stability of a phenol-enriched olive oil during storage

Research Article

Stability of a phenol-enriched olive oil during storage

Manuel Suarez, Maria-Paz Romero, Tomas Ramo and Maria-Jose Motilva

Food Technology Department, XaRTA-TPV, Escuela Tecnica Superior de Ingenierıa Agraria, University of

Lleida, Lleida, Spain

The use of an emulsifier to stabilize the phenolic compounds added in the preparation of an enriched olive

oil was evaluated. Two emulsifiers, lecithin andmonoglyceride, were studied. The results showed lecithin to

be the most convenient, due to the increase in the value of the oxidative stability of the phenol-enriched oils

in relation to the enrichments preparedwithmonoglycerides. After that, the shelf life of the prepared oils was

evaluated during a period of 256 days of storage at 258C in the dark. Oil quality parameters, total phenolic

content, bitterness index and oxidative stability were studied during the storage period. Additionally, the

phenolic composition and antioxidant capacity (by using the ORAC assay) were evaluated at the end of the

storage. The phenolic enrichment of the oils allowed the shelf life of the oils to be extended compared with

the control (virgin olive oil without phenol addition), delaying the appearance of peroxides and improving

their oxidative stability. In addition, the higher content of phenolic compounds in the oils at all stages of

storage is desirable in order to increase the intake of these beneficial compounds.

Practical applications: The preparation of phenol-enriched olive oils with a higher phenolic content

than the commercial virgin olive oils is of special interest to increase the ingestion of these healthy

compounds the daily intake of which is limited due to the high caloric value of olive oil. There are two key

points in the development of this product: (i) the dispersion and stabilization of the phenol extract in the

oil matrix and (ii) the stability of the phenols in the prepared oils to guarantee the phenol concentration

during their shelf life. It is important to study the use of emulsifiers to determine if they allow an

improvement in the dispersion of the phenolic extract, and their stabilization in the final product. In

addition, the emulsifiers could mask the bitter taste of the enriched oils, which is desirable to increase

consumer acceptance of the enriched oil.

Keywords: emulsifiers / phenol enrichment / polyphenols / olive oil / ORAC

Received: August 1, 2010 / Revised: January 10, 2011 / Accepted: March 2, 2011

DOI: 10.1002/ejlt.201000432

: Supporting information available online

1 Introduction

The increasing interest of consumers in healthy, natural

products, manufactured without the use of synthetic anti-

oxidants, has led the food industry to search for new sources

of compounds that could be used as additives. In this

search, phenolic compounds, which are widely spread in

plants, have earned attention due to their wide spectra of

properties [1, 2].

Virgin olive oil, the consumption of which is believed to

have a direct influence on the reduction of coronary heart

diseases, is characterized by its content of phenolic com-

pounds that the refined olive oils do not contain [3, 4]. In

addition to their in vitro activities [5, 6], the identification of

these compounds and relatedmetabolites in plasma and urine

samples after the intake of virgin olive oil with high phenolic

content has pointed to their bioavailability [7, 8], reinforcing

virgin olive oil as natural source of healthy compounds.

However, some studies have suggested that only 55–66%

of the phenolic content of virgin olive oil is absorbed by

humans [9]. This, together with the limited daily intake of

Correspondence: Dr. Maria-Jose Motilva, Food Technology Department,

XaRTA-TPV, Escuela Tecnica Superior de Ingenierıa Agraria, University of

Lleida, Av/Alcalde Rovira Roure 191, 25198 Lleida, Spain

E-mail: [email protected]

Fax: þ34 973 702596

Abbreviations: AAPH, 2,20-azobis(2-methylpropionamide) dihydrochloride;

ORAC, oxygen radical absorbance capacity; UPLC, ultra-performance

liquid chromatography; 3,4-DHPEA-EDA, dialdehydic form of elenolic

acid linked to hydroxytyrosol; p-HPEA-EDA, dialdehydic form of elenolic

acid linked to tyrosol

894 Eur. J. Lipid Sci. Technol. 2011, 113, 894–903

� 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

olive oil (due to its high caloric content) reduces the import-

ance of this product as a source of phenols compared with

other foods like fruit and vegetables.

Enrichment of foods, such as milk and fruit juices, has

been proposed over the years to supplement the natural dose

of some compounds in foods. Therefore, it is possible to

increment the intake of some specific compounds like vita-

mins or antioxidants that could have been lost during the

processing [10]. A potential enrichment of olive oil with its

own polyphenols could improve the limited ingestion of these

compounds, increasing their daily intake without the draw-

back of a higher calorie intake.

Olive-cake, the main by-product from the virgin olive oil

extraction process, has been shown to be a rich source of these

compounds, and could be used to obtain an extract which

contains the main phenolic compounds from virgin olive oil.

In addition, the development of new extraction procedures,

such as accelerated solvent extraction (ASE), allows a

reduction of the extraction time and solvent consumption

while increasing the efficiency of extraction [5].

The dispersion of phenolic extracts (dissolved in an aque-

ous media) into lipid matrices is complicated due to the high

instability of this kind of emulsions, in which there is a natural

trend towards the separation of the oil and water phases [11].

Therefore, the small droplets of water dispersed in the oil can

aggregate when they are close to each other. In addition, the

absence of strong repulsive forces in the continuous oil phase

could promote droplet aggregation [12]. The use of emulsi-

fiers has been proposed in order to prevent these problems.

Thus, lecithin and monoglycerides are commonly used in the

food industry in the formulation of different products, such as

butters, baby food, and ice-creams among others [13]. Their

use is aimed to improve the emulsification process in oil-in-

water (o/w) and water-in-oil (w/o) suspensions and to avoid

the separation of components in food formulas. Some authors

suggested that a co-surfactant (in general a short-chain alco-

hol such as propanol or ethanol) is necessary to allow the

formation of this type of microemulsions [14].

One of the main causes of the loss of quality in virgin

olive oil during storage is oxidative degradation, and the

importance of the role of phenolic compounds in preventing

this oxidation in the oil has been demonstrated [15]. A

previous study with virgin olive oils from the Arbequina culti-

var showed a noticeable decrease in the concentration of the

phenolic fraction, mainly in secoiridoids derivatives, after

12 months of storage. On the other hand, lignans were the

most stable phenolic compounds [16]. Different studies have

confirmed this phenolic loss during long-term storage (up to

21 months) [17, 18] with more notable losses after 6 months

of storage [19]. Oxidation seems to produce the transform-

ation secoiridoids and the appearance of their oxidized forms

when virgin olive oil is submitted to an accelerated treatment

at 608C for up to 7 weeks.

The goal of this study was firstly to evaluate the use of

emulsifiers (lecithin or monoglycerides) in the preparation of

a nutraceutical olive oil enriched with phenols, to improve the

dispersion and stabilization of the phenolic extract in the oil

matrix. Once the best option was selected, and taking into

account the sensitivity of the phenols to storage, a study of the

shelf life of the prepared enriched olive oil was carried out to

establish the influence of the emulsifier. To this end, different

parameters were analyzed during a storage period of 256 days

at 258C in the dark. These were: (i) quality parameters; (ii)

total phenolic content and phenol composition; (iii) antiox-

idant activity and (iv) bitterness index, to find out the long

term effects of these enrichments in the oils. This is important

to guarantee the phenolic content of the nutraceutical olive oil

and determine the date of the recommended consumption

that would ensure a specific content.

2 Materials and methods

2.1 Samples

The samples of virgin olive oil used as the matrix in the

phenolic enrichment were from the Siurana Protected

Denomination of Origin (Catalonia, Spain), which is pro-

duced exclusively from the Arbequina cultivar. On the other

hand, the samples of olive cake, used to obtain the phenolic

extract, were taken from a commercial olive oil mill from the

olive-growing area of Les Garrigues (Catalonia, Spain),

which works by the two-phase centrifugation system.

These samples were taken at the decanter outlet and liquid

nitrogen was immediately added to avoid oxidative damage.

The samples were stored at �408C until the extraction of the

phenolic compounds.

The lecithin used was EmulpurTM (Cargill, Barcelona,

Spain) and the monoglyceride mixture wasMyverol 18–04 K

from Quest International (Naarden, The Netherlands).

2.2 Chemicals and reagents

Apigenin, apigenin-7-O-glucoside, luteolin, luteolin 7-O-glu-

coside, oleuropein, rutin, tyrosol, and vanillin were purchased

from Extrasynthese (Genay, France). Hydroxytyrosol was pur-

chased from Seprox Biotech, S.L. (Madrid, Spain). Caffeic,

p-coumaric and vanillic acids and fluorescein were purchased

from Fluka Co. (Buchs, Switzerland) and (þ)-pinoresinol

was acquired from ArboNova (Turku, Finland). The dialde-

hydic form of elenolic acid linked to hydroxytyrosol (3,4-

DHPEA-EDA), the dialdehydic form of elenolic acid linked

to tyrosol (p-HPEA-EDA) and the lignan acetoxypinoresinol

were not available commercially and were isolated from

virgin olive oil by semi-preparative HPLC [20]. Methyl-b-

cyclodextrin (RMCD) was from Aldrich (Steinheim,

Germany), 2,20-azobis(2-amidino-propano) dihydrochloride

(AAPH), and Trolox were from Acros Organics (Geel,

Belgium).

Acetonitrile (HPLC grade), ethanol, hexane, and acetic

acid were all provided by Scharlau Chemie (Barcelona,

Eur. J. Lipid Sci. Technol. 2011, 113, 894–903 Storage phenol-enriched olive oil 895


Spain). Water was Milli-Q quality (Millipore Corp, Bedford,

MA, USA).

2.3 Extraction of phenolic compounds from freeze-dried olive cake

The phenolic extract was obtained from freeze-dried olive-

cake using an accelerated solvent extractor (ASE 100)

(Dionex, USA) following the method described in our

previous paper [5]. Briefly, 10 g of freeze-dried olive-cake

and 5 g of diatomaceous earth were mixed and introduced in

a 100-mL extraction cell where the flush volume was set at

60%. Ethanol:water (80:20 v/v) at 808C was used as the

extraction solvent and two static cycles of 5 min were pro-

grammed in each extraction. The extract was rotary evapor-

ated to eliminate all the ethanol and then freeze-dried

(Lyobeta, Telstar, Spain) and stored at �808C.

The composition of phenolic compounds of the extract

was determined by UPLC-ESI-MS/MS (Additional

Information). Basically, it included secoiridoids and deriva-

tives such as 3,4-DHPEA-EDA, 3,4-DHPEA-EA (oleuro-

pein aglycone), elenolic acid, and ligstroside derivative. This

group of compounds represents 89.4% of the total phenolic

content of the extract. The quantities of phenyl alcohols

(3.5%) (hydroxytyrosol and tyrosol) and flavonoids (6.0%)

(rutin, apigenin, luteolin, apigenin-7-O-glucoside and luteo-

lin-7-O-glucoside) are also of special interest.

2.4 Phenolic enrichment of virgin olive oil

To carry out the phenolic enrichment of the olive oil, 7 mg of

olive-cake phenolic extract/mL oil and 0.3% (p/v) of emulsi-

fier (either lecithin or monoglycerides) were dissolved

in ethanol:water (50:50 v/v) and were incorporated into a

small amount of virgin olive oil (approximately 25% of the

final volume). The mixture was sonified (Sonifier 150D,

Branson, USA) for 20 s to allow the complete dissolution

of the phenolic extract and emulsifier in the oily matrix.

After that, the remaining oil was added and, all together,

was mixed using a Polytron (Kinematica, Littau,

Switzerland) for 1 min to obtain a homogeneous mixture.

A cold bath was used to counteract the increase in tempera-

ture generated during this step of the dispersion/enrichment

process. Finally, the olive oil was filtered through a paper

filter Ahlstrom (Ahsltrom S.A., Barcelona, Spain) and bot-

tled in dark bottles of 30 mL which were stored at 258Cduring a period of 256 days. Nitrogen was added in the

headspace of each bottle to avoid oxidation. The control

olive oil (without added phenolic compounds) was also sub-

mitted to the process of mixing and filtering to have equal

conditions in all the olive oils under study. All the oils were

prepared by duplicate.

To study the shelf life of the enriched oil, different

parameters were analyzed at 0, 20, 50, 90, 120, 172, and

256 days after the preparation of the enriched oil.

2.5 Analysis of control and enriched olive oils

2.5.1 Quality parameters

The quality parameters of the olive oils were analyzed accord-

ing to the European Union Commission Regulation EEC/

2568/91. The peroxide value, which indicates the primary

oxidation of the oil, was expressed as milliequivalents of active

oxygen per kilogram of oil. Free fatty acid, which indicates the

amount of free fatty acid in the oil, was expressed as percentage

of oleic acid. Finally, K270, relatedwith the secondary oxidation

of the oil, was expressed as absorbance at 270 nm.

2.5.2 Total phenolic content (Folin-Ciocalteau test)

The extraction of the total phenolic compounds from the oils

was carried out following the method described by Suarez

et al [21] by triple extraction of an oil-in-hexane solution with

methanol:water (60:40 v/v). After that, the total phenolic con-

tent of the extract was estimated using the Folin-Ciocalteau

reagent measuring the absorbance at 725 nm [22]. The results

were expressed using caffeic acid as the reference compound

(mg of caffeic acid per kg of oil).

2.5.3 Oxidative stability

The Rancimat test (Metrohom, Herisau, Switzerland) was

used to evaluate the oxidative stability of the enriched olive

oils. An air flow of 20 L/h and a temperature of 1208C were

set to enhance the oxidation of the olive oil (ISO 6886:1996).

The conductivity of the samples was continuously measured

and the induction time, the time needed to reach the break

point in this curve, was measured. All the samples were

analyzed in duplicate and a control (virgin olive oil without

addition of phenolic extract) was incorporated into each

experimental set. The results were expressed in hours and

increased proportionally with the stability of the sample.

2.5.4 Bitter index (K225)

Bitter index is a parameter that provides an estimation of the

presence of compounds with a strong bitter attribute due to

the correlation of these compounds with the absorbance

at 225 nm [23]. We followed the method reported by

Artajo et al. [20]. Briefly, 1 g of oil dissolved in 4 mL of

hexane was passed through a C18 column (Waters Sep-Pak

cartridges), previously activated with 6 mL of methanol and

6 mL of hexane. A clean-up of the cartridges was done using

10 mL of hexane and, finally, the compounds were eluted

with 25 mL of methanol:water (50:50 v/v).

2.5.5 Phenol extraction of the olive oils

The phenolic compounds of the olive oils were extracted

following the method described in our previous paper [21].

896 M. Suarez et al. Eur. J. Lipid Sci. Technol. 2011, 113, 894–903


Briefly, 20 mL of methanol/water (80:20 v/v) were added

to 45 g of oil and homogenized for 2 min with a Polytron.

After that, the two phases were separated by centrifuging

at 640 g for 10 min, and the hydroalcoholic phase was evap-

orated to obtain a syrupy consistency at 318C and purified by

liquid–liquid extraction with acetonitrile. The acetonitrile

solution was rotary evaporated to dryness, dissolved in

5 mL of methanol and kept at �408C until the chromato-

graphic analysis.

2.5.6 Identification of phenolic compounds by UPLC-ESI-MS/MS

The phenolic compounds were analyzed by UPLC-MS/MS.

The system consisted of an AcQuityTM UPLC equipped

with a binary pump system Waters (Milford, MA, USA)

using an AcQuity UPLCTM BEH C18 column (1.7 mm,

100 mm � 2.1 mm id). The analyses were carry out at

308C, using a flow rate of 0.4 mL/min with milliQ water:-

acetic acid (100:0.2 v/v) as solvent A and acetonitrile as

solvent B. Chromatographic separation was done using the

method optimized in our previous paper [21].

The UPLC was coupled to a TQTM mass spectrometer

(Waters, Milford, MA, USA). The software used was

MassLynx 4.1. Ionization was done by ESI in the negative

mode and the data were collected in the selected reaction

monitoring (SRM) mode. The ionization source parameters

were capillary voltage 3 kV, source temperature 1508C and

desolvation gas temperature 4008C with a flow rate of 800 L/

h. Nitrogen (99.99% purity, N2 LC-MS nitrogen generator,

Claind, Como, Italy) and argon (�99.99% purity, Alphagaz,

Madrid, Spain) were used as the cone and collision gases,

respectively. 10 mg/L of each compound were directly

infused into the equipment to obtain the best instrumental

conditions [5].

Commercial standards were used to obtain calibration

curves for the respective compounds. When no commercial

compounds were available, semi-preparative HPLC was

used to purify them [20]. Secoiridoids were quantified using

3,4-DHPEA-EDA and p-HPEA-EDA on the basis of the

chemical similarity of each compound with the standard

chosen for its quantification [20]. The results are expressed

as mg of phenol/kg of oil.

2.5.7 Antioxidant activity of enriched olive oil byORAC assay

The oxygen radical absorbance capacity (ORAC) assay was

based on the methodology reported by Prior et al. [24] with

some modifications. The assays were carried out on a

FLUORstar optima spectrofluorometric analyzer (BMG

Labtechnologies GmbH; Offenburg, Germany) in 96-well

microplates, using an excitation filter at 485 nm and an

emission filter at 520 nm. Trolox was used as the reference

substance while AAPH was used as the initiator of the reac-

tion. The ORAC values were calculated based in the area

under curve (AUC) results, with the data expressed as micro-

moles of Trolox equivalents per 100 g of oil.

To prepare the samples, 0.15 g of oil was diluted in

hexane until the range delimited by the Trolox was reached.

After drying under nitrogen flow, the samples were dissolved

in a solution of RMCD 7% in acetone:water (50:50 v/v) in

order to allow their correct solubility. Trolox was also pre-

pared in RMCD 7% while fluorescein and AAPH solutions

were prepared using a phosphate buffer. The reactions mix

consisted in 20 mL of either diluted olive oil or Trolox,

125 mL of 68 nM fluorescein solution, and 50 mL of

74 mM AAPH solution. RMCD 7% was used as a blank.

The experiments were carried out at 378C.

2.6 Data treatment

All the experiments were carried out in triplicate. The data

were analyzed using Statgraphics plus v.5.1 software

(Manugistics Inc., Rockville,MD,USA). Data were analyzed

by the ANOVA test with a significance level of 0.05. The

principal components analysis (PCA) was conducted to

observe correlations between the variables.

3 Results and discussion

3.1 Influence of the emulsifier in the effectiveness ofthe phenolic enrichment

The enrichment of olive oil with olive-cake extract could be

considered a water/oil emulsion in which small droplets of the

hydro-ethanolic solution of the extract are dispersed in the

oil. These droplets are surrounded by the emulsifier and form

the so-called reverse micelles [25]. In order to study the

influence of emulsifiers on the stabilization of these emul-

sions, different oils were prepared adding either lecithin or

monoglycerides. The effectiveness of the phenol enrichment

was monitored by the evaluation of the oxidative stability of

the prepared oils with the Rancimat test. Moreover, enriched

oil without emulsifier was also prepared to compare the

resulting data. The choice of these two emulsifiers was done

according with the literature and their frequent use in the

food industry for the preparation of emulsions [13], setting

their final amount in the oil similarly to the quantities usually

used in fats and oils in the food industry. Thus, a percentage

of 0.30% of emulsifier (weight/volume) was maintained in all

the enrichments. The effect of the addition of emulsifier on

the oxidative stability of the enriched oils is shown in Fig. 1.

As can be seen, the addition of the olive-cake extract

significantly increased the oxidative stability of the oils com-

pared with the control in all the cases. Concerning the

enriched oils, while the use of lecithin allowed an improve-

ment in the oxidative stability compared with the value

obtained without emulsifier (approximately a 12% incre-

ment), the use of monoglycerides produced the opposite



effect, decreasing the induction time (approximately 18%

reduction). This could be explained by the ability of lecithin

to stabilize the added phenolic compounds in the oil matrix

due to its amphiphile behavior, forming reverse micelles that

contain the hydro-ethanolic solution of the extract. On the

other hand, the decrease observed with monoglycerides

revealed some interference between the phenol extract and

this emulsifier that led to the lower stability of the enriched

oil. This result agreed with those by McSweeney et al. [26],

who found that the use of monoglycerides in baby foods

decreased their heat stability. They argued that this could

be explained by the reaction between the proteins and the

monoglycerides, which generated a protein displacement.

In our case, the presence of substances apart from the phe-

nolic compounds in the olive-cake extract could be the cause

of this phenomenon. Thus, monoglycerides could be inter-

acting with these components producing the loss of stability

of the enriched oils. This reduction of oxidative stability

could also be related to the self-oxidation of the

monoglycerides.

As a consequence, the results suggested the choice of

lecithin as the best emulsifier to be used in the enrichment

of the olive oils due to its improvement in the final oxidative

stability of the enriched oils.

3.2 Phenolic composition of the phenol enriched oils

Once lecithin was shown to be the most suitable emulsifier to

carry out the enrichments, an experimental design was devel-

oped to measure its effect on the phenolic composition of

enriched olive oils. Thus, four olive oils were prepared:

(i) control olive oil (without enrichment); (ii) enriched olive

oil with olive-cake extract using lecithin as an emulsifier;

(iii) enriched olive oil with olive-cake extract; (iv) enriched

olive oil with lecithin. The phenolic composition of the four

oils was determined by UPLC-ESI-MS/MS and the results

are shown in Table 1. As can be seen, the addition of

the olive-cake extract significantly increased the amount of

phenolic compounds in the oils in relation to the virgin olive

oil used as the basis for the preparation of the enriched oil (Oil

I). However, the use of lecithin produced a decrease in the

concentration of these phenols in the oils. This can be

observed by comparing oils I and IV and III and II, respect-

ively, where the oils with lecithin presented a lower value of

phenolic compounds. This could be explained by the amphi-

pilic behavior of lecithin, which could have reduced the

extraction of phenols by the methanolic solution prior to

chromatographic analysis.

3.3 Evaluation of the stability of the enriched oilduring storage

3.3.1 Quality parameters

Firstly, the quality parameters of the four olive oils were

analyzed throughout storage (256 days) to determine their

shelf life. As can be seen, in almost all cases, the parameters

where within the limits established by the European

Regulation. Thus, it is possible to see that the peroxide value

increased during the storage of the oil (Fig. 2a). Initially, all of

them had similar levels of peroxide. However, after 256 days,

the peroxide value of the control oil was far beyond the limit

established (�20) while the enriched oils were still in the

range of 18–19 meqO2 active/kg oil. This indicated a delay in

the peroxidation of the enriched oils due to the incorporation

of the olive-cake extract and, therefore, an increase in the time

that these oils could maintain their properties. This could be

due to the higher amount of phenolic compounds in the

enriched oils, as these compounds have a high antioxidant

activity, and can oxidize themselves thus preventing the oxi-

dation of the oils. The fact that oil IV, which only contained

lecithin, also had a lower peroxide value, compared with the

control, could be attributed to the contribution of the phos-

pholipids (the main constituents of soy lecithin) in the oxi-

dative stability of the oils [27]. Nevertheless, no significant

synergistic effect was detected by the combination of both the

olive-cake extract and lecithin.

By contrast, a slight increase in the acidity, expressed as a

percentage of free oleic acid, was observed in oils prepared

with olive cake (Fig. 2b). However, the acidity values were

low and remained within the range for extra virgin olive oil

(�0.8%).

Finally, although the value of the K270 increased with the

storage of the oils, all of them were under the limit for extra

virgin olive oil at the end of this period (�0.22) (Fig. 2c). It

can be seen that the enriched oils had a slightly higher value

compared with the control. Nevertheless, Koprivnjak et al [28]

observed similar behavior in their study of olive oils enriched

with lecithin and found out that this increase was pro-

portional to the amount of lecithin incorporated, concluding

that there might have been secondary oxidation compounds

and commercial conjugated trienes in the commercial lecithin

they used.

0 5 10 15 20It (hours)

MonoglycerideLecithinNo emulsifierControl

a

b

b

c

Figure 1. Effect of the addition of emulsifier on the oxidative stability

of the enriched olive oils. Values expressed as hours (n ¼ 3).



3.3.2 Total phenolic content and related parameters:Oxidative stability and bitterness

The total phenolic content, bitterness index, and oxidative

stability are strongly interrelated parameters. Therefore, a

change in one of these parameters has a direct influence

on the others. Figure 3 shows that these three factors

decreased with storage time. The decrease in the total phe-

nolic content and the oxidative stability were very similar,

while the change observed in the value of the bitterness index

was slightly lower.

The total phenolic content of the stored oils, quantified by

the Folin-Ciocalteau, decreased proportionally with storage,

as can be seen in Fig. 3a. Similarly to what was observed in the

chromatographic identification of the polyphenols, those

samples which contained lecithin had a lower total phenolic

content. This could be due to the formation of small drops of

the hydroethanolic solution of the extract surrounded by

lecithin molecules that prevented them from reacting with

the Folin-Ciocalteau, resulting in the underestimation of the

total phenol content of oils prepared with lecithin. Our results

agree with those obtained by Koprivnjak et al [28], who

observed a decrease in the total phenolic content directly

proportional to the amount of lecithin added to the oil.

Considering that phenolic compounds are one of the com-

ponents most responsible for the healthy properties of olive

oil, it is desirable to maintain the highest possible levels of

these compounds. For this reason, the use of the olive-cake

extract, which allowed a 1.5 and 3-fold increment of the total

phenolic content, with and without the use of lecithin,

respectively, compared with the control, is a very attractive

way of extending the shelf life of the oils.

As mentioned above, the evolution of the bitterness index

(K225) during storage was directly proportional to the total

phenolic content (Fig. 3b). This way, oil IV presented the

lowest value and oil III, the highest. This agreed with our

previous studies that showed that phenolic compounds from

olive oil, especially secoiridoid derivatives, which are themost

abundant phenolic group of the olive-cake extract, are the

most responsible for its bitter taste [20]. As a consequence,

the use of lecithin in the preparation of the phenol enriched

olive oils could be desirable as a strategy to mask the incre-

ment in the bitter attribute produced by the phenolic com-

pounds and, therefore, improve the acceptance of the oil by

the consumer.

Finally, as can be clearly seen in Fig. 3c, the oxidative

stability behaved like the total phenolic content of the oils,

decreasing during storage. The higher value of oxidative

stability of the enriched oils can be related to the incorpora-

tion of phenolic compounds, which are known to have high

Table 1. Phenol composition of enriched olive oils. Oil I: control (olive oil without phenol addition); Oil II: olive oil plus olive-cake phenol extract

plus lecithin; Oil III: olive oil plus olive-cake phenol extract; Oil IV: olive oil plus lecithin. Values expressed in ppm of compound in the oil.

Phenolic compound (mg/kg oil) Oil I Oil II Oil III Oil IV

Hydroxytyrosol 1.59 � 0.21 4.39 � 0.84 3.28 � 0.52 0.32 � 0.02

Tyrosol 1.08 � 0.19 1.85 � 0.26 1.00 � 0.06 0.50 � 0.04

Total phenyl alcohols 2.7 6.2 4.3 0.8

Vanillin 0.37 � 0.02 0.45 � 0.02 0.48 � 0.06 0.16 � 0.01

Vanillic acid 0.17 � 0.03 0.63 � 0.11 0.42 � 0.01 0.00 � 0.00

p-coumaric acid 0.09 � 0.01 0.60 � 0.03 0.33 � 0.00 0.00 � 0.00

Total phenolic acids 0.6 1.7 1.2 0.2

3,4-DHPEA-EDAa) 180 � 12 393 � 19 458 � 23 58 � 0.7

3,4-DHPEA-ACa) 0.34 � 0.05 0.27 � 0.05 0.29 � 0.05 0.16 � 0.02

3,4-DHPEA-EAa) 92.1 � 6.8 97.6 � 3.4 112.9 � 12.4 98.6 � 0.4

p-HPEA-EDAb) 8.92 � 1.03 11.42 � 0.69 5.61 � 0.81 1.54 � 0.04

p-HPEA-EAb) 16.31 � 2.24 21.65 � 1.87 17.69 � 1.12 9.87 � 0.74

Total secoiridoid derivatives 298 524 595 111

Pinoresinol 1.77 � 0.39 2.65 � 0.34 0.98 � 0.12 0.65 � 0.03

Acetoxypinoresinol 13.03 � 0.31 7.56 � 0.41 6.86 � 0.04 6.18 � 0.36

Total lignans 14.8 10.2 7.8 6.8

Luteolin 2.23 � 0.16 4.50 � 0.92 4.20 � 0.21 0.38 � 0.04

Apigenin 0.37 � 0.12 0.68 � 0.04 0.58 � 0.09 0.20 � 0.03

Total flavonoids 2.6 5.2 4.8 0.6

Total phenols 319 547 613 177

3,4-DHPEA-EDA: dialdehydic form of elenolic acid linked to hydroxytyrosol; p-HPEA-EDA: dialdehydic form of elenolic acid linked to

tyrosol; p-HPEA-EA: aldehydic form of elenolic acid linked to tyrosol; 3,4-DHPEA-AC: 4-(acetoxyethyl)-1,2-dihydroxybenzene; 3,4-

DHPEA-EA oleuropein aglycone;a) Quantified with the calibration curve of 3,4-DHPEA-EDA.b) Quantified with the calibration curve of p-HPEA-EDA.



antioxidant properties. Thus, the enriched oils had an average

enhancement of oxidative stability of 65% compared with the

value of the control oil, which is desirable for extending the

shelf life of this product.

3.3.3 Principal component analysis

Once the information from the analysis of all the parameters

in the oils had been collected, a statistical study was carried

out to see the distribution that followed the data. Principal

component analysis (PCA) was considered the best option

due to its usefulness in establishing relationships between

variables. Thus, Fig. 4 shows the plot of the two first principal

components that explain 81.5% of the total variance in the

data. As can be clearly seen in the general plot of the

parameters, there is a positive relationship between the total

phenolic content, the bitterness index and the oxidative

stability and a negative relation of these three parameters

with the peroxide value. On the other hand, the plot of the

data helps us to understand the evolution of the parameters

during storage. Thus, samples initially had a higher value of

oxidative stability, total phenolic content and K225 and low

peroxide value, appearing in the upper left quadrant of the

figure and, as the storage time increased the sample moved

diagonally to the bottom right quadrant (the peroxide value

increasing and the other parameters decreasing).

In addition, the use of PCA allows us to successfully

place all the samples into three groups: one made up of

the control oils (Oil I), another of the control oils with lecithin

(Oil IV), and the last one made up of the oils enriched with

the olive-cake extract (Oil II and Oil III). Thus, it was not

possible to differentiate the enriched oils with lecithin from

the oils without lecithin. This could suggest that, in general

trends, there is no significant difference from the application

of lecithin to the oils in terms of analytical determinations

during storage.

0

5

10

15

20

25

30

O

meq

2/ k

g oi

l

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

% O

leic

aci

d

Days

0

0.05

0.1

0.15

0.2

0.25

250200150100500

K27

0

(a)

(b)

(c)

Figure 2. Evolution of the quality parameters of the enriched olive

oil during the storage in dark bottles at 258C. 2a: peroxide value; 2b:

acidity; 2c: K270. Oil I: control (^); Oil II: control plus olive-cake

extract plus lecithin (&); Oil III: control plus olive-cake (~); Oil IV:

control plus lecithin ( Þ:

0

2

4

6

8

10

12

14

250200150100500Days

Hou

rs

00.050.1

0.150.2

0.250.3

0.350.4

0.450.5

K22

5

0

100

200

300

400

500

600

oil

mg

caff

eic

acid

/kg (a)

(b)

(c)

Figure 3. Evolution of the total phenolic content (a), K225 (b) and

oxidative stability (c) of the enriched olive oil during the storage in

dark bottles at 258C. Oil I: control (^); Oil II: control plus olive-cake

extract plus lecithin (&); Oil III: control plus olive-cake (~); Oil IV:

control plus lecithin ( Þ:



3.3.4 Antioxidant capacity of the phenol enriched oilsby ORAC assay

The antioxidant capacity of the oils was analyzed by the

ORAC assay at the beginning (fresh oils) and the end of

storage (256 days) in the four oils under study. As can be

seen in Fig. 5, storage significantly decreased the antioxidant

capacity of all the oils. This can be related to the loss of

phenolic compounds, which was proportional to the storage

time of the oil. With regard to the type of oil, it can be seen

that the decrease in the antioxidant capacity of the oils without

olive-cake extract was lower than that suffered when it was

incorporated in the oil (40% reduction in front of 65%, respect-

ively). This loss of phenols related with the initial phenol con-

tent in oil was also observed by Gomez-Alonso et al. [17] who

saw that the reduction of the total phenolic compounds

ranged from 43 to 73%, the losses being greater in samples

with higher initial contents. In addition, the non-significant

difference in the value of the antioxidant capacity between

samples with andwithout lecithin observed in our study could

mean that the addition of lecithin has no significant influence

on the antioxidant capacity of the oils, this only being

dependent on the addition of the phenolic extract.

3.3.5 Effect of the storage in the phenoliccomposition of the oils

Finally, individual phenolic compounds were quantified at

the end of storage (256 days) to find out the long-term effect

on the phenol enrichment. In general, a decrease in the total

amount of phenolic compounds was observed at the end of

storage similarly to that previously observed in the total

phenolic quantification by the Folin-Ciocalteau. The con-

centration of phenolic acids (p-coumaric and vanillic acid

and vanillin) remained almost constant during storage. This

result agreed with those obtained by Morello et al [16], who

saw no significant difference in the phenolic acids after

12 months of storage of commercial virgin olive oil from

the Arbequina cultivar. Due to the richness in secoiridoid

derivatives of the olive-cake extract used in the present study,

special attention was paid to this group of phenolic com-

pounds. As can be seen in Fig. 6, oleuropein derivatives (3,4-

DHPEA-EDA and 3,4-DHPEA-EA) significantly decreased

in a similar manner in all the oils, both enriched and controls,

during storage. As a consequence, the amount of hydroxy-

tyrosol increased significantly in parallel with this. On the

other hand, with regard to ligstroside derivatives, while p-

HPEA-EDA and p-HPEA-EA decreased in the oils without

olive-cake extract (Oils I and IV), they increased at the end of

storage in oils enriched with olive-cake. This could be

explained by the presence of some precursors of these com-

pounds, such as dimer structures of ligstroside derivatives, in

the extract that could be releasing these during storage. In the

study by Morello et al [16], an increase in the amount of p-

HPEA-EA as consequence of the oil storage was also

observed, similarly to the one obtained in the present study.

Similar results were also obtained by Boselli et al [29].

Finally, the concentration of tyrosol increased in all the oils

following the same behavior as hydroxytyrosol.

4 Conclusions

In this study, lecithin was identified as the best emulsifier to

prepare olive oils enriched with an olive-cake phenol extract,

based on the improved oxidative stability in the prepared oils

compared with those obtained when monoglycerides where

used as emulsifiers. This could be due to the amphiphylic

I1

I2I3

I4

I5

I6

I7

I1 I2

I3 I4

I5I6

I7

II1

II2

II3II4

II5

II6II7

II1

II2

II3

II4

II5

II6

II7

III1

III2

III3 III4

III5

III6

III7

III1III2

III3

III4

III5

III6

III7

IV1 IV2IV3

IV4

IV5

IV6

IV7

IV1 IV2

IV3

IV4

IV5

IV6 IV7

543210-1-2-3-4-5-6

Factor 1: 51.89%

-5

-4

-3

-2

-1

0

1

2

3

4

Fact

or2:

29.6

1%

PeroxideAcidity

K270

Phenols

K225

Rancimat

Figure 4. Principal components analysis of the enriched olive oils

based on the main important factors (Rancimat: oxidative

stability; K225: bitterness index; Phenols: total phenolic content;

Acidity; K270; Peroxide: peroxide value). Oil I: control (oil without

enrichment); Oil II: olive oil plus olive-cake extract plus lecithin; Oil

III: olive oil plus olive-cake extract; Oil IV: olive oil plus lecithin.

Numbers from 1 to 7 correspond with increasing time of storage.

µmol

sTro

lox

equi

v/10

0 g

oil

0

500

1000

1500

2000

2500

3000

IVOilIIIOilIIOilIOil

µ

Figure 5. Changes in the antioxidant capacity between fresh

enriched oils and after the storage (256 days). Oil I: control (oil

without enrichment); Oil II: olive oil plus olive-cake extract plus

lecithin; Oil III: olive oil plus olive-cake extract; Oil IV: olive oil plus

lecithin. Values in ppm of compound in the oil.



character of lecithin, which allowed the hydroethanolic

particles in the oil to be stabilized. The enriched oils

presented higher oxidative stability values and higher

total phenolic contents than the control. Although no signifi-

cant effect was obtained from using lecithin to either

increase the antioxidant activity based on the ORAC

value or the oxidative stability by the Rancimat test of

the resulting oils, its use limited the increase in the bitterness

that occurred as a consequence of the phenolic enrichment.

This is one of the most important parameters for the

acceptance of these oils by the consumers and increases

the need to reconsider this emulsifier as an additive in the

development of oils with high concentrations of phenolic

compounds which would otherwise be rejected due to their

bitter taste.

This work was supported by the Spanish Ministry of Education

and Science financing the project AGL2009-13517-C03-02 and

the grant received by Manuel Suarez (BES-2006-14136).

We wish to thank the company Moli dels Torm S.L. (Els Torms,

Lleida, Catalonia, Spain) for the supply of the olive-cake samples

to obtain the phenolic extracts.

The authors have declared no conflict of interest.

3,4-DHPEA-EDA

0

100

200

300

400

500

600

Oil IVOil IIIOil IIOil I

p-HPEA-EDA

0

2

4

6

8

10

12

14

16


3,4-DHPEA-EA

0

20

40

60

80

100

120

140


p-HPEA-EA

0

5

10

15

20

25

30


0

1

2

3

4

5

6

7

8

9

10


Hydroxytyrosol

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5


Tyrosol

mg/

kg o

ilm

g/kg

oil

mg/

kg o

il

Figure 6. Changes in the amount of secoiridoid derivatives between fresh enriched oils and after storage (256 days). Oil I: control (oil without

enrichment); Oil II: olive oil plus olive-cake extract plus lecithin; Oil III: olive oil plus olive-cake extract; Oil IV: olive oil plus lecithin. Values in

ppm of compound in the oil.



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stability of a phenol-enriched olive oil during storage

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