella, G. C.; Compagnone, D. Selective Voltammetric Analysis of O-Diphenols from Olive Oil Using Na2MoO4 as Electrochemical Mediator. Electroanalysis 2012, 24 (4), 889–896. 20 ACS Paragon Plus Environment

Page 20 of 37

Page 21 of 37


490 491

(24)

Mannino, S.; Brenna, O.; Buratti, S.; Cosio, M. S. A New Method for the Evaluation of the “Antioxidant Power” of Wines. Electroanalysis 1998, 10 (13), 908–912.

492 493 494 495

(25)

Galato, D.; Ckless, K.; Susin, M. F.; Giacomelli, C.; Ribeiro-do-Valle, R. M.; Spinelli, A. Antioxidant Capacity of Phenolic and Related Compounds: Correlation among Electrochemical, Visible Spectroscopy Methods and Structure–antioxidant Activity. Redox Rep. 2001, 6 (4), 243–250.

496 497 498

(26)

Del Carlo, M.; Sacchetti, G.; Di Mattia, C.; Compagnone, D.; Mastrocola, D.; Liberatore, L.; Cichelli, A. Contribution of the Phenolic Fraction to the Antioxidant Activity and Oxidative Stability of Olive Oil. J. Agric. Food Chem. 2004, 52 (13), 4072–4079.

499 500 501

(27)

Riihimäki, L. H.; Vainio, M. J.; Heikura, J. M. S.; Valkonen, K. H.; Virtanen, V. T.; Vuorela, P. M. Binding of Phenolic Compounds and Their Derivatives to Bovine and Reindeer BetaLactoglobulin. J. Agric. Food Chem. 2008, 56 (17), 7721–7729.

502 503

(28)

Ozdal, T.; Capanoglu, E.; Altay, F. A Review on Protein–phenolic Interactions and Associated Changes. Food Res. Int. 2013, 51 (2), 954–970.

504 505 506

(29)

Salminen, H.; Heinonen, M.; Decker, E. A. Antioxidant Effects of Berry Phenolics Incorporated in Oil-in-Water Emulsions with Continuous Phase β-Lactoglobulin. J. Am. Oil Chem. Soc. 2009, 87 (4), 419–428.

507 508 509 510

(30)

Stojadinovic, M.; Radosavljevic, J.; Ognjenovic, J.; Vesic, J.; Prodic, I.; Stanic-Vucinic, D.; Cirkovic Velickovic, T. Binding Affinity between Dietary Polyphenols and β-Lactoglobulin Negatively Correlates with the Protein Susceptibility to Digestion and Total Antioxidant Activity of Complexes Formed. Food Chem. 2013, 136 (3-4), 1263–1271.

511 512

(31)

Dubeau, S.; Samson, G.; Tajmir-Riahi, H.-A. Dual Effect of Milk on the Antioxidant Capacity of Green, Darjeeling, and English Breakfast Teas. Food Chem. 2010, 122 (3), 539–545.

513 514

(32)

Pripp, A. H.; Vreeker, R.; van Duynhoven, J. Binding of Olive Oil Phenolics to Food Proteins. J. Sci. Food Agric. 2005, 85 (3), 354–362.

515 516

(33)

Amorati, R.; Valgimigli, L. Modulation of the Antioxidant Activity of Phenols by NonCovalent Interactions. Org. Biomol. Chem. 2012, 10 (21), 4147–4158.

517 518

(34)

Pierpoint, W. The Enzymic Oxidation of Chlorogenic Acid and Some Reactions of the Quinone Produced. Biochem. J 1966, 567–580.

519 520 521

(35)

Bassil, D.; Makris, D. P.; Kefalas, P. Oxidation of Caffeic Acid in the Presence of L-Cysteine: Isolation of 2-S-Cysteinylcaffeic Acid and Evaluation of Its Antioxidant Properties. Food Res. Int. 2005, 38 (4), 395–402.

522 523

(36)

Enache, T. A.; Amine, A.; Brett, C. M. a; Oliveira-Brett, A. M. Virgin Olive Oil Ortho-Phenols-Electroanalytical Quantification. Talanta 2013, 105, 179–186.

524 525

(37)

Romsted, L. S.; Bravo-Díaz, C. Modeling Chemical Reactivity in Emulsions. Curr. Opin. Colloid Interface Sci. 2013, 18 (1), 3–14.

526 527

(38)

Oehlke, K.; Heins, A.; St??ckmann, H.; Schwarz, K. Impact of Emulsifier Microenvironments on Acid-Base Equilibrium and Activity of Antioxidants. Food Chem. 2010, 118 (1), 48–55.

528 529 530

(39)

Sasaki, K.; Alamed, J.; Weiss, J.; Villeneuve, P.; L??pez Giraldo, L. J.; Lecomte, J.; FigueroaEspinoza, M. C.; Decker, E. A. Relationship between the Physical Properties of Chlorogenic Acid Esters and Their Ability to Inhibit Lipid Oxidation in Oil-in-Water Emulsions. Food Chem. 21 ACS Paragon Plus Environment


2010, 118 (3), 830–835.

531 532 533 534

(40)

Viljanen, K.; Kylli, P.; Hubbermann, E. M.; Schwarz, K.; Heinonen, M. Anthocyanin Antioxidant Activity and Partition Behavior in Whey Protein Emulsion. J. Agric. Food Chem. 2005, 53 (6), 2022–2027.

535 536 537

(41)

Genot, C. Distributions of Phenolic Acid Antioxidants between the Interfacial and Aqueous Regions of Corn Oil Emulsions-a Commentary. Eur. J. Lipid Sci. Technol. 2015, 117 (11), 1684–1686.

538 539

(42)

Paiva-Martins, F.; Gordon, M. H.; Gameiro, P. Activity and Location of Olive Oil Phenolic Antioxidants in Liposomes. Chem. Phys. Lipids 2003, 124 (1), 23–36.

540 541 542

(43)

Souilem, S.; Kobayashi, I.; Neves, M. A.; Jlaiel, L.; Isoda, H.; Sayadi, S.; Nakajima, M. Interfacial Characteristics and Microchannel Emulsification of Oleuropein-Containing Triglyceride Oil–water Systems. Food Res. Int. 2014, 62, 467–475.

543 544

(44)

Druaux, C.; Le Thanh, M.; Seuvre, A.-M.; Voilley, A. Application of Headspace Analysis to the Study of Aroma Compounds-Lipids Interactions. J. Am. Oil Chem. Soc. 1998, 75 (2), 127–130.

545 546

(45)

Benavente-Garcı ́a, O.; Castillo, J.; Lorente, J.; Ortuño, A.; Del Rio, J. . Antioxidant Activity of Phenolics Extracted from Olea Europaea L. Leaves. Food Chem. 2000, 68 (4), 457–462.

547 548

(46)

Baldioli, M.; Servili, M.; Perretti, G.; Montedoro, G. F. Antioxidant Activity of Tocopherols and Phenolic Compounds of Virgin Olive Oil. J. Am. Oil Chem. Soc. 1996, 73 (11), 1589–1593.

549 550

(47)

Lavelli, V. Comparison of the Antioxidant Activities of Extra Virgin Olive Oils. J. Agric. Food Chem. 2002, 50 (26), 7704–7708.

551 552

(48)

Morales, M. T.; Rios, J. J.; Aparicio, R. Changes in the Volatile Composition of Virgin Olive Oil during Oxidation: Flavors and Off-Flavors. J. Agric. Food Chem. 1997, 45 (7), 2666–2673.

553 554

(49)

Ambrosone, L.; Mosca, M.; Ceglie, A. Impact of Edible Surfactants on the Oxidation of Olive Oil in Water-in-Oil Emulsions. Food Hydrocoll. 2007, 21 (7), 1163–1171.

555


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556

Figure captions

557

Figure 1. Cyclic Voltammograms (CVs) recorded in the solutions containing the antioxidants, the

558

antioxidants plus β-lactoglobulin (BLG), and the antioxidants plus BLG plus maltodextrins (MD) at

559

different concentrations.

560 561

Figure 2. Partitioning of the olive phenolic antioxidants (%, w/w in aqueous phase) as affected by

562

emulsion formulation. MD0, no added maltodextrins; MD1, with 1% maltodextrins; MD15, with

563

15% maltodextrins. Fisher’s LSD test was applied for multiple comparisons (p = 0.05). Capital

564

letters are used to compare partitioning of different antioxidants in the same molecular

565

environment; small letters are used to compare partitioning of the same antioxidant in different

566

molecular environments.

567 568

Figure 3. Antiradical activity (% of radical quenched) of the aqueous phase of the emulsions (I) and

569

of the same aqueous solutions as the ones used for the cyclic voltammetry measurements (II).

570

MD0, no added maltodextrins; MD1, 1% maltodextrins; MD15, 15% maltodextrins. Fisher’s LSD

571

test was applied for multiple comparisons (p = 0.05). Capital letters are used to compare

572

antiradical activity of different antioxidants in the same molecular environment; small letters are

573

used to compare antiradical activity of the same antioxidant in different molecular environments.

574 575

Figure 4. Anodic peak potential (Ep,a) of the solutions containing the olive phenolic antioxidants

576

plus β-lactoglobulin plus maltodextrins (MD) at different concentrations plotted versus the

577

oxidative stability of the corresponding emulsions, expressed as area under the curve (AUC) of

578

hydroperoxides after 7 (LOH-7, red), 16 (LOH-16, green) and 28 (LOH-28, blue) days under

579

accelerated oxidation conditions. Fitted linear regressions are reported for significant correlations 23 ACS Paragon Plus Environment


580

(see Table 2). 0, no added maltodextrins; 1, 1% maltodextrins; 15, 15% maltodextrins. See

581

reference (13) for the accelerated oxidation tests.

582 583

Figure 5. Antioxidant partitioning in the acqueous phase of emulsions containing the olive phenolic

584

antioxidants and maltodextrins (MD) at different concentrations, plotted versus the oxidative

585

stability of the corresponding emulsions, expressed as area under the curve (AUC) of

586

hydroperoxides after 7 (LOH-7, red), 16 (LOH-16, green) and 28 (LOH-28, blue) days under

587

accelerated oxidation conditions. Fitted linear regressions are reported for significant correlations

588

(see Table 2). 0, no added maltodextrins; 1, 1% maltodextrins; 15, 15% maltodextrins. See

589

reference (13) for the accelerated oxidation tests.

590 591

Figure 6. Antiradical activity of the acqueous phase of the emulsions (I) and of the solutions (II)

592

containing the olive phenolic antioxidants and maltodextrins (MD) at different concentrations

593

plotted versus the oxidative stability of the corresponding emulsions, expressed as area under the

594

curve (AUC) of hydroperoxides after 7 (LOH-7, red), 16 (LOH-16, green) and 28 (LOH-28, blue) days

595

under accelerated oxidation conditions. Fitted linear regressions are reported for significant

596

correlations (see Table 2). 0, no added maltodextrins; 1, 1% maltodextrins; 15, 15% maltodextrins.

597

See reference (13) for the accelerated oxidation tests.

598 599

Figure 7. Antiradical activity of the separated aqueous phase of the emulsions (I), antiradical

600

activity of the solutions (II) and anodic peak potential (Ep,a) of the solutions (III) containing the olive

601

phenolic antioxidants plus β-lactoglobulin plus maltodextrins (MD) at different concentrations

602

plotted versus the oxidative stability of the corresponding emulsions, expressed as area under the

603

curve (AUC) of hexanal after 28 days under accelerated oxidation conditions. Fitted linear 24 ACS Paragon Plus Environment

Page 24 of 37

Page 25 of 37


604

regressions are reported for significant correlations (see Table 2). 0, no added maltodextrins; 1, 1%

605

maltodextrins; 15, 15% maltodextrins. See reference (13) for the accelerated oxidation tests.

606 607

Figure 8. Results of the regression analysis of the oxidative stability (expressed as area under the

608

curve, AUC, of hexanal after 28 days under accelerated oxidation conditions) of emulsions

609

containing the olive phenolic antioxidants and maltodextrins (MD) at different concentrations, as a

610

function of the antiradical activity of their aqueous phase and of the anodic peak potential (Ep,a) of

611

the corresponding solutions. Plot of observed values versus calculated values (I) and contour plot

612

of the AUC of hexanal after 28 days as a function of the anodic peak potential of the antioxidant

613

and of antiradical activity of the aqueous phase of emulsions (II). 0, no added maltodextrins; 1, 1%

614


615 616

Figure 9. Antioxidant partitioning in the acqueous phase of emulsions containing the olive phenolic

617

antioxidants and maltodextrins (MD) at different concentrations, plotted versus the oxidative

618

stability of the corresponding emulsions, expressed as area under the curve (AUC) of nonanal after

619

16 (C9-16, green) and 28 (C9-28, blue) days under accelerated oxidation conditions. Fitted linear

620

regressions are reported for significant correlations (see Table 2). 0, no added maltodextrins; 1, 1%

621


622



Table 1. Cyclic voltammetry parameters measured at a 3mm glassy carbon electrode for 10-4 M solutions of pure olive phenolic compounds, along with their binary and ternary solutions in presence of β-lactoglobulin (BLG), plus maltodextrins (MD). Antioxidant BLG MD (%) Ep,a (mV) Ip,a/Ip,c Ip,a (µA) ∆E (mV) LSD LSD Mean Mean testb test 0 92±14 B,b 6.21±1.04 B,a 46 1.23 ● 0 106±8 B,ab 6.42±0.50 B,a 34 1.34 oleuropein ● 1 90±15 B,b 5.06±0.71 A,b 44 1.68 ● 15 118±4 B,a 3.85±0.38 A,c 54 1.69

tyrosol

● ● ●

0 0 1 15

446±25 476±32 520±18 530±9

A,b A,b A,a A,a

6.25±0.45 5.30±0.45 3.75±0.20 3.58±0.08

B,a C,b B,c AB,c

0 78±16 B,b 7.28±0.26 A,b 39 1.07 ● 0 74±3 C,b 8.73±0.24 A,a 25 1.13 hydroxytyrosol ● 1 83±9 B,b 5.59±0.18 A,c 48 1.04 ● B,d 53 0.91 15 131±4 B,a 3.22±0.12 a , Ep,a, anodic potential; Ip,a, anodic current intensity; Ip,c, cathodic peak current; ∆E, difference between anodic and cathodic peak potentials; -, no added beta-lactoglobulin; ●, with 10-5 M betalactoglobulin; MD, maltodextrin. b , Fisher’s LSD test for multiple comparisons (p = 0.05). Capital letters are used to compare parameters of different antioxidants in the same molecular environment; small letters are used to compare parameters of the same antioxidant in different molecular environments.


Page 26 of 37

Page 27 of 37


Table 2. Pearson correlation matrix of the parameters measured for each emulsion type Variablesa LOH-7 LOH-16 LOH-28 C6-16 C6-28 C9-16 C9-28 Ep,a 0.805 0.898 0.840 0.477 0.809 0.332 0.476 Ip,a -0.163 -0.438 -0.471 0.059 -0.148 0.123 0.001 Partitioning 0.580 0.851 0.841 0.345 0.633 0.225 0.376 Quenching (solution) -0.692 -0.845 -0.814 -0.566 -0.805 -0.375 -0.484 Quenching (emulsion) -0.828 -0.741 -0.643 -0.244 -0.716 -0.316 -0.438 MD% -0.372 0.094 0.186 -0.621 -0.239 -0.739 -0.658 a , LOH-7,LOH-16,LOH-28: area under the curve of hydroperoxides after 7, 16 and 28 days of accelerated oxidation respectively; C6-16,C6-28: area under the curve of hexanal after 16 and 28 days of accelerated oxidation respectively; C9-16,C9-28: area under the curve of nonanal after 16 and 28 days of accelerated oxidation respectively; Ep,a, anodic potential; Ip,a, anodic current intensity; MD, maltodextrins.



2x10

-5

1x10

-5

oleuropein I oleuropein+BLG oleuropein+BLG+MD1% oleuropein+BLG+MD15%

tyrosol tyrosol+BLG tyrosol+BLG+MD1% tyrosol+BLG+MD15%

Page 28 of 37

OH-tyrosol III OH-tyrosol+BLG OH-tyrosol+BLG+MD1% OH-tyrosol+BLG+MD15%

II

0x100

-1x10

-5

-300

0

300

600

900

-300

0

E (mV)

300

600

900

E (mV)

Figure 1 – Paradiso et al.


-300

0

300

E (mV)

600

900


Antioxidant partitioning (%)

Page 29 of 37




50

80

I

MD0 MD1 MD15

Page 30 of 37

II

A,-

A,a

40

60

A,-

A,-

A,A,-

A,b

30

B,-

B,-

A,c

40

B,-

20

B,C,- B,-

20

10

C,a

B,-

C,b

C,c

0

oleuropein

tyrosol

hydroxytyrosol

0

oleuropein



tyrosol

hydroxytyrosol


Hydroperoxides (AUC)

Page 31 of 37



Hydroperoxides (AUC)




Page 32 of 37

Page 33 of 37


50

I

1

15

15

40

II

1

0

0

15 1

30

0 15

15 0

1

1

1 15

0

0 1

1

20

0

15

0

15 15

15 10 0

10 0

15

1

0

15

1

15

1

0

10 15

10

15

0

1 15

1 15

1 0

15 1

0

0 0

10 20 30 40 Antiradical activity - emulsion (%)

50

10

20 30 40 50 60 Antiradical activity - solution (%)



70

Hexanal - 28 days (AUC)




Page 34 of 37

Page 35 of 37


I

300

C6-28 < -400 -400 – -200 -200 – 0 0 – 200 > 200

500

0

250 400

200 Epa

15

150

1

100 50

0

200

1

1 15

300

0

15

100

0

5

0

50

100 150 200 Hexanal - 28 days calculated

250

300

10

15

20

25

30

quenching Antiradical activity (%)



35

40

II


oleuropein hydroxytyrosol tyrosol



Page 36 of 37

Page 37 of 37


For Table of Contents Only – Paradiso et al.


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

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