olive phenolic compound in ow emulsions

7/31/2019 Olive Phenolic Compound in OW Emulsions

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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 antiox-

idants 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 . Oxidativestability

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: [email protected]

Food Biophysics (2011) 6:295302

DOI 10.1007/s11483-010-9195-7


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systems to the generation of undesirable off-flavors and off-

odors 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 multi-

component and multiphasic systems can be considered an

interfacial phenomenon affected by the presence of antiox-

idant and pro-oxidant compounds and by the interactions

between the various ingredients of the system.18

The

prese nce 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 dispersedsystems.

Several amphyphilic compounds take part to the com-

position 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 charac-

terized 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 oil-

based 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) andwas characterized by the following average composition: 62%

-lactoglobulin, 12% glycoproteins, 5% -lactoalbumin, 5%

humidity, fats


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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-in-

water 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


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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 concentra-

tion in the water phase.

Besides surface tension, interfacial tensions measure-

ments were carried out to determine the affinity of these

molecules toward the olive oilwater interfaces and the

results, expressed as interfacial pressure =0, where 0is the interfacial tension value of the olive oil/buffer system

(23.11.1 mNm1) are shown in Figure 2. In general, itcan 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

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. Bytaking 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 M

1103 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 com-

pared 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.

Fig. 2 Surface pressure of antioxidant-buffered solutions contain-

ing different concentrations of oleuropein, syringic acid, and tyrosol

and refined olive oil

Concentrations [M] Tyrosol (mNm1) Syringic Acid (mNm1) Oleuropein (mNm1)

1106 73.30.5 69.82.3 61.70.2

1105 74.20.7 76.30.1 61.82.5

5105 74.30.2 75.90.1 60.80.3

1104 73.50.3 74.11.0 62.30.5

5104 74.60.3 76.40.1 53.80.5

110

3 74.70.1 76.20.3 56.70.3

Table 1 Air/water surface

tensions of tyrosol, syringic

acid, and oleuropein, tested in

a concentration range of

11061103 M

298 Food Biophysics (2011) 6:295302


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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 antioxidant-

surfactant 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 concen-

trations, 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

effect of the antioxidant addition on the dispersion degree is

strongly dependent on the emulsifier used for the stabiliza-

tion 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 emulsionscan 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 5 104 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 hydro-

peroxides 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)

Food Biophysics (2011) 6:295302 299


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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 individ-

ually 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


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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 hydro-

peroxides shown in Figure 4a.

In the WPC-stabilized emulsions, as already evidenced

in the formation of hydroperoxides, the production ofsecondary 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 antiox-

idative 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 oxidativestabilization 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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