EFFECT OF STORAGE PERIOD AND EXPOSURE CONDITIONS

ON THE QUALITY OF BOSANA EXTRA-VIRGIN OLIVE OIL

VINCENZO VACCA1, ALESSANDRA DEL CARO1,3, MARCO POIANA2 and

ANTONIO PIGA1
1
Dipartimento di Scienze Ambientali Agrarie e Biotecnologie Agro-Alimentari
Università degli Studi
Viale Italia 39, 07100 Sassari, Italy
2
Dipartimento di Biotecnologie per il Monitoraggio Agroalimentare ed Ambientale
Università degli Studi Mediterranea
Piazza San Francesco di Sales 4, 89061
Gallina, Reggio Calabria, Italy

Accepted for Publication December 1, 2005

ABSTRACT

Changes in quality parameters, antioxidant compounds, oxidative stabil-

ity and antioxidant activity during 18 months of storage of a monovarietal
extra-virgin olive oil from Bosana cultivar, and exposed to light and dark,
were studied. Analysis of data showed that all the parameters underwent
significant changes during storage: free acidity, peroxide and ultraviolet (UV)
spectrophotometric indexes remained below the limits reported in the EEC
Regulations 2568/91 and 1989/03, and these are: ⱕ0.8% for acidity,
ⱕ20 meq O2/kg for peroxide and ⱕ2.50 and ⱕ0.22 for K232 and K270,
respectively. Phenol and a-tocopherol content decreased during storage (42.0
and 29.6%, respectively) while chlorophylls and carotenoids underwent a
decrease until 8 months of storage (49% and 30%, respectively); after that, the
values remained constant. Oxidative stability and antioxidant activity had not
changed dramatically during 18 months. Phenols were significantly correlated
to the antioxidant activity of the oil, while oxidative stability measured by
Rancimat did not show any correlation with carotenoids, chlorophylls,
phenols and a-tocopherol. Regarding exposure conditions, storage in the dark
was better in retaining the quality of the oil, as expected.

3
Corresponding author. TEL: +39-079-229346; FAX: +39-079-229273; EMAIL: delcaro@uniss.it

Journal of Food Quality 29 (2006) 139–150. All Rights Reserved.

© 2006, The Author(s) 139
Journal compilation © 2006, Blackwell Publishing
140 V. VACCA ET AL.

INTRODUCTION

Olive oil plays a special role in the category of vegetable oils for many
important reasons. One is the balanced fatty acid composition reported by
many studies (Fedeli and Testolin 1991; Hill and Giacosa 1992; Martin-
Moreno et al. 1994; Roche et al. 2000), which rank this product as first among
dietary fats. Moreover, specific sensory characteristics and the presence of
other important components in the unsaponifiable fraction, which accounts for
the nutritive and health-giving properties, distinguish extra-virgin oil from
other edible vegetable oils (Khor et al. 1998; Visioli and Galli 1998; Owen
et al. 2000). These characteristics are mostly intrinsic to the oil, although some
result from the extraction systems used which do not involve refining pro-
cesses, and therefore leave the unsaponifiable fraction unchanged. On the
other hand, many other factors influence the quality of the oil, such as the
olive-ripening stage when picked, the olive variety, cultural and harvesting
practices, the health of the drupe, the interval between harvest and processing,
and pedoclimatic conditions (Montedoro and Servili 1992). As in other foods,
the quality of the oil decreases during storage. The reduction in shelf life is
attributable to lipid oxidation mechanisms which lead to rancidity. It is known
that these reactions are catalyzed by light and heat and are partly slowed down
by compounds belonging to the unsaponifiable fraction (phenolic compounds,
carotenoids, tocopherols) (Gutierrez Gonzales-Quijano et al. 1977; Lercker
and Capella 1997; Aparicio et al. 1999; Gutierrez et al. 2001; Servili and
Montedoro 2002; Del Carlo et al. 2004).
The antioxidant effects of extra-virgin olive oil seem to be a result
of the phenolic compounds, as the oleuropein, hydroxytyrosol, tyrosol,
4-hydroxyphenylacetic acid, caffeic acid, vanillic acid, syringic acid, etc.
(Kris-Etherton et al. 2002; Tuck and Hayball 2002), of which content depends
on the cultivar, climate and degree of ripeness of the fruit. Also, carotenoids
and a-tocopherol are important antioxidants because they protect oil from
auto- and photooxidation (Psomiadou and Tsimidou 2002a).
In the last years, the production of monovarietal extra-virgin olive
oils has increased because of their chemical and sensory characteristics
(Psomiadou et al. 2000; Aparicio and Luna 2002; Morello et al. 2004), and
Italy now has more than 30 protected designation of origin extra-virgin olive
oils. As in Sardinia most of the O. europea L. trees belong to “Bosana” cultivar
(used to produce extra-virgin olive oils) and as so far there are no studies about
storage of oils obtained from this cultivar, a work has been carried out to
determine the influence of storage period and different exposure conditions
(light and dark) on quality parameters, phenols, carotenoids, a-tocopherol,
chlorophylls, oxidative stability and antioxidant activity of this monovarietal
oil in order to assess its shelf life. Extra-virgin olive oil was analyzed until 18
 STORAGE AND EXPOSURE EFFECT ON BOSANA QUALITY 141

months because most producers consider 12–18 months as the maximum

storage time, starting from bottling to consumption.

MATERIALS AND METHODS

Sampling
A homogeneous sample of 10 q of olives of the cultivar Bosana harvested
in December 2002 with no defects and at an optimal stage of ripening (70%
just turned dark-colored and the rest green), was processed in an industrial oil
mill using a three-phase centrifugation system, after defoliation and washing
of the drupes. The olives were crushed with a fixed-hammer metal crusher
(model MO 30, OMT, Grassina, Florence, Italy) and the olive paste was
kneaded for 35 min at 30C. The paste was then centrifuged (model GAMMA
1, OMT) at a flow rate of 600 kg/h, using 300 L/h of water. The oil phase was
further clarified in an automated discharge vertical centrifuge (model Elma
2500, OMT).
The oil obtained was stored in hermetically sealed 60-mL colorless trans-
parent glass bottles. Bottles were filled up leaving a headspace of 3 mL and
exposed to either direct sunlight or in the dark at room temperature 22 ± 3C for
18 months. Analyses were carried out as soon as the olive oil arrived at the lab
and after 2, 4, 6, 8, 12 and 18 months of storage.

Free Acidity
This was determined by the methods reported in EEC Regulation 2568/91
of the European Union Commission (EUC). Data obtained were expressed as
gram of oleic acid per 100 g of oil (% oleic acid).

Peroxide Index
We used the method described in EEC Regulation 2568/91 of the EUC.
Data obtained were expressed as milliequivalent of O2 per 1000 g of oil
(meq O2/kg oil).

Ultraviolet Light Absorption K232 and K270

These indexes were determined by the methods described in EEC Regu-
lation 2568/91 of the EUC. K232 and K270 represent the extinction coefficients
of diene (K232) and triene (K270) conjugates of polyunsatured fatty acids, and
they are also indicative of oil oxidation, especially the K270 index, responsible
for the presence of primary and secondary oxidation products, more than K232.
142 V. VACCA ET AL.

Samples were analyzed in a 10-mm cuvette, using a HP 8453 spectrophotometer

(Hewlett–Packard, Palo Alto, CA).

Total Phenol Content

Phenolic compounds were extracted following the procedure of
Montedoro et al. (1992). The phenol content was determined spectrophoto-
metrically (Spectrophotometer model 8453, Hewlett–Packard) at 760 nm, and
the concentration was expressed as milligram of gallic acid per kilogram of oil
(mg gallic acid/kg oil).

Carotenoids and Chlorophylls

These were determined using a method proposed by Mincione et al.
(1996). About 2.5 g of oil was added to 25 mL of isooctane for spectropho-
tometry. The spectrophotometric reading was made at 436 and 670 nm for
carotenoids and chlorophylls, respectively. The specific absorptions were com-
pared to the curves obtained measuring the specific absorptions of solutions at
known concentrations of b-carotene and chlorophylls soluble in oil measured
in isooctane. Data were expressed as milligram of b-carotene per kilogram of
oil (mg/kg), and milligram of chlorophylls per kilogram of oil (mg/kg).

Antioxidant Activity
Antioxidant activity was measured in the phenol extract, by the method
described by Brand-Williams et al. (1995), using a decoloration curve of the
stable radical 2,2 diphenyl-1-picrylhydrazyl (DPPH·). Fifty microliters of the
sample were made to react with 3 mL of a 6 ¥ 10-5 M solution of DPPH·for 1 h
at 515 nm and 25C, in order to obtain a decrease in absorbance by the radical
DPPH·. As the graph of absorbance versus time showed that the decrease
followed a fourth-order kinetic (r 2 = 0.99), it was possible to express the
antioxidant activity as -OD-3/min mg 103, i.e., with the following equation:

1 A3 − 1 A30 = −3kt

where A0 is the initial absorbance and A is the absorbance at rising time t

(Manzocco et al. 1998).

a-Tocopherol
This was extracted by the method reported by Psomiadou et al. (2000),
using a Waters 625 high-performance liquid chromatography equipped with a
spectrofluorometric detector FL3000 (Thermo Separation, Muskegon, MI) at
 STORAGE AND EXPOSURE EFFECT ON BOSANA QUALITY 143

294 and 330 nm for excitation and emission, respectively, a LiChrospher

column 60Si with a 250 ¥ 4 mm i.d. (Agilent Technologies, Palo Alto, CA), a
mobile phase of n-hexane-isopropyl alcohol (99:1, v/v) and a flow rate of
1.2 mL/min. Results were expressed as milligram of a-tocopherol per kilo-
gram of oil.

Oxidative Stability
This was measured with a Rancimat apparatus (Metrohm Co., Basilea,
Switzerland) using an oil sample of 3 g at 120C and 20 L/h airflow. Stability
was expressed as oxidation induction time (h) (Laübli and Bruttel 1986).

Statistical Analysis
All the analyses were carried out using two containers for each sample,
making two determinations for each container, for a total of four analyses.
Data were evaluated by a two-factor completely randomized factorial design
using MSTAT-C software (Russell D. Freed, MSTAT-C Director, Crops and
Soils Sciences Department, Michigan State University; Version 2.0). Storage
period and exposure conditions (light or dark) were chosen as variables.
Means, where appropriate, were separated by Duncan’s multiple range test for
P ⱕ 0.05 and P ⱕ 0.01.

RESULTS AND DISCUSSION

Results of factorial analysis of variance (ANOVA) on the effects of the

variables storage and exposure on the principal quality parameters are reported
in Table 1.
With regard to free acidity, it was observed that this significantly
increased in the twelfth month of storage, but remained below the limits
reported in EEC regulation 1989/2003, which prescribes a value inferior to 0.8
for a virgin olive oil. Besides, a significant difference was found between
samples stored in the light and those kept in the dark; in fact, free acidity was
higher in the former because light negatively affects olive oil quality (Vekiari
et al. 2002).
During storage, the peroxide index underwent an initial increase in the
fourth month, and then it slowly decreased in the eighteenth month (Vekiari
et al. 2002). This may be because of the fact that the newly formed oxidation
products (we left a bottle headspace) were further converted to secondary
products (Vekiari et al. 2002). This phenomenon was significantly more pro-
nounced in the samples kept in the light, compared to those stored in the dark.
A lesser formation of secondary products in samples stored in the dark may
explain this higher value.
144 V. VACCA ET AL.

TABLE 1.
INFLUENCE OF STORAGE PERIOD AND EXPOSURE ON MAIN QUALITY PARAMETERS
OF BOSANA EXTRA-VIRGIN OLIVE OIL

Source of variation Free acidity Peroxide value UV spectrophotometric

(% oleic acid) (meq O2/kg oil) indexes

K232 K270

Storage period (month)

0 0.33cd 6.56c 1.785b 0.124e
2 0.25e 8.23° 1.884° 0.144d
4 0.38b 8.07a 1.874a 0.161c
6 0.32d 7.25b 1.884a 0.172b
8 0.33cd 6.64bc 1.893a 0.172b
12 0.37b 6.08cd 1.887a 0.184a
18 0.40a 5.91d 1.889° 0.173b
Significance ** ** ** **
Exposure
Light 0.36a 6.72b 1.878a 0.183a
Dark 0.32b 7.20a 1.864a 0.140b
Significance ** ** ns **

Data followed by different letters for each source of variation and column are significantly different by
Duncan’s multiple range test.
** Significant at P ⬍ 0.01.
UV, ultraviolet; ns, not significant.

Ultraviolet (UV)-spectrophotometric indexes increased significantly

during storage with a different pattern and depending on the index, but these
did not exceed limits reported in EEC regulation 1989/2003 (ⱕ2.50 and ⱕ0.22
for K232 and K270, respectively). It is noteworthy to see that the K232 index
showed a significant increase only after 2 months of storage, while later on,
these values did not change. On the other hand, the K270 showed a constant
significant growth except at the last inspection time. The K270 values were
affected by the exposure conditions, with higher values reported in the samples
stored in the light than in those kept in the dark, probably because of the fact
that the presence of chlorophylls in the oil acts as an antioxidant in the dark
(Kiritsakis and Dugan 1984). It is well known that light affects olive oil quality
(Lercker and Capella 1997; Vekiari et al. 2002), making possible an increase
in the triene formation, measured by K270, more than in the diene.
Table 2 reports the effects of storage and exposure on chlorophylls, caro-
tenoids, total phenols, a-tocopherol content, oxidative stability and antioxi-
dant activity.
Chlorophylls decreased significantly in 8 months of storage as widely
reported in the literature (Morello et al. 2004) with a loss of nearly 50% of the
 TABLE 2.
INFLUENCE OF STORAGE PERIOD AND EXPOSURE ON CHLOROPHYLLS, CAROTENOIDS, TOTAL PHENOLS, a-TOCOPHEROL,
OXIDATIVE STABILITY AND ANTIOXIDANT ACTIVITY OF BOSANA EXTRA-VIRGIN OLIVE OIL

Source of variation Chloropyhlls Carotenoids Phenols (mg a-tocopherol Oxidative Antioxidant activity
(mg/kg) (mg/kg) gallic acid/kg oil) (mg/kg) stability (h) (-OD-3/min mg 103)

Storage period (months)

0 17.74a 9.31a 182.59a 252.50a 5.72a 0.121ab
2 16.83a 7.07bc 177.04a 231.00b 5.21bcd 0.124a
4 14.77b 7.55b 161.75b 217.00c 5.32abcd 0.116bcd
6 11.82c 7.01c 136.48c 217.00c 5.05d 0.114cd
8 9.09d 6.54cd 130.13c 215.25c 5.15cd 0.116bc
12 8.93d 6.28d 114.10d 201.50d 5.61ab 0.095e
18 9.32d 6.32d 105.48e 177.75e 5.52abc 0.111d
Significance ** ** ** ** * *
Exposure
Light 9.56b 6.77b 140.06b 201.43b 5.06b 0.119a
Dark 15.73a 7.54a 147.82a 230.57a 5.68a 0.109b
Significance ** ** ** ** ** *

Data followed by different letters for each source of variation and column are significantly different by Duncan’s multiple range test.
* Significant at P ⬍ 0.05.
STORAGE AND EXPOSURE EFFECT ON BOSANA QUALITY

** Significant at P ⬍ 0.01.
145
146 V. VACCA ET AL.

initial value. As expected, exposure to light resulted in a higher decrease in the

initial values, than those that occurred in samples stored in the dark. In fact, as
reported in literature, chlorophylls are known to act as photosensitizers during
light exposure (Psomiadou and Tsimidou 2002b).
Carotenoids showed a trend very similar to that of chlorophylls with a
decrease until 8 months of storage, but the loss was 30% of the initial value
(Morello et al. 2004). It was noted as a protective effect of the dark against
depletion. In literature, carotenoids are reported to protect oil from photo-
oxidation (Aparicio et al. 1999), but they are also particularly vulnerable in the
presence of oxygen (Psomiadou and Tsimidou 2002a). This explains their
decrease during storage.
Phenolic compounds constantly decreased during storage; samples stored
in the dark revealed a significantly higher value than those stored in the light
(Table 2). The literature reports that the reduction of the total phenol content
during oil storage is a result of oxidation and hydrolytic activity (Psomiadou
and Tsimidou 2002a). Also in this case, light affected total phenol content,
which is significantly lower in samples stored in the light than those in the
dark.
The a-tocopherol content gradually decreased throughout storage with
losses of about 30%, confirming data reported in literature (Manzi et al. 1998;
Psomiadou and Tsimidou 2002a), apart from what was observed by Morello
et al. (2004), which reported a total disappearance of a-tocopherol content
throughout storage. Samples kept in the dark showed significantly higher
values than samples stored in the light (Table 2). Many studies report the very
important role of a-tocopherol as an antioxidant in the oxidative stability of
virgin olive oils (Baldioli et al. 1996; Servili et al. 1996; Morello et al. 2004),
so its decrease is because of the fact that a-tocopherol protects oil against
oxidation (Psomiadou and Tsimidou 2002b).
Oxidative stability of the samples analyzed showed a decrease up to the
sixth month. After that, the values did not change significantly from those at
the beginning of storage. Samples kept in the dark showed a significantly
longer induction time than those stored in the light. The a-tocopherol content
was not significantly correlated to oxidative stability (Table 3), confirming
data reported in literature (Baldioli et al. 1996). With regard to phenol content
and contrary to what were reported in several works (Baldioli et al. 1996;
Aparicio et al. 1999; Del Carlo et al. 2004), there was no observed positive
correlation between oxidative stability and total phenol content (in our study,
only for samples stored in the light with a value of 0.85, P ⬍ 0.05, data not
shown). Our results confirm what was reported in an Italian work (Pizzale
et al. 1999) where, in evaluating the oxidative stability of foods lipids by
Rancimat, the authors did not find correlations with total phenol content. It is
important to remember that oxidative stability of extra-virgin olive oil is
 STORAGE AND EXPOSURE EFFECT ON BOSANA QUALITY 147

TABLE 3.
CORRELATION COEFFICIENTS AMONG OXIDATIVE STABILITY AND ANTIOXIDANT
ACTIVITY AND THE PARAMETERS CAROTENOIDS, PHENOLS AND a-TOCOPHEROL OF
BOSANA EXTRA-VIRGIN OLIVE OIL

Carotenoids Phenols a-tocopherol Oxidative Antioxidant

stability activity

Oil stability 0.37 0.00 0.03 1.00

Antioxidant activity 0.56 0.76* 0.61 -0.33 1.00

* Significant at P ⬍ 0.05.

mainly linked to its fatty acid composition (especially the ratio of oleic to
linoleic acid) but are also important minor components as tocopherols, caro-
tenoids, etc. Therefore, induction period is the result of the fatty acid compo-
sition and the simultaneous activity of various prooxidant and antioxidant
factors endogenous in the oils (Psomiadou and Tsimidou 2002a). Some
researchers state that oxidative stability measured by Rancimat is not a stan-
dard quality parameter, but it is a useful tool to gain information about the shelf
life of virgin olive oils (Aparicio et al. 1999); others say that it is useful only
to evaluate differences in oils obtained with different technological processes
(Lercker et al. 1991). So we can suppose that total phenol content itself cannot
explain the variations observed in our data, especially for the last two sam-
plings. Therefore, this point needs to be studied more deeply to explain this
phenomenon.
As regards antioxidant activity, a significant decrease was observed
during storage. Samples stored in the light retained higher antioxidant activity
than those kept in the dark. Correlations between antioxidant activity and
parameters reported in Table 2 showed significance only for total phenols
(Table 3) (Del Carlo et al. 2004). Antioxidant activity, determined by the
method previously described, was not correlated to oxidative oil stability
(Pizzale et al. 1999). We can suppose that the lack of correlation in the samples
during storage is because of the fact that polyphenols undergo hydrolysis
reactions during aging, as reported in other works (Del Carlo et al. 2004).

CONCLUSIONS

In conclusion, the results of our study revealed that the extra-virgin olive
oil of Bosana olives is a shelf-stable oil. In fact, after 18 months of storage, the
oil can be still considered extra-virgin, thus conforming to the shelf life stated
in the label. This in part could be attributed to the high original content of
148 V. VACCA ET AL.

antioxidant compounds, such as polyphenols, a-tocopherol and carotenoids,

which are able to slow down the lipid oxidation in the oil. As expected, the
results were better when oil samples were stored in the dark, thus confirming
what was reported in the literature. In consideration of these results, the aim of
future research could be to study the different behaviors of single olive oil
polyphenols during storage to better evaluate their antioxidant activity and
their participation to oxidative stability of extra-virgin olive oils.

ACKNOWLEDGMENT

This research was supported by the Università degli Studi di Sassari

Project 60%-2003 “Influenza della denocciolatura e delle condizioni di con-
servazione sulla shelf-life di oli extravergini di oliva monovarietali.”

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