International Journal of Food Microbiology 75 (2002) 111 118

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Microbiological activity in stored olive oil

G. Ciafardini *, B.A. Zullo
Department of Animal, Plant, and Environmental Sciences, Agriculture Faculty, University of Molise,
Via De Sanctis, I-86100, Campobasso, Italy
Received 17 June 2001; accepted 5 November 2001

Abstract

The disappearance of the bitter taste of newly produced olive oil during storage is due to the enzymatic hydrolysis of the
bitter-tasting secoiridoid compound known as oleuropein. Current knowledge attributes the enzymatic hydrolysis of the
oleuropein to the b-glucosidase present in the olives. The present study, however, has demonstrated for the first time that
oleuropein present in olive oil can be hydrolysed by b-glucosidase from the yeasts Saccharomyces cerevisiae and Candida
wickerhamii. The enzymatic analyses carried out directly on the untreated olive oil and on sterilized olive oil inoculated with the
above-mentioned yeasts proved the b-glucosidase activity through the hydrolysis of both the synthetic substrate p-nitrophenyl-
b-D-glucopyranoside (PNPG) and the oleuropein. The absence of lipases in the isolated S. cerevisiae and C. wickerhamii
examined lead us to believe that the yeasts contribute in a positive way towards the improvement of the organological quality of
the oil without altering the composition of the triglycerides. D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Oleuropein; Olive oil; Microbial activity

1. Introduction during preservation and olive oil contains different

Olives are usually used for direct consumption as
table olives or they are processed for olive oil extrac-
tion. The nutritional quality of the olive oil depends
on the type and concentration of substances like
tocopherols, carotenoids and phenolic compounds.
The tocopherols and carotenoids are of great impor-
tance to human health because of their antioxidant and
free-radical antagonistic properties (Gross, 1991; Has-
sapidou and Manoukas, 1993; Claude et al., 2000;
Pulido et al., 2000). The phenolic compounds prevent
the oxidization of the triglycerides of the olive oil
*
Corresponding author. Tel.: +39-874-404689; fax: +39-874-
404678.
E-mail address: ciafardi@unimol.it (G. Ciafardini).
classes of phenolic compounds as phenol acids, phe-
nol alcohols, flavonoids and secoiridoids (Macheix et
al., 1990). 3,4(dyhydroxyphenyl)ethanol, p(hydroxy-
phenyl)ethanol, luteolin-7-glucoside, rutin, cyanidin
and delphinidin glycoside are the main phenol alco-
hols and flavonoids, whereas oleuropein and deme-
thyloleuzopeim are the predominant secoiridoid
compounds of olive oil. Secoiridoids are the most
concentrated primary antioxidants of olive oil and
their nutritional properties have been recognized (Vis-
ioli et al., 1995). In fact, secoiridoids and their
derivative compounds inhibit blood platelet aggrega-
tion and are involved in the synthesis of thromboxane
in human cells (Petroni et al., 1995). Virgin olive oil
and table olives are the only sources reported, in
human diets, of these protective nutrients against the
risk of thromboses. The concentration of secoiridoid

0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 1 6 0 5 ( 0 1 ) 0 0 7 3 9 - 5
112 G. Ciafardini, B.A. Zullo / International Journal of Food Microbiology 75 (2002) 111118
glucoside in olive oil is dependent upon the olive
variety and the ripening state of the fruit (Goupy et al.,
1991). The high concentration of oleuropein gives an
excessively bitter taste to the newly produced olive
oil; however, during the decanting period, the phys-
icochemical properties of the olive oil improve,
acquiring greater clarity and new tastes. From scien-
tific information available, it is not possible to estab-
lish whether or not, during the olive oil decanting
process, the oleuropein is hydrolysed by enzymes
from the olives (Angerosa et al., 1995; Bota et al.,
2001; Montedoro et al., 1993) or whether enzymes
produced by microorganisms present in olive oil are
involved. The aim of the present research has been to
discover whether microorganisms are present in olive
oil during the decanting process and whether their
presence might influence the hydrolysis of the oleur-
opein through the production of b-glucosidase.
2. Materials and methods
2.1. Olive oil
The olive oil studied was extracted from the
Leccino olive variety obtained from olives pro-
cessed at low temperatures in a traditional plantation
situated in central Italy. The newly produced olive oil
was divided into three stainless-steel containers each
containing 100 kg of product and preserved at a
constant temperature of 15 C. Samples were taken
from two taps, one located at the bottom of the vessel
and the other at the top, about 10 cm from the surface
of the oil. The trials were carried out in 5 months
during the oils sedimentation time, taking 300 ml
samples at the beginning of the experiment (time zero)
and subsequently once a month, from each tap of the
three containers. The samples taken aseptically with a
pipette were then divided into three groups and
chemical, microbiological and enzymatic analyses
were conducted immediately. At the same time, from
three industrial containers of a capacity of 10,000 kg
filled with the same type of oil as used for the
experiment and preserved at 15 C, samples of sedi-
ment were taken using the same procedure. The
routine chemical analyses of acidity, peroxides, poly-
phenols and other chemical parameters requested for
the commercial classification of the olive oil were
made according to the European Communitys 2568/
91 Regulation (EC, 1991).
2.2. Microbiological analysis
Bacteria were enumerated in Plate Count Agar
Standard (PCAS) (Oxoid, Basingstoke, Hampshire,
England) and MRS agar medium (Oxoid) for the
lactic acid bacteria cultivation. The plates with the
PCAS medium were surface-inoculated with 0.2 ml of
olive oil or oil sediments diluted by appropriate 10-
fold dilution in olive oil sterilised at 110 C for 20 min
and incubated aerobically for 5 days at 28 C. MRS
Agar was inoculated in the same way, sealed in plastic
bags with a vacuum pump (KNF, Sigma, St. Louis,
MO, USA) and incubated for 5 days at 32 C. The
moulds were enumerated using Oxytetracyclin Glu-
cose Yeast Extract Agar medium (Oxoid) containing
100 mg/ml of gentamicin and chloramphenicol,
respectively. The colonies of moulds were counted
after 7 days of incubation at 28 C. The yeasts were
analyzed using sabouraud medium (Oxoid), incubated
for 7 days at 28 C. The samples of newly produced
olive oil were also examined by light field microscopy
at 600  magnification.
2.3. Yeast identification
The yeast isolates were identified according to
conventional methods used in yeast taxonomy (Kurtz-
man and Fell, 1998). The classification included 50
isolates taken at random from the colonies of yeasts
obtained from the plates with higher dilution. Each
test was repeated three times for each yeast isolated.
The ability to produce lipase was tested according to
Marquina et al. (1992) by using the Gorodkowa
medium (van der Walt and Yarrow, 1984) with 5%
(v/v) olive oil, emulsified by vigorous shaking. After
10 days of incubation, the plates were flooded with
saturated copper sulfate and the appearance of colony
with a bluish colour was taken as being indicative of a
positive lipolytic activity. The b-glucosidase activity
was evaluated according to Kengen et al. (1993).
2.4. Oleuropeinolytic activity
The oleuropeinolytic activity was carried out on
the 50 isolates used in the taxonomy test. The trials
 G. Ciafardini, B.A. Zullo / International Journal of Food Microbiology 75 (2002) 111118
were performed by using thin-layer chromatography
(TLC) as reported by Ciafardini et al. (1994). The
tests were carried out in microwell plates (standard 96
well; Cole Parmer, Chicago, IL) by inoculation of 0.2
ml of liquid Sabouraud medium containing 0.1% (wt/
vol) of oleuropein (Extrasynthese, Geney, France)
with single colony of yeast. After 7 days of incubation
at 28 C, samples were taken from each well, centri-
fuged at 10,000  g for 10 min, and analyzed by TLC
performed on precoated silica gel, 60 plates, 0.26-mm
thick (Merck, Darmstadt, Germany). The spots were
detected under UV light at 254 nm (transilluminator
mod. T2202; Sigma) and revealed by spraying of
Folin-Ciocalteus reagent (Merck), which reacts with
oleuropein. The solvent mixture used to develop the
TLC plates was n-propanol benzyl alcohol 88%
formic acid water (50:72:20:20).
2.5. b-Glucosidase activity in olive oil
The activity of b-glucosidase in the olive oil was
determined by the hydrolysis of p-nitrophenyl b-D-
glucopyranoside (PNPG) (Sigma). The enzymatic
hydrolysis of PNPG induced from the b-glucosidase
was established with a DECA PROBE/PR 200 instru-
ment (Hoefer Scientific Instruments, San Francisco,
CA, USA) by placing one strip of blotting paper
Fig. 1. Microdrops of vegetation water and solid particles observed with a light microscope at 600  magnification in the newly produced olive
oil. The arrows show the microorganisms and the solid particles entrapped in the microdrops of vegetation water suspended in the olive oil.
113
soaked with 1 ml of 0.1 M phosphate buffer (pH 7)
containing 0.4% (w/v) of PNPG in each reaction
groove filled with the olive oil samples. The instru-
ment was set up on a vertical rotating mixer at 45 rpm
and the samples were incubated overnight at 30 C.
After the incubation period, the olive oil samples were
removed from the instrument and, after being
weighed, the blotting paper strips were transferred
into an 10 ml of empty Pyrex test tube where they
were homogenized with a Turrax mod. T25 homog-
enizer (IKA-Milan, Italy), in the presence of 1 ml of
0.1 M phosphate buffer (pH 7). Finally, the liquid
fraction containing p-nitrophenol was recuperated
through centrifugation at 5500  g per 10 min and
analyzed with a mod. 941 spectrophotometer (Kon-
tron Instrument, Milan, Italy) at 410 nm. The data
obtained were compared to the calibration curve set
up by using p-nitrophenol (Sigma). The tests were
performed with unsterilised and sterilised newly pro-
duced olive oil. The sterilised olive oil fraction was
obtained by using a nitrocellulose filter membrane
with a porosity of 0.2 mm (Minisart NML-Sartorius,
Gottingen, Germany). A part of the sterilised olive oil
was used as a control, while another part was enriched
respectively with 2% (wt/vol) commercial b-glucosi-
dase from almonds (Fluka, Buchs, Switzerland), 27%
water, holding 2 units/mg (1 unit correspond to 1
114 G. Ciafardini, B.A. Zullo / International Journal of Food Microbiology 75 (2002) 111118

2.6. Microbial oleuropein hydrolysis in olive oil

The oleuropein hydrolysis was evaluated as re-

ported before, by using newly produced olive oil,
sterilized through filter membrane, enriched with 2%
(wt/vol) of yeast biomass with 27% humidity pro-
ducers of b-glucosidase, consisting of a mixture of
Saccharomyces cerevisiae and Candida wickerhamii
in a ratio of 1:1 (wt/wt), isolated from the previous
tests. Both the sterilized newly produced olive oil
(control) and the yeast-enriched olive oil, after being
homogenized with a Turrax for 5 min, were trans-

Table 1
Yeast species isolated from olive oil and comparison of some
characteristics
Saccharomyces Candida
Fig. 2. Yeasts found in the sediments of olive oil during storage. cerevisiae wickerhamii
Urease
Esterase
b-glucosidase + +
mmol of glucose liberate from salicin per minute at pH Lipase
5 and 37 C) and 2% (wt/vol) of yeast biomass (27% Growth on sugars:
water) producers of b-glucosidase isolated from the D(+)-Galactose + +
Maltose
previous tests. The test was performed three times Me a-D-glucoside +
with four repetitions of each treatment. D(+)-Trehalose +,
Melibiose +,
Lactose
D(+)-Cellobiose +
D(+)-Melezitose , +
D(+)-Raffinose +
Inulin
D( )-Arabinose D
Starch
D(+)Xylose +
D( )-Mannitol ,D +
Sugars fermented:
D(+)-Glucose + +
D(+)-Galactose +,
Maltose
Me a-D-glucoside
Sucrose +
D(+)-Trehalose
Melibiose +,
Lactose
D(+)-Cellobiose +
D(+)-Melezitose
D(+)-Raffinose +,
Inulin
Fig. 3. Yeasts found in the olive oil during storage. x, Samples Starch
taken from the upper part of the vessel; n, samples taken from the +, positive reaction; , negative reaction ; D, positivity found after
bottom part of the vessel. Data are means F standard deviation. a week.
 G. Ciafardini, B.A. Zullo / International Journal of Food Microbiology 75 (2002) 111118 115

extract by HPLC (HP 1050) equipped with a Diode

Array 168 detector, operated at 280 nm. The thermo-
stat column at 35 C was a C-18 in inverse phase
SuperEcosil-LC-18 characterised by a length of 25 cm
and an internal diameter of 4.6 mm; the thickness of
the stationary phase was of 5 mm. The mobile phase of
constant composition resulted in being constructed of
a mixture composed of 50% (vol/vol) water with 1%
of glacial acetic acid (vol/vol) and of 50% (vol/vol)
methanol with 1% of glacial acetic acid (vol/vol). As
reference standard, pure commercial oleuropein was
used (Extrasynthese).

3. Results

The chemical analyses carried out during the

decanting process permitted the classification of the
olive oil studied as an extravirgin olive oil according
to the European Communitys 2568/91 Regulation
(EC, 1991) (data not shown). By microscopy, it was
observed that newly produced olive oil contains
Fig. 4. Reduction of oleuropein content in the samples inoculated numerous solid particles and microdrops of olive
with the two species of yeasts isolated from olive oil. (A) vegetation water containing, trapped within, a high
Uninoculated Sabouraud medium with 0.1% of oleuropein analyzed number of microorganisms that remain during the
after 7 days of incubation at 28 C (Control). (B) Sabouraud
medium containing 0.1% of oleuropein inoculated with S. cerevisiae
and analyzed after 7 days of incubation at 28 C. (C) Sabouraud
medium containing 0.1% of oleuropein inoculated with C.
wickerhamii and analyzed after 7 days of incubation at 28 C.
The arrows show the start point of the TLC analysis.

ferred respectively into 1000 ml Pyrex flasks, placed

on a rotating agitator and incubated at 30 C for 30
days at a constant agitation of 25 rpm. At the moment
in which the test was set up (zero time) and at
intervals of 15 days, 1 ml of sample was withdrawn
from each flask and frozen at 20 C until being
analysed. At the end of the test, the samples of olive
oil were defrosted and centrifuged at 12,000  g per
10 min and the oleuropein was extracted with 1 ml of
a mixture of methanol and water in the ratio of 60:40.
After vortexing for 20 min, the watery phase was
separated by centrifugation at 5500  g and trans- Fig. 5. Release of p-nitrophenol under the following conditions: (1)
Olive oil sterilised with nitrocellulose filter membrane; (2) sterilised
ferred into a new test tube and then filtered through
olive oil enriched with commercial b-glucosidase from almonds; (3)
nitrocellulose membrane (Minisart NML-Sartorius) sterilised olive oil enriched with a suspension of b-glucosidase-
with a porosity of 0.2 mm. The hydrolysis of the producing yeasts; (4) normal unsterilised newly produced olive oil.
oleuropein was shown by analysing 50 ml of phenolic Data are means F standard deviation.
116 G. Ciafardini, B.A. Zullo / International Journal of Food Microbiology 75 (2002) 111118
Fig. 6. HPLC analysis of the oleuropein from newly produced olive
oil inoculated with b-glucosidase producing yeasts. (A) Oleuropein
standard; (B) filter membrane-sterilized olive oil analysed after 1
month of incubation; (C) filter membrane-sterilized olive oil
enriched with b-glucosidase producing yeasts analysed after 1
month of incubation.
entire period of olive oil preservation (Fig. 1). The
results of the microbiological analyses carried out on
the sediments demonstrated a high number of yeasts
especially in the samples taken during the first 10 days
of decantation of the olive oil (Fig. 2); in the same
samples of sediment, the presence of bacteria or
moulds was not found. The microbiological analyses
carried out directly on the stored olive oil highlighted
the presence of yeasts but not of bacteria and moulds.
The number of yeasts was reduced significantly dur-
ing the first month of sedimentation of the oil,
whereas successively, the rate of reduction was more
gradual with slightly higher count observed in the oil
situated in the top part of the containers (Fig. 3). The
yeasts isolated from the olive oil, characterised on the
basis of the carbohydrate fermentation pattern and
other physiological characteristics, were classed as S.
cerevisiae and C. wickerhamii in the ratio of 3:1
(Table 1). The enzymatic characterization of the yeasts
classified allowed us to verify, in all of the isolates,
the absence of the activity of urease, lipase and
esterase and the capability to synthesise b-glucosidase
active both on the synthetic substrate PNPG (Table 1)
and on the glucoside oleuropein (Fig. 4). The enzy-
matic analyses carried out directly on the oil allowed
us to verify that in the newly produced olive oil, a b-
glucosidase is active, able to hydrolyse the synthetic
substrate PNPG (Fig. 5). Trials carried out using
sterile olive oil enriched with commercial b-glucosi-
dase or with yeast producers of b-glucosidase high-
lighted in both cases the enzymatic hydrolysis of the
synthetic substrate PNPG (Fig. 5). The analyses
carried out with the HPLC of the phenolic com-
pounds, extracted from the samples of olive oil
inoculated with the b-glucosidase producing yeasts,
showed a high content of oleuropein in the sterile
newly produced olive oil, whereas in the olive oil
inoculated with the yeasts, the bitter-tasting glucoside
was hydrolysed during 30 days of incubation until it
completely disappeared (Fig. 6). The same result was
obtained by inoculating the olive oil with the com-
mercial b-glucosidase extracted from almonds (data
not shown).
4. Discussion
Olive oil is usually consumed after a storage period
of a few months in order to allow the solid particles
and microdrops of vegetation water to deposit at the
bottom of the vessel. The physicochemical character-
istics of the olive oil together with the taste all
improve during the sedimentation time. In fact, some
types of newly produced oil are very bitter since they
are rich in oleuropein, whereas after a few months
preservation, the bitter taste completely disappears
 G. Ciafardini, B.A. Zullo / International Journal of Food Microbiology 75 (2002) 111118
following the hydrolysis of the oleuropein. However,
up until now, the oil has been greatly studied from a
chemical and nutritional aspect and the enzymatic
activity found within it were attributed to the enzymes
from the fruits (Montedoro et al., 1993; Bota et al.,
2001). Our research, however, has demonstrated that a
rich microflora exists in newly produced olive oil that
is active during the entire decanting process. In fact,
Fig. 1 shows the presence of solid particles and
microdrops of vegetation water containing entrapped
cells of yeasts and other components of the fruits. The
yeasts present in the material suspended through
decantation slowly migrate towards the bottom of
the vessel to form the sediment, nevertheless conserv-
ing their vitality both in the olive oil and in the
sediments during the entire decanting period (Figs. 2
and 3). The strong reduction in the number of yeasts
observed in the first month of decantation can partly
be attributed to the rapid sedimentation of the coarse
material rich in microorganisms suspended in the
newly produced olive oil (Fig. 3). The yeasts activity
in olive oil can condition the physicochemical and
organoleptic properties of the final product through
the production of b-glucosidase. The data shown in
Fig. 5 demonstrate that the b-glucosidase active in
newly produced olive oil can be derived from the
olive fruit (Amiot et al., 1989; Montedoro et al., 1993;
Angerosa et al., 1995; Bota et al., 2001) and also
from the activity of the microorganisms (Ciafardini et
al., 1994; Ciafardini and Zullo, 2000). In fact, the
same tests demonstrate that pure culture of both the
classed yeasts are able to hydrolyse oleuropein in vitro
(Fig. 4), whereas in olive oil, the PNPG can be
hydrolysed from b-glucosidase of a vegetable origin
as well as from microbial origin (Fig. 5). Furthermore,
the analyses carried out on HPLC demonstrated that
the yeasts present in the oil contribute towards the
debittering process of the olive oil during the decant-
ing period. In fact, Fig. 6 demonstrates that the yeasts
producing b-glucosidase inoculated into the sterile
olive oil are capable of completely degrading the
oleuropein present within it in a month of incubation.
The absence of lipases in the species S. cerevisiae and
C. wickerhamii present in the oil studied lead us to
believe that the identified yeasts can contribute in a
positive way towards the improvement of the quality
of the oil without altering the quality of the compo-
sition of the triglycerides (Table 1); nevertheless, it
117
cannot be excluded that lipolytic yeasts, mould or
bacteria might be associated with the olive oil habitat.
From a practical aspect, the results of this research
open an interesting chapter in the sector of olive oil
production. In fact, as an alternative to the present
system of the industrial filtration of the newly pro-
duced olive oil, the system of natural sedimentation of
the oil can rationally be developed, rediscovering in a
modern context a technique used by the ancient
Romans. Furthermore, through adjusted microbiolog-
ical tests, it will be possible to monitor the type and
the activity of the olive oil microflora and therefore
guide the natural structuring of the oil during the
sedimentation phase, towards the final aim of produc-
ing top quality olive oil.
Acknowledgements
The authors express their thanks to Prof. F. Fava
and Dr. D. Di Gioia of the Faculty of Engineering,
DICASM, Bologna (Italy) for the support offered in
the oleuropein analysis.
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