ARTICLE IN PRESS

FOOD
MICROBIOLOGY
Food Microbiology 23 (2006) 6067
www.elsevier.com/locate/fm

Lipase production by yeasts from extra virgin olive oil

G. Ciafardini, B.A. Zullo, A. Iride
Department of Animal, Plant and Environmental Sciences, Agriculture Faculty, Molise University, Via De Sanctis, I-86100 Campobasso, Italy
Received 16 August 2004; accepted 4 January 2005

Abstract

Newly produced olive oil has an opalescent appearance due to the presence of solid particles and micro-drops of vegetation water
from the fruits. Some of our recent microbiological research has shown that a rich micro-ora is present in the suspended fraction of
the freshly produced olive oil capable of improving the quality of the oil through the hydrolysis of the oleuropein. Present research
however has, for the rst time, demonstrated the presence of lipase-positive yeasts in some samples of extra virgin olive oil which can
lower the quality of the oil through the hydrolysis of the triglycerides. The tests performed with yeasts of our collection, previously
isolated from olive oil, demonstrated that two lipase-producing yeast strains named Saccharomyces cerevisiae 1525 and Williopsis
californica 1639 were able to hydrolyse different specic synthetic substrates represented by p-nitrophenyl stearate, 4-nitrophenyl
palmitate, tripalmitin and triolein as well as olive oil triglycerides. The lipase activity in S. cerevisiae 1525 was conned to the whole
cells, whereas in W. californica 1639 it was also detected in the extracellular fraction. The enzyme activity in both yeasts was
inuenced by the ratio of the aqueous to the organic phase reaching its maximum value in S. cerevisiae 1525 when the water added
to the olive oil was present in a ratio of 0.25% (v/v), whereas in W. californica 1639 the optimal ratio was 1% (v/v). Furthermore, the
free fatty acids of olive oil proved to be good inducers of lipase activity in both yeasts. The microbiological analysis carried out on
commercial extra virgin olive oil, produced in four different geographic areas, demonstrated that the presence of lipase-producing
yeast varied from zero to 56% of the total yeasts detected, according to the source of oil samples.
The discovery of lipase-positive yeasts in some extra virgin olive oils leads us to believe that yeasts are able to contribute in a
positive or negative way towards the organological quality of the olive oil.
r 2005 Elsevier Ltd. All rights reserved.

Keywords: Lipase; Microbial enzyme; Olive oil; Yeast

1. Introduction and from the European Communitys 1513/2001 Reg-

Lipases (EC-3.1.1.3) include the hydrolase family of
enzymes (Brockerhoff and Jensen, 1974; Macre and
Hammond, 1985), that possess the unique feature of
acting as an interface between the aqueous and non-
aqueous (i.e. olive oil) phases and this feature distin-
guishes them from esterase. In damaged olives, before
processing and during transformation, the lipases may
be responsible for the high olive oil acidity due to the
hydrolysis of the triglycerides. High acidity exerts a
great inuence on olive oil stability (Frega et al., 1999)
Corresponding author. Tel.: +39 874 404 689;
fax: +39 874 404 855.
E-mail address: ciafardi@unimol.it (G. Ciafardini).
ulation (EC, 2001) less than 0.8% of total free fatty
acids, expressed as oleic acid, is requested in the
production of extra virgin olive oil. Studies carried out
up till now have proved that the lipases originating from
the fruits of the olive are the main causes of the acidity
of the newly produced olive oil while no information is
available on the presence of lipase-positive micro-
organisms in the oil and on the role of the lipases of
microbial origin on the increase in acidity of the olive oil
during storage. In fact, only recently has it been
discovered that freshly produced olive oil is contami-
nated by a rich micro-ora, capable of conditioning the
physico-chemical and organoleptic characteristics of
the oil through the production of enzymes (Ciafardini
and Zullo, 2002a,b). Among the micro-organisms we

0740-0020/$ - see front matter r 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fm.2005.01.009
 ARTICLE IN PRESS
G. Ciafardini et al. / Food Microbiology 23 (2006) 6067
isolated several strains of yeasts identied as Sacchar-
omyces cerevisiae, Candida wickerhamii, Williopsis cali-
fornica and Candida boidinii (Ciafardini and Zullo,
2002a; Ciafardini et al., 2004). Some of the identied
yeast species demonstrated the ability to improve the
organoleptic characteristics of the freshly produced
extra virgin olive oil, through the production of a b-
glucosidase active on the bitter glucoside noted as
oleuropein (Ciafardini and Zullo, 2002a). Nevertheless,
microbiological research carried out so far on extra
virgin olive oil is still somewhat limited and, from the
scientic data available, it is not possible to establish
whether, other than the species of yeasts useful in
stabilizing the organoleptic characteristics of the oil,
others capable of compromising the oils quality exist. In
particular, it is not known if the yeasts present in the
freshly produced olive oil can modify, in determined
conditions, some parameters responsible for the quality
of extra virgin olive oil. One of these parameters concern
the total free fatty acids content of the oil which can
increase following the activity of some lipases which
hydrolyse the triglycerides. The aim of the present
research has been to discover the presence of lipase-
producing yeasts in extra virgin olive oil.
2. Materials and methods
2.1. Cell culture
The studies on lipolytic activity were carried out with
various substrates using yeasts from our collection
(DAPES collection) isolated from extra virgin olive oil
and previously identied as C. boidinii, C. wickerhamii,
S. cerevisisae and W. californica (Ciafardini and Zullo,
2002a; Ciafardini et al., 2004). The yeast cultures were
grown separately in 1 l asks containing Malt, Yeast,
Glucose, Peptone medium (MYGP) with the following
composition: yeast extract (Biolife, Milan, Italy) 3 g,
malt extract (BBL, Cockeysville, Maryland, USA) 3 g,
beef extract powder (BBL, Cockeysville, Maryland,
USA) 5 g, D-glucose (Merck, Darmstadt, Germany)
10 g, distilled water 1000 ml, pH 5.5 (Kurtzman and
Fell, 1998). After 3 days of incubation at 30 1C, yeasts
were separated by centrifugation at 5000 g for 10 min
using a Hettich centrifuge, mod. Universal 32, (Hettich
Instruments, Tuttlingen, Germany) and were nally
used for the tests described below.
2.2. Enzyme assay
The lipase activity was evaluated by measuring the
free fatty acids produced by triacylglycerol hydrolysis or
nitrophenol produced from p-nitrophenyl stearate (Sigma
Chem. Co. St. Louis, Missouri, USA) and 4-nitrophenyl
palmitate (Fluka Chem., Buchs, Switzerland). Reaction
61
mixtures were prepared by adding, respectively, equal
amounts of triolein (Sigma) tripalmitin (Fluka) and
highly rened olive oil of low acidity (Sigma) to 0.1 M
sodium phosphate buffer (pH 7.0) containing 5% (v/v)
arabic gum.
Triolein and tripalmitin dissolved in an equal amount
of undecane (Sigma) and untreated olive oil were melted
at 40 1C and then emulsied as a 100 mM of substrate
solution in phosphate buffer with 5% arabic gum, for
20 s with a sonier apparatus (Branson, B20 Sonier) as
reported by Murphy et al. (1989). Positive controls
contained the above lipid emulsion and 200 ml of type
XIII lipase produced by Pseudomonas species (25 units
mg solid; Sigma). Cell biomass was added to 2 ml of
lipid emulsion at 40 mg ml
1. Reaction mixtures were
incubated at 30 1C on a shaking water-bath at 200 rpm
for up to 48 h. Samples were removed and the lipid
portion was analysed as reported by Meyers et al.
(1996). The lipase activity evaluated in the presence of
synthetic chromogenic substrate was carried out using p-
nitrophenyl stearate and 4-nitrophenyl palmitate at the
concentration of 1 mmol l
1 dissolved in 1 ml of
undecane. The reactions carried out in biphasic system
were 1 ml of substrate in undecane forming the organic
phase and 1 ml of culture suspension with 40 mg ml
1 of
cell biomass forming the aqueous phase. The concentra-
tion of nitrophenol, obtained after 24 h incubation at
30 1C, was evaluated measuring the absorbance of the
aqueous fraction at 410 nm with a mod. 941 spectro-
photometer (Kontron Instrument, Milan, Italy).
2.3. Preparation of cell-free extracts
Lipase activity was determined using two lipase-
producing strains of the DAPES collection namely W.
californica 1639 and S. cerevisiae 1525. Cells were grown
for 5 days at 30 1C in asks containing 600 ml of MYGP
broth. At the end of the incubation time, cells were
harvested by centrifugation for 20 min at 6500g (Het-
tich, Universal 32 centrifuge, Germany), whereas the
supernatant was sterilized through micro-ltration using
a sterile, syringe-driven lter unit with 0.2 mm of
porosity (Millipore Co., Bedford, Massachusett, USA)
and used as extracellular portion. Two grams of each
yeasts biomass were washed twice in sterile 50 mM
phosphate buffer (pH 7), then were divided into two
equal portions; the rst was used as whole cell, while the
second was suspended in 2 ml of the above sterile buffer
and sonicated for 10 min at 70 W using 1 min pulse
(Branson, mod. B20 Sonifer, USA).
The lipase test was carried out in test tubes with a
screw-on-cap, inoculating 10 ml of ler sterilized olive
oil with sterile water (control), with the extracellular
portion consisting of supernatant where yeasts were
cultivated, with the non-sonicate whole cell, and with
the intracellular portion consisting of lter sterilized
 ARTICLE IN PRESS
62 G. Ciafardini et al. / Food Microbiology 23 (2006) 6067
supernatant obtained by the sonication of yeasts. The
concentration of the inoculum was equal to 10% (w/v)
of the oily mass. Samples were incubated at 30 1C for 3
days and, at the end of the incubation time, the total
fatty acids were evaluated as oleic acid with the
titrimetric method reported by the European Commu-
nitys 1513/2001 Regulation (EC, 2001).
2.4. Effect of volume ratio of aqueous to organic phase on
enzyme activity
The enzymatic activity, in the presence of increasing
doses of water, was evaluated using lipase-producing
yeasts W. californica 1639 and S. cerevisiae 1525. The
trials were carried out using 50 ml sterile test tubes each
containing 200 mg of yeast biomass reported above. A
mass of 40 g of lter sterilized olive oil and sterile
distilled water was added to each test tube in the ratio of
0%, 0.25%, 1%, 5% and 10% (w/w). At the same time
the control was set up using the same ratio of oil/water
without yeasts. The trials were repeated four times and
samples, which were incubated at 30 1C for a month,
were agitated daily for 1 min with a vortex. The
enzymatic activity was evaluated every week by measur-
ing the acidity of the olive oil using the same titrimetric
method reported above.
2.5. Effect of olive oil acidity on lipase activity
The inoculation trials were carried out using two
freshly produced olive oils originating from the Lecci-
no olive variety, characterized by an acidity equal to
0.42% and 5% expressed as oleic acid. Both types of oil
were rst centrifuged at 6000g for 20 min and then
sterilized through nitrocellulose lters with a porosity of
0.2 mm (Minisart NML-Sartorius, Gottingen, Germany).
Then 1000 ml of samples were transferred into sterile
Pyrex asks with screw caps. The inoculum was prepared
using two lipase-producing yeasts, represented by S.
cerevisiae 1525 and W. californica 1639 strains and a
lipase-negative strain represented by C. wickerhamii 1542.
Yeasts were grown in MYGP medium as reported above.
Next the biomass was suspended in sterile water in a ratio
of 0.45% (w/v) and used to inoculate the olive oil. Both
oils present in the asks were inoculated with the above-
mentioned yeasts using an inoculating dose equal to 1%
(v/v), while in the control the inoculum consisted only of
sterile water. Furthermore, the trial consisted of four
repetitions for each type of treatment. After being lightly
mixed manually for 1 min, the samples were incubated at
30 1C for 20 days. The total yeasts number was evaluated
at the beginning of the experiment (zero time) and
subsequently once every 4 days using the plate dish
method described before (Ciafardini and Zullo, 2002a)
and also the free fatty acids of the inoculated olive oil
using the titrimetric method previously reported.
2.6. Evaluation of lipase-producing yeasts in commercial
extra virgin olive oil
The microbiological analyses were carried out using
samples of commercial extra virgin olive oil produced
from the Molise area (central Italy). The total yeasts
number was evaluated using MYGPagar medium using
the previously reported procedure (Ciafardini and Zullo,
2002b). After 5 days of incubation, the yeast colonies
were rst counted and then 400 colonies were randomly
chosen from each sample and were used to set up a
double master where the same colonies were transferred
onto MYGPagar and in CHROMagar medium (Tornai-
Lehoczki et al., 2003). After 3 days of incubation at
30 1C, the colonies developed on the MYGPagar were
extracted with all the substrate cut into cubes with a
bistoury and transferred to a sterile plastic test tube with
a cap, containing 0.1 ml of sterile water and 10 ml of
extra virgin olive oil sterilized through nitrocellulose
lters with a porosity of 0.2 mm (Minisart NML-
Sartorius, Gottingen, Germany). After briey mixing
with a vortex mod. Maxi Mixer (FAVS, Bologna, Italy),
samples were incubated at 30 1C for 3 days. During
incubation, samples were agitated daily for a minute with a
vortex. At the end of the incubation period, samples were
centrifuged at 4500g for 15 min and free fatty acids were
evaluated on the olive oil fraction using the titrimetric
method as previously reported. The values of each colony,
exceeding by 20% the average acidity of the uninoculated
control, were recorded as positive. The same colonies of
the second master, inoculated in CHROMagar medium,
were analysed after 7 days of incubation at 30 1C noting
the morphological characteristics and colour of the lipase-
positive and negative colonies.
2.7. Scanning electron microscopy (SEM) observation of
yeasts present in commercial olive oil
Samples of 1.5 ml of freshly produced commercial
olive oil from the Molise area (Central Italy) were
transferred into sterile micro-centrifuge plastic test
tubes. Centrifugation was carried out with an ALC
international micro-centrifuge mod. 4204 (Milan, Italy),
using 4500g per 10 min. The supernatant was discarded,
whereas the pellet was suspended in 1 ml of 0.1 M
phosphate buffer pH 7 and treated for SEM observation
using the same procedure reported previously (Ciafar-
dini and Marotta, 1988). Finally, samples were observed
with an SEM Zeiss DSM 940 (Zeiss, Rome, Italy).
3. Results
The laboratory tests carried out, using yeasts from the
DAPES collection isolated from olive oil, allowed us to
single out two strains of lipase-producing yeasts
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G. Ciafardini et al. / Food Microbiology 23 (2006) 6067 63

belonging to the S. cerevisiae and W. californica species. concentration of free fatty acids present in olive oil
In fact, both strains of S. cerevisiae 1525 and W. samples. Trials carried with extra virgin olive oil
californica 1639 hydrolysed the synthetic cromogen containing 0.4% of initial acidity, showed that after 20
substrates consisting of, other than triolein and tripal- days of incubation the acidity increased until reaching,
mitin, p-nitrophenyl palmitate and 4-nitrophenyl stea- respectively, 1.1% for S. cerevisiae 1525 and 1.5% in W.
rate. Yeasts, capable of hydrolysing the specic californica 1639, whereas times of induction of the
synthetic substrates, increased the acidity of the oil
following the hydrolysis of the triglycerides promoted by
lipases produced by both strains of yeasts mentioned
above (Table 1). Based on their ability to hydrolyse
several synthetic substrates and olive oil triglycerides
both strains S. cerevisiae 1525 and W. californica 1639
were chosen for further study using olive oil as a source.
Laboratory research carried out on the two lipase-
producing yeasts proved that in the S. cerevisiae 1525
strain the lipase activity was associated only with the
whole cells or the fragments of sonicate cells, whereas in
the W. californica 1639 strain the enzyme activity was
also detected in extracellular materials (Fig. 1). The
lipase activity of both yeasts increased with the
increasing volume ratio of aqueous to organic phase.
In fact, in the S. cerevisiae 1525 strain the lipolytic
activity increased in the olive oil as the aqueous fraction
increased until reaching its maximum value when the
water was present in a ratio of 0.25% (v/v) (Fig. 2).
Equally, in the W. californica 1639 strain the lipolytic
activity increased according to the concentration of
Fig. 1. Production of lipase in S. cerevisiae 1525 and W. californica
water but the maximum values were reached when the
1639. , whole cells; , sonicated yeast cells; , supernatant obtained
water added to the oil was present in a ratio of 1% (v/v) from centrifugation of sonicated yeast cells; &, supernatant obtained
(Fig. 3). The lipase activity of the S. cerevisiae 1525 and from the yeasts growth medium; , acidity of the uninoculated oil
W. californica 1639 strains was inuenced by the (control). Bars are mean (SD).

Table 1
Lipase activity in representative yeast species isolated from extra virgin olive oil

Species DAPES Collectiona Lipase activity with different substrates

p-Nitrophenyl stearate 4-Nitrophenyl palmitate Triolein Tripalmitin Olive oil

S. cerevisiae
1445





1456





1468





1476





1525 + + + + +

W. californica
1639 + + + + +
1652





C. wickerhamii
1542





1655





1656




1657

C. boidinii
1638

+, lipase positive;
, lipase negative.

a
DAPES, Collection of Department of Animal, Plant, and Environmental Sciences.
 ARTICLE IN PRESS
64 G. Ciafardini et al. / Food Microbiology 23 (2006) 6067
Fig. 2. Lipase activity of the S. cerevisiae 1525 in the presence of
different water concentrations. &, 0%; , 0.25%; , 0.5%; , 1%, ,
5%, , 10%. Bars are mean (SD).
Fig. 3. Lipase activity of the W. californica 1639 in the presence of
different water concentrations. &, 0%; , 0.25%; ,0.5%; , 1%, ,
5%, , 10%. Bars are mean (SD).
enzymatic activity were 12 days for S. cerevisiae 1525
and 4 days for W. californica 1639 (Fig. 4). When,
however, olive oil with an initial 5% acidity was used, in
20 days of incubation the acidity increased more sharply
until reaching 13.8% in S. cerevisiae 1525 and 18.5% in
W. californica 1639, while the induction period of the
enzymatic activity was found to be shorter than the
previous one reaching 8 days in the case of S. cerevisiae
1525 and less than 4 days in the case of W. californica
Fig. 4. Lipase activity and survival (empty symbol) of yeasts
inoculated in olive oil with low acidity. m, Fatty acids produced with
lipase-positive S. cerevisiae 1525; , fatty acids produced with lipase-
positive W. californica 1639; , fatty acids produced with lipase-
negative C. wickerhamii 1542; ~, fatty acids present in the
uninoculated oil (control). Bars are mean (SD).
Fig. 5. Lipase activity and survival (empty symbol) of yeasts
inoculated in olive oil with high acidity. m, Fatty acids produced with
the lipase-positive S. cerevisiae 1525; , fatty acids produced with
lipase-positive W. californica 1639; , fatty acids produced with lipase-
negative C. wickerhamii 1542; ~, fatty acids present in the
uninoculated oil (control). Bars are mean (SD).
1639 (Fig. 5). On the contrary, the lipase-negative C.
wickerhamii 1542 strain did not modify the acidity in
either type of olive oil whereas the microbiological
analysis of both olive oil types showed a high survival of
yeasts during the whole incubation period (Figs. 4 and
5). Research carried out on commercial olive oils has
allowed us to observe for the rst time, through the use
of an electronic microscope, the presence of yeasts in the
oil. In fact, samples of the freshly produced, commer-
 ARTICLE IN PRESS
G. Ciafardini et al. / Food Microbiology 23 (2006) 6067
cial, extra virgin olive oil appeared slightly opaque due
to the presence of suspended solid particles and micro-
drops of vegetation water. The SEM observation
demonstrated the presence of forms of yeasts in
suspended particles where the yeasts resulted both
entrapped in the solid particles (Fig. 6) or on the surface
of the solid suspended particles (Fig. 7). The micro-
biological analysis, carried out on samples of commer-
cial extra virgin olive oil produced in four different
geographical areas of the same region, demonstrated the
presence of lipase-producing yeasts that are active on
the triglycerides of the olive oil. In fact, even if the total
number of yeasts remained almost constant in the
various samples of oil analysed, the physiological group,
consisting of the lipase-producing yeasts, varied from
Fig. 6. SEM observation of yeasts entrapped in solid particles
suspended in extra-virgin olive oil.
Fig. 7. SEM observation of budding yeasts (arrows) present in the
suspended materials of extra virgin olive oil.
65
0% to 56% according to the source of the oil samples
(Table 2). The yeast colonies cultivated on CHROMa-
gar according to the geographical origin of samples
demonstrated high biodiversity with four types of
colony colour, while the lipase-positive yeasts assumed
only two types of colony colour: creamy reddish purple
or wrinkled purple colony (Table 2).
4. Discussion
Recently our microbiological research demonstrated
the presence of a rich micro-ora in extra virgin olive oil
which remains vital during the entire decanting process
(Ciafardini and Zullo, 2002b). Some of these micro-
organisms increase the quality of the oil as they reduce
the bitter taste through the enzymatic hydrolysis of the
bitter glucoside known as oleuropein (Ciafardini and
Zullo, 2002a). The present research demonstrated for
the rst time that forms of lipase-producing yeasts also
exist in extra virgin olive oil, which are potentially
harmful to the quality of the oil since they increase the
acidity through the hydrolysis of triglycerides. The
similar enzymatic specicity towards the ester substrates
showed by S. cerevisiae 1525 and W. californica 1639
strains (Table 1) is in agreement with the data
concerning Candida rugosa reported by Shaw et al.
(1989). In fact, as observed for C. rugosa, this result is
possibly due to the contemporary presence of different
forms of lipolytic enzyme produced by both yeast
strains. The further study made on the two lipase-
producing yeasts demonstrated that lipolytic activity
carried out by W. californica 1639, compared to S.
cerevisiae 1525, was found to be more active in various
trials (Figs. 4 and 5). In fact, in respect to the whole cell-
conned lipases of S. cerevisiae 1525, the water-
dissolved extracellular lipases produced by W. californi-
ca 1639 (Fig. 1) have a great interaction with the
substrate present in the organic phase, to form an acyl-
enzyme complex in the interface area which is the rst
step of lipase reaction (Brockerhoff and Jensen, 1974).
The hydrolytic activity of the lipases produced by S.
cerevisiae 1525 showed a trend of water dependence
similar to that of W. californica 1639. Nevertheless, in
this case also since water participated in the diacylation
of the above-mentioned acyl-enzyme complex, which
should also proceed at the interface (Wang et al., 1988),
the different optimal aqueous/organic ratio for lipase
activity can be attributed to the whole cell or extra
cellular type of lipases produced by the two strains
(Figs. 2 and 3). As practical aspect, such results
demonstrate that the lipases produced from the two
species of yeasts can be activated in the olive oil where
the quantity of water of approx. 0.25% is normally
present as vegetation water in all of the newly produced
oil mass, while 1% is reached in the lower layers of the
 ARTICLE IN PRESS
66 G. Ciafardini et al. / Food Microbiology 23 (2006) 6067

Lip+
oily mass during the sedimentation period. The free

Bluish (%)
fatty acids present in the olive oil are good inducers for
lipase production in both S. cerevisiae 1525 and W.

Lip

californica 1639. In fact, in agreement with the results

10



reported for C. rugosa by Obradors et al. (1993) and
Lakshmi et al. (1999), from the data reported in Figs. 4
Lip+
and 5 it is possible to observe that in 20 days of

White (%)

incubation, the yield of free fatty acids was increased in

Lip

23 proportion to the initial acidity of the olive oil

18
10
8
inoculated with both yeasts. The SEM observation and
the microbiological analysis of the commercial extra
virgin olive oil conrm our previous nding concerning
Wrinkled purple (%)

the presence of yeast in newly produced olive oil. In fact

Lip+

100
88

from Figs. 6 and 7 it is possible to observe different

forms of yeasts dividing by budding. Furthermore, the
presence of cells in the growing phase conrm the
viability of yeasts in extra virgin olive oil (Ciafardini and
Lip

34
56

Zullo, 2002b). Instead, the data reported in Table 2

demonstrates for the rst time the presence of a high
percentage of lipase-producing yeasts from the oil
Creamy reddish purple (%)

originating from two geographical areas. These results

Colony colour of yeasts

are very important from a technical aspect as they

Lip+c

demonstrate that the olive oils, rich in lipase-producing

12

yeasts, are more subject to going rancid, especially if the

storage temperature is favourable to yeast activity. This
Total yeasts and colony colour characteristics of yeast forms evaluated in commercial extra virgin olive oil

research demonstrates the importance of knowing the

chemical and microbiological composition of the newly
Lip
b

produced olive oil in order to maintain quality. In fact, a

67
52
56
36

high presence of lipase-producing yeasts in olive oil needs

Lipase-producing

attention as it can negatively inuence the shelf life of the

product through an increase in acidity in the olive oil.
yeasts (%)

Acknowledgments


34
56

The authors express their thanks to Dr. L. Maiuro of

(Log cfu/ml)a
Total yeasts

5.0970.22
6.3970.02
5.3570.18
5.1870.17

the Electronic Microscopy Center, Agriculture Faculty,

Campobasso (Italy), for the support offered in the SEM
observation.
Extra virgin olive oil from different geographic areas

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