Sensors and Actuators B 76 (2001) 158165

Hydroperoxide determination by a catalase OPEE: application to

the study of extra virgin olive oil rancidication process
L. Campanella*, M.P. Sammartino, M. Tomassetti, S. Zannella
Department of Chemistry, ``La Sapienza'' University of Rome, p.le A. Moro, 5-00185 Rome, Italy

Abstract

A new application to authentic matrices of a catalase organic phase enzyme electrode (OPEE) recently developed by the present authors
is described in this communication. This biosensor was obtained by coupling an amperometric gas diffusion electrode for the oxygen, made
of teon, and the catalase enzyme immobilised in kappa-carrageenan gel.
The response of the catalase biosensor to cumene hydroperoxide and to terbutylhydroperoxide, working directly in n-decane or toluene,
was studied. The hydroperoxide content of the extra virgin olive oil was monitored during an articial rancidication process. One
interesting result obtained is the qualitative correlation observed between the above trend of the hydroperoxide content pool and that of the
peroxide number; moreover, a qualitative inverse correlation trend was found between both the last two indicators and the content of
polyphenols in the olive oil which are known to be the most important natural antioxidant agents contained in olive oil. # 2001 Elsevier
Science B.V. All rights reserved.

Keywords: Hydroperoxides; Determination; Catalase OPEE; Olive oil rancidication

1. Introduction on the catalase enzyme and able to check hydrogen peroxide

Olive oil is certainly one of the most typical and important
food matrixes in several European countries, and is also of
great interest in food and commodity chemistry. We thus
spent the last year's research in developing innovative
techniques for the analysis of the minor components of
extra virgin olive oil such as polyphenols, which however
are very important as they affect the chemical and organo-
leptic properties of the oil. In view of the very low solubility
of olive oil in water and its high solubility in organic non
polar solvents, we recently developed a new OPEE capable
of operating in the latter type of solvent [1]; moreover, this
type of approach was recently followed also by other work-
ers [2], and not only for olive oil but also in the analysis of
other hydrophobic substances or substances that are com-
paratively insoluble in water [36]. The new biosensor
allows us to check two new indicators of the oil's character-
istics: authenticity [7,8] and condition of preservation [9];
both indicators can be checked using a tyrosinase biosensor
dipped directly in n-hexane solutions [1,79].
As our results were greeted with noticeable interest, we
expanded this research line, developing a new OPEE based
*
Corresponding author. Tel.: 39-06-49913744;
fax: 39-06-49913725.
and working in several organic solvents (chloroform, chlor-
obenzene, toluene, ethyl acetate) [10]. In the present
research in this eld we investigated the possibility of using
the new OPEE to determine more complex hydroperoxides
such as terbutylhydroperoxide and cumene hydroperoxide in
non-polar organic solvents like toluene and n-decane. Lastly,
we tested if it was possible to use the catalase OPEE to
monitor the trend of the pool of the hydroperoxides content
during an articial rancidication process of an extra virgin
olive oil.
2. Experimental
2.1. Apparatus
The biosensor was constructed using an electrode for
oxygen determination supplied by Universal Sensor Inc.
(US) New Orleans (USA), of the gaseous diffusion ampero-
metric type, i.e. consisting of a platinum cathode and an
Ag/AgCl/Cl anode and equipped with a gas-permeable
membrane. The original plastic cap of the electrode cannot
be used in organic solvents as it is rapidly attacked by them.
The original cap was therefore replaced with a specially
made teon cap of the same size. Also the original rubber

0925-4005/01/$ see front matter # 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 5 - 4 0 0 5 ( 0 1 ) 0 0 6 1 5 - 3
 L. Campanella et al. / Sensors and Actuators B 76 (2001) 158165
O-ring of the cap, used to x the gas-permeable membrane,
was replaced by a suitable teon ring [1,11]. Measurement
was performed using a Metrohm G41 VA potentiostatic
detector connected to a model 868 Amel recorder and a
Mitek-MK 5001 digital multimeter. The gas-permeable
membranes were supplied by Radelkis (Budapest). D-
9777 type dialysis membranes were supplied by Sigma-
Aldrich (Milan).
The measurements were performed in a 15 cm3 capacity
glass cell, maintained at a constant temperature of 208C
using a model VC 20B Julabo thermostat (Germany).
The measuring cell was stirred using a magnetic micro-
stirrer, equipped with a magnetic anchor supplied by Velp
Scientica (Italy).
2.2. Reagents
The bovine liver catalase (21,000 U mg 1 of solid) was
supplied by Sigma (Milan), potassium monobasic phos-
phate, potassium dibasic phosphate and potassium chloride,
supplied by Carlo Erba (Milan).
Kappa-carrageenan and cumene hydroperoxide, 80% (p/
p) in cumene, from Fluka (Switzerland).
Terbutylhydroperoxide, 5.5 mol l 1 in n-decane, from
Sigma (Milan).
Terbutylhydroperoxide, 3.0 mol l 1 in toluene, from
Fluka.
2.3. Solvents
The toluene for RPE analysis was supplied by Carlo Erba
(Milan); the n-decane for gas chromatography by Fluka
(Switzerland).
The water content of the organic solvents used, expressed
as a percentage by weight, was determined using a model
DL18 Karl Fischer automatic titrator supplied by Mettler
(Switzerland); it was found to be 0.002% by weight for
toluene and 0.007% by weight for n-decane.
The solutions of cumene hydroperoxide in toluene, pre-
pared fresh each day, were obtained from the standard
solution supplied by Fluka. An aliquot was accurately
weighed out so as to obtain nal concentrations of
0.90 mol l 1.
Likewise, the solutions of terbutylhydroperoxide in n-
decane or in toluene were obtained by diluting with decane
or toluene starting from the standard solution supplied by
Sigma or Fluka in such a way as to obtain concentrations of a
80.0 mmol l 1 in n-decane or in toluene.
2.4. Enzyme
The enzyme catalase (hydrogen-peroxide oxidoreduc-
tase) catalyzes the reaction in which two molecules of
hydrogen peroxide are converted to oxygen and water:
H2 O2 H2 O2 ! O2 2H2 O
159
The enzyme (for example, from beef liver) has a molecular
weight of about 250,000, a molecular activity of 9  104 s 1
(at pH 7, at 208C and H2O2 10 2 mol l 1). The Michaelis
constant for hydrogen peroxide is 1.1 mol l 1 (at pH 7 and
308C).
The enzyme can utilise also alkylhydrogen peroxide in the
place of hydrogen peroxide as substrate and several organic
substances can replace the second hydrogen peroxide mole-
cule as hydrogen donor (ethanol, formiate, thiol and pyr-
ogallol).
With the reference to the simplied scheme for the
reaction mechanism [12], rate constant values of k1 6
106 mol l 1 s 1 and k4 1:5  107 mol l 1 s 1 (at pH 7,
and 20258C) were found [12].
3. Methods
3.1. Enzyme immobilisation
The enzyme was immobilized by entrapment in kappa-
carrageenan gel [13]: starting from a 2% by weight aqueous
solution of polysaccharide stratied in a Petri dish, a gela-
tinous disk was obtained from which several 5 mm diameter
diskettes were cut. The diskettes thus obtained were then
dehydrated for 5 days at a low temperature (48C) in order to
prevent bacterial pollution. A dried diskette was placed in a
cylindrical container of suitable diameter to which was
added 25 ml of enzymatic solution obtained by dissolving
about 10 mg of catalase in 25 ml phosphate buffer,
0.06 mol l 1 at pH 7. The container was then sealed and
stored for 3 days at a temperature of 48C, during which the
enzymatic solution was completely absorbed by the gel and
the latter dried completely. The diskette thus obtained was
then used for biosensor assembly.
3.2. Biosensor assembly
The amperometric electrode was placed in a solvent-
resistant teon cap lled with an electrolytic solution con-
sisting of phosphate buffer (1/15 mol l 1, pH 6.6) and
potassium chloride (0.1 mol l 1). The bottom of the cap
was closed by the O2 selective gas-permeable membrane; a
kappa-carrageenan gel diskette on which the enzyme was
immobilized was sandwiched between the latter and a
dialysis membrane, both of which were secured by teon
rings.
The complete biosensor diagram is shown in Fig. 1.
3.3. Measurements with the catalase biosensor
The enzyme used to construct the biosensor for hydro-
peroxide analysis is catalase, which catalyzes the following
reaction:
catalase
Hydroperoxide ! Corresponding alcohol 12 O2
160 L. Campanella et al. / Sensors and Actuators B 76 (2001) 158165
Fig. 1. Gaseous diffusion amperometric electrode and biosensor assembly:
(a) electrode body; (b) internal solution (phosphate buffer 1/15 mol l 1 pH
6.6 and KCl 0.1 mol l 1); (c) reference electrode (Ag/AgCl) (anode); (d)
platinum electrode (cathode); (e) teflon cap; (f) gas-permeable membrane;
(g) teflon O-ring; (h) dialysis membrane; (i) catalase immobilized in
kappa-carrageenan.
The electrode, assembled as described in the preceding
section, is immersed in the cell containing 10 ml of organic
solvent under stirring until signal stabilization occurs, which
corresponds to the stationary state set up when the dissolved
O2 is reduced at the same rate at which atmospheric oxygen
is dissolved in the solution. Several ml (251000 ml) of the
substrate solution are then added and the resulting signal
variation recorded until a new stationary state is reached,
which is characterized by an increase of the reduction
current of the O2, produced during the enzymatic reaction.
By plotting the difference in signal between the first sta-
tionary state and the one following the addition against the
hydroperoxide concentration in the measuring cell it is
possible to construct a calibration curve that may be used
to determine the concentration of the analyte tested in the
unknown samples. Generally, the time required for biosen-
sor equilibration is about 510 min and for one measurement
about 15 min.
3.4. Determination of hydroperoxide content during the
process of artificial rancidification of extra virgin olive oil
The oil samples were articially oxidized using the active
oxygen method (AOM) [14] developed by the American Oil
Chemist Society (AOCS).
The aim of the AOM method is to determine the stability
of a fat or oil versus rancidication by ascertaining the
length of time required to reach a peroxide number equal to
100 mEq of O2 per kilogram of oil, under given experi-
mental conditions. The aim of our research was instead to
monitor the rancidication process by determining the time
trend of hydroperoxide concentration and correlating it with
the concentration trend of the peroxides which, together
with the hydroperoxides, are produced by oxidation of fatty
acids. This is similar to the procedure adopted in previous
research carried out in our laboratory [9] aimed at relating
peroxide formation to the variation of the content of poly-
phenols, which are the most important natural substances
present in extra virgin olive oil and which have strong
antioxidant properties.
The specications of the method, performed using the
simple apparatus illustrated in a previous paper [9], require
the use of pyrex glass tubes 25 mm in diameter and 200 mm
high; each tube is tted with a neoprene or silicone plug in
which two 4 mm diameter holes are bored to allow aerating
capillaries, also made of pyrex glass, to be inserted; 25 ml
of oil are transferred to the tubes, taking care to avoid any
contact between the sample and the end of the tube where
the air ow exit is located. The sealed tube containing the
oil sample is heated over a water bath at the temperature of
boiling water for a period of 5 min. The tube is then placed
in a silicone oil bath and maintained at a constant tem-
perature of 988C. The bath is thermoregulated using a
calibrated Vertex immersed in the bath itself. The air ow
in the glass capillaries, which is essential for effective
oxidation, is regulated by means of a owmeter set at
140 cm3 min 1; a constant ow is vital for reproducibility
of results.
During the articial randicication process, oil samples
were taken at xed time intervals and their hydroperoxide
content determined immediately using the catalase biosen-
sor, together with the number of peroxides, as outlined in the
following section.
3.5. Determination of peroxide number in extra virgin olive
oil
This well-known method consists essentially in thiosul-
phate titration to determine the iodine released from KI
solution as the result of oxidation due to the peroxides
contained in the sample [15]. The peroxide number,
expressed as mEq active O2 per kilogram of oil, is calculated
as follows:
 
T
Peroxide number V   1000
M
where V is the volume in milliliter of the sodium thiosulphate
solution used for the titration; T the normality of the sodium
thiosulphate solution used; M the weight in grams of the
sample to be analysed.
4. Results
The main aim of the present research was to investigate
the possible use of the catalase OPEE to monitor the ``pool''
of hydroperoxides produced during the process of articial
rancidication of extra virgin olive oil.
In previous works [10,16], an analysis had already been
made of the response of the catalase biosensor versus
hydrogen peroxide in different organic solvents (chloro-
form, chlorobenzene, ethyl acetate, toluene).
 L. Campanella et al. / Sensors and Actuators B 76 (2001) 158165 161

Table 1
Repeatability and main analytical data referring to the calibration curves of
cumene hydroperoxide over the linear range, obtained during the first day
of life of the catalase biosensora,b

Run Slope Intercept Linear range Correlation

1
no. (a.u. mmol l) (a.u.) (mmol l 1) coefficient

1 0.80 8.2 9.250.4 0.9551

2 0.72 4.2 9.250.4 0.9976
3 1.00 8.4 9.250.4 0.9960
a
Experimental conditions: enzyme immobilized in kappa-carrageenan
gel, solvent toluene.
b
Equation of the mean straight line Y (0.80  0.05)X (7  2)
r2 0.9934.

in n-decane and using as reference solutions standard solu-

Fig. 2. Chemical formulae of terbutylhydroperoxide, cumenehydroper-
oxide and benzoylperoxide.
tions of terbutylhydroperoxide in n-decane, since prelimin-
ary experiments aimed at characterizing biosensor response
had shown that these operating conditions ensure excellent
performance in terms of sensitivity, linearity range and
biosensor `lifetime'; furthermore, olive oil is highly soluble
in n-decane.
Table 1 lists all the more signicant analytical data as well
as the reproducibility of repeated calibration curves versus
cumene hydroperoxide obtained using the catalase OPEE
with toluene as solvent, while Fig. 3 contains histograms
showing the trends in calibration curve slope and linear
range throughout the biosensor's `lifetime'.

In the present work, the main focus is on the determina-

tion of the `lifetime' and trend of the catalase biosensor
response to hydroperoxides having a more complex mole-
cular structure, such as terbutylhydroperoxide and cumene
hydroperoxide (see Fig. 2), operating in non polar and thus
highly hydrophobic solvents, such as n-decane and toluene,
in which these peroxides are highly soluble and in which
also olive oil is highly soluble. These two organic hydro-
peroxides were selected as they are readily available on the
market and also because, in view of their greater molecular
complexity, they were expected to be more representative
versus hydroperoxides formed during the process of fat
rancidication than for example hydrogen peroxide.
The biosensor was then used to check the increase in
hydroperoxide content that occurs during the process of
rancidication of extra virgin olive oil.
The preliminary part of the research was thus devoted to
investigating the time trend of the catalase biosensor
response versus cumene hydroperoxide, operating in
toluene. The behaviour of the same biosensor was then
studied in the determination of terbutylhydroperoxide, using
n-decane as solvent, but without however neglecting to
check the possibility of operating also in toluene. Further-
Fig. 3. Trend: (a) of slope; (b) linear range of calibration curves of cumene
more, the determination at xed time intervals of the hydro- hydroperoxide obtained daily throughout the lifetime of the catalase
peroxide content of extra virgin olive oil subjected to a biosensor, with the enzyme immobilized in kappa-carrageenan gel, solvent
process of articial rancidication was performed, operating toluene.
162 L. Campanella et al. / Sensors and Actuators B 76 (2001) 158165

Table 2
Repeatability and main analytical data referring to the calibration curves of
terbutylhydroperoxide over the linear range, obtained during the second
day of life of the catalase biosensora,b,c

Run Slope Intercept Linear range Correlation

1
no. (a.u. mmol l) (a.u.) (mmol l 1) coefficient

1 6.5 4.7 0.111.2 0.9939

2 7.9 6.2 0.111.2 0.9993
3 9.5 5.0 0.111.2 0.9960
a
Experimental conditions: enzyme immobilized in kappa-carrageenan
gel, solvent n-decane.
b
Equation of the mean straight line Y (8  1)X (5.0  0.8)
r2 0.9996.
c
Comparison of mean analytical data working in toluene. Equation of
the mean straight line Y (17.1  0.7)X (4.0  0.1) linear range
1.06.0 mmol l 1 r2 0.9998.

The catalase biosensor's response to terbutylhydroperox-

ide was investigated operating in both n-decane and toluene.
As toluene was the operating solution, despite the good
response sensitivity found, a rather narrow linear range was
observed, as well as a diminished reproducibility and above
all a rather short `lifetime' (only 23 days). The preferred
solvent in this case was thus n-decane. For this reason daily
calibration curves versus terbutylhydroperoxide were con-
structed, operating in n-decane. Table 2 shows the more
signicant analytical data and the repeatability of the cali-
bration curves obtained using the catalase OPEE versus
terbutylhydroperoxide, operating in n-decane, while Fig. 4
contains histograms showing the trends referring to sensi-
tivity (calibration curve slope) and linear range of the
response throughout the biosensor's `lifetime'. Lastly, the
main analytical data obtained using the same hydroperoxide
but operating in toluene are also shown in Table 2. The
equations of the calibration curves are shown in Tables 1 and
2, and were obtained by performing successive calibrations
in the course of the same day. Between calibrations, the
biosensor was simply thoroughly rinsed in the same organic
solvent in which the measures were performed. The histo-
grams shown in Figs. 3 and 4 refer instead to calibrations
performed on successive days. In these tests, as in the
preceding ones, at the end of each day's measures, the
biosensor was thoroughly rinsed, rst in the same organic
solvent in which the measures were performed, and then in
phosphate buffer. The teon cap on which the enzymatic
membrane was mounted was kept at 48C in a humid
atmosphere until the following day. Both in the tests
repeated daily, and in those repeated on successive days,
the enzymatic membrane used was in any case always the
same.
Of course, in repeated tests over a period of several
months, a large number of membranes were prepared and
the biosensor was re-assembled in the same way many times.
The equations of the calibration curves carried out using
different membranes, all prepared in the same way as
described above, gave a repeatability that was usually
Fig. 4. Trend: (a) of slope; (b) of linear range of calibration curves of
terbutylhydroperoxide obtained daily throughout the lifetime of the
catalase biosensor; enzyme immobilized in kappa-carrageenan gel; solvent
n-decane.
comparable with that shown, respectively, in Tables 1 and
2, i.e. with that of the calibrations obtained using the same
membrane, for a given substrate and a given solvent.
Table 3 shows a comparison of the response time, the
lifetime and the wide of the linear range, on the day during
the biosensor's `lifetime' on which the highest is the
response sensitivity, obtained using standard solutions of
terbutylhydroperoxide or cumene hydroperoxide or hydro-
gen peroxide, respectively, with the catalase biosensor,
when operating in toluene or in other organic solvents. As
can be seen, both the solvent and the complexity of the
substrate have a considerable inuence on biosensor lin-
earity; on the other hand, also biosensor sensitivity is
inuenced (as shown by a comparison between the two
angular coefcients in Tables 1 and 2). Precisely for this
reason, as already mentioned, it was preferred, in measures
performed on real samples, to refer to biosensor response
to terbutylhydroperoxide, which is certainly more reper-
esentative than hydrogen peroxide, versus more complex
hydroperoxides formed during the rancidication process
of the olive oil.
Lastly, the catalase biosensor was used to determine the
hydroperoxide content formed during the process of arti-
cial rancidication of the extra virgin olive oil. For this
purpose, the experimental apparatus for the articial oxida-
tion of oil described in the preceding section was used.
The hydroperoxide content of extra virgin oil, determined
at set time intervals throughout the entire process, was
expressed, with the reference to the amperometric signal,
 L. Campanella et al. / Sensors and Actuators B 76 (2001) 158165 163

Table 3
Main analytical data obtained on the day of maximum response sensitivity in the determination of cumene hydroperoxide or terbutylhydroperoxide, or
hydrogen peroxide, operating in toluene, or n-decane, or chloroform

Analysed compound Solvent Response time (min) Lifetime (days) Linear range (mmol l 1)

Cumene hydroperoxidea Toluene 5 10 4.8143.0

t-Butylhydroperoxidea Toluene 7 1.06.0
t-Butylhydroperoxideb n-Decane 5 16 0.212.5
Hydrogen peroxidec Toluene 4 0.030.2
Hydrogen peroxidec Chloroform 3 15 0.030.2
a
II day.
b
III day.
c
I day.

Table 4
Variation in hydroperoxide content over time during the process of
rancidification of extra virgin olive oil, as determined by the catalase
biosensora

Time (h) Hydroperoxide content

(mEq/kg oil) RSD%  10 (n  5)

0 0
6 0
16 1.0
22 2.3
28 2.8
33 5.7
40 6.0
45 17
50 50
a
Experimental conditions: enzyme immobilized in kappa-carrageenan
gel, solvent n-decane. Hydroperoxides expressed as mEq of terbutylhy-
droperoxide/kg of oil.

detected in solutions of known titre of terbutylhydroper-

oxide in n-decane, as mEq of terbutylhydroperoxide per
kilogram of oil.
Table 4 shows the experimental values obtained using the
catalase biosensor referring to the determination of hydro-
peroxide content in the oil during the process of articial
rancidication of extra virgin olive oil.
Fig. 5 illustrates the trend in hydroperoxide content
observed during the process of rancidication of the oil,
comparing it with the trends in peroxide number and poly-
phenol content obtained (using the same experimental ran-
cidication apparatus) and using respectively a tyrosinase
OPEE developed in previous research [13] for the determi-
nation of polyphenol content [9], while for peroxide number
determination, conventional iodometric titration method
[15] was used, consisting in using thiosulphate to titrate
the iodine released through the oxidation of a solution of
potassium iodide by the peroxides formed in the oil due to
rancidication. During the rancidication process an
increase occurs in both hydroperoxide and peroxide con-
centration as primary oxidation products, accompanied by a Fig. 5. Qualitative direct or inverse correlation of the trend of
hydroperoxide content, as determined by catalase OPEE, of peroxide
decrease in polyphenols content, i.e. the main substances number, as determined by titration, and of polyphenols content, as
possessing antioxidant properties naturally present in extra determined by tyrosinase OPEE, during the process of artificial
virgin olive oil. rancidification of extra virgin olive oil.
164 L. Campanella et al. / Sensors and Actuators B 76 (2001) 158165
The time taken to achieve maximum development of
hydroperoxides and peroxides and the simultaneous drastic
reduction in polyphenols is about 50 h under the process
conditions described.
5. Discussion
The study of the catalase biosensor for the purpose of
hydroperoxide assay was initially aimed at characterizing
biosensor response to hydroperoxides of greater complexity
than hydrogen peroxide, such as terbutylhydroperoxide and
cumene hydroperoxide, operating in a non polar organic
solvent. The determination in the organic phase of hydro-
peroxides produced by the oxidation of lipids is actually of
importance in the evaluation not only of the stability of food
fats, but also of the activity of free radicals in vivo and in
vitro [17,18], although indirect spectrophotometric and
chromatographic methods of hydroperoxide determination
do exist [18], as well as direct enzymatic [19] or polaro-
graphic [20] methods. Nevertheless, these methods are
usually rather tedious. Compared with them determination
using biosensors operating in non aqueous medium is cer-
tainly simpler and faster; this is a great advantage when the
species to be determined is highly reactive, as hydroper-
oxides usually are. In hydroperoxide determination using the
catalase OPEE interference by organic peroxides was also
found to be absent. The latter are often present together with
hydroperoxides in real matrixes to be analysed. For this
purpose tests were run using benzoylperoxide (see Fig. 1)
over a concentration range of 0.110 mmol l 1 in order to
ascertain whether there was any biosensor response to the
peroxides and thus the possible interference of the latter in
hydroperoxide determination. It was found experimentally
that the biosensor is not sensitive to the presence of this
species over the concentration range adopted.
The proper functioning of the biosensor in n-decane
solution (lifetime exceeding 16 days, almost constant linear
range, sensitivity not always constant throughout the `life-
time', but which nevertheless remains high enough to allow
hydroperoxides to be measured correctly) made it possible
to monitor hydroperoxide content throughout the process of
articial rancidication of extra virgin olive oil. The results
live up to the expectations as a substantial increase was
observed in hydroperoxide concentration after about 50 h
treatment. This length of time is in agreement with the
results obtained in previous similar tests, in which peroxide
number variation was determined by volumetric titration and
polyphenol content using a tyrosinase biosensor [9].
6. Conclusions
In conclusion, hydroperoxide determination in real com-
plex matrixes (such as olive oil) may thus be performed
quickly (15 min) and enough precisely (RSD%  10)
using the catalase biosensor, operating in an organic solvent
such as n-decane, in which olive oil is highly soluble.
Moreover, the results show that the assay of the hydroper-
oxide ``pool'' may be used as a new indicator of the process
of rancidication of extra virgin olive oil and thus of its state
of conservation. Lastly, the histogram trends shown in Fig. 5
further support the antioxidant action exerted by the poly-
phenols, present in large quantities in extra virgin olive oil,
in inhibiting auto-oxidation phenomena, which are typical
above all of highly unsaturated fats.
Acknowledgements
The authors wish to thank the National Research Council
(CNR) of Italy, in particular, the ``National Group for the
Defence from Chemical Industrial and Ecological Risks''
(GNDRCIE) and the Targeted Projects in `Solid State
Electronic Materials' (MADESS), lastly the Consorzio
Interuniversitario ``La Chimica per l'Ambiente'' (INCA)
for nancial support.
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