Determination of killer activity in yeasts isolated from the elaboration of seasoned green table olives –

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Determination of killer activity in yeasts isolated from the elaboration of seasoned green table olives
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Determination of killer activity in yeasts isolated from the elaboration of
seasoned green table olives
Alejandro Hernández, Alberto Martín, María G. Córdoba, María José Benito,
Emilio Aranda, Francisco Pérez-Nevado
Nutrición y Bromatología, Escuela de Ingenierías Agrarias, Universidad de Extremadura, Ctra. de Cáceres s/n. 06071 Badajoz, Spain
Received 10 January 2007; received in revised form 31 July 2007; accepted 6 November 2007
In this work 51 yeasts strains isolated from seasoned green table olives and belonging to the
genera were characterized by their killer activity in different conditions. Killer activity of isolates was analyzed in a medium
with different pH’s (3.5 to 8.5) and NaCl concentrations (5, 8, and 10%). At every pH tested, all the genera studied had killer strains, although the
smallest percentages of killer yeasts were found at the highest pH (8.5). The presence of 5 and 8% NaCl increased the detected killer percentage, but
the highest salt concentration (10%) decreased it. The interaction between the reference killer yeasts and yeasts isolated from olives was analyzed
Most isolates were killer-sensitive to one or more killer reference strains. Only 2 of the 51 strains tested were considered killer-neutral. Cross-
reaction trials between isolates and spoilage yeasts showed that, of the isolates, nine killer strains, belonging to
Debaryomyces hansenii
veromyces marxianus
Pichia anomala
Pichia guilliermondii
, and
Saccharomyces cerevisiae
, had the broadest spectra of action against yeasts that
cause spoilage. These killer yeasts and the toxins that they produce are candidates for further investigation as suppressors of indigenous olive tab
yeast growth. The results confirmed the highly polymorphic expression of the killing activity, with each strain showing different killer activitie
This method may thus be very useful for simple and rapid characterization of yeast strains of industrial interest.
© 2007 Elsevier B.V. All rights reserved.
Killer; Yeast; Brine; Fermentation; Olives
1. Introduction
The presence of yeasts in different kinds of table olive
fermentation is common (
Marquina et al., 1992; Llorente et al.,
1997; Kotzekidou, 1997; Tassou et al., 2002; Durán Quintana
et al., 2003; Hernández et al., 2006
). The predominant yeast
species that have been isolated from Greek-style black olives are
Torulaspora delbrueckii
Debaryomyces hansenii
, and
coccus laurentii
Kotzekidou, 1997
). Other workers (
et al., 1992
) have isolated
Pichia membranifaciens
and related
species as the dominant yeasts from spontaneous fermentations
of olive brines from Portugal as the dominant yeasts. In studies
performed by
Marquina et al. (1997)
with olive brines from
seven locations in Morocco, the most ubiquitous and abundant
species were
T. delbrueckii
Candida boidinii
P. membra-
. Various studies have attributed to the yeast population
the role of contributing to the sensorial characteristics of table
olives (
Garrido et al., 1995; Sánchez et al., 2000
). Since these
micro-organisms could have an important effect on the quality of
this product, their study could help in the selection of starter
cultures to be used in the elaboration of table olives. Yeasts such
Candida krusei
, and
P. membranifaciens
), for example, are not considered spoilage yeasts and
could be used as starter cultures (
Durán Quintana et al., 1979
However, other surveys have associated the presence of
yeasts with different kinds of olive spoilage (
Vaughn et al., 1969
and 1972; Durán Quintana et al., 1979 and 1986
). If film-
forming yeasts are not controlled, they can rapidly oxidize the
desirable acidity in the storage brines of Sicilian-style and
Spanish-type olives.
Vaughn et al. (1972)
found that
vailable online at
International Journal of Food Microbiology 121 (2008) 178

Corresponding author. Tel.: +34 924 286200; fax: +34 924 286201.
E-mail address:
(F. Pérez-Nevado).
(F. Pérez-Nevado).
0168-1605/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
myces cerevisiae
(originally identified as
S. oleaginosus
charomyces kluyveri
, and
Pichia anomala
(originally identified
Hansenula anomala
) can cause softening and gas-pocket
formation in olives. Also, pink yeasts identified as
Rhodotorula minuta
, and
Rhodotorula rubra
slow softening of olive tissue (
Vaughn et al., 1969
). The pre-
sence of various yeast strains of
S. cerevisiae
P. anomala
been related to


(bloater) spoilage in spontaneous
fermentation in black olives (
Durán Quintana et al., 1979
). Other
species that have been related to this alteration are
Pichia sub-
Hansenula subpelliculosa
myces thermotolerans
Kluyveromyces veronae
Candida saitoana
Torulopsis candida
Torulopsis norvegica
D. hansenii
, and
Pichia fermentans
Different methods have been used to control spoilage pro-
duced by yeasts in table olives. The most commonly used
practice in this industry of controlling the pH and salt level of
the brine is insufficient to avoid these problems (
Lamzira et al.,
). Studies with an essential oil prepared from garlic had a
major effect in controlling the yeast population, but the orga-
noleptic characteristics of the product were affected (
et al., 1997
). Trials with pH adjusted to 4, added potassium
sorbate, and
Lactobacillus plantarum
inoculation have found
that bloater spoilage was reduced drastically (
Asehraou et al.,
2002; Lamzira et al., 2005
). The use of yeasts as starter culture
could protect the product against spoilage yeasts, selecting, for
example, killer yeasts which are known to be able to control
spoilage in the preservation of food. Killer yeasts can produce
toxic proteins or glycoproteins (so-called killer toxins) that can
cause death in other sensitive (killer-sensitive) yeast strains. The
killer phenotype appears to be widely distributed within many
yeast genera (
Schmitt and Breinig, 2002
) some of them isolated
from a great variety of fermentation food processes (
Llorente et
al., 1997; Regodón et al., 1997; Gulbiniene et al., 2004
). It is
affected by diverse ambient conditions like pH, including
temperature, and the presence of salt (
Woods and Bevan, 1968;
Llorente et al., 1997; Marquina et al., 2001; Buzzini et al., 2004;
Izgü and Altinbay, 2004
). Moreover, its detection depends
strongly on the sensitive strains used

the killing ability of
different compounds may be underestimated or may even
remain unnoticed depending on the selection of the appropriate
sensitive strain and other experimental conditions. For this
reason, the high variability of the killer phenomenon in nature
provides an exceptional tool for the discrimination of yeasts at
the strain level. Also, the use of these yeasts as a biocontrol
method may improve table olives by reducing the requirement
for salt or such chemical preservatives as sorbic acid or similar.
The aim of the present work was to study the killer activity of
yeast strains isolated from seasoned green table olives. Since
salt addition is a common practice in the production of tra-
ditional fermented table olives, the effect of NaCl addition on
the killer expression was analyzed, as also was the effect of pH.
In addition, the killer activity and sensitivity of isolates to
different killer reference and wild yeast strains was analyzed
with a view to the potential use of these yeasts as biocontrol
agents against spoilage yeasts.
2. Materials and methods
2.1. Yeast strains isolated from green table olives
Seventy-four indigenous yeast strains isolated from seasoned
table olive fermentation by
Hernández et al. (2006)
were tested.
Table 1
List of pre-selected strains used in the survey
Species        Strain  Origin      Species        Strain  Origin
Candida                         Pichia
C. inconspicua
FM47  Olive brine
P. anomala
FM5   Olive brine
C. lusitaniae
FM59  Olive brine
P. anomala
FM14  Olive brine
C. maris
FM15  Olive brine
P. anomala
FM23  Olive brine
C. maris
FM46  Olive brine
P. anomala
FM25  Olive brine
C. maris
FM66  Olive brine
P. anomala
FM30  Olive brine
C. maris
FM67  Olive brine
P. anomala
FM31  Olive brine
C. zeylanoides
FM10  Olive brine
P. anomala
FM34  Olive brine
C. zeylanoides
FM71  Olive brine
P. anomala
FM36  Olive brine
Cryptococcus                     P. anomala
FM37  Olive brine
C. humicola
FM29  Olive brine
P. anomala
FM38  Olive brine
C. humicola
MP11  Fresh olives
P. anomala
FM40  Olive brine
Debaryomyces                    P. anomala
FM52  Olive brine
D. hansenii
FM1   Olive brine
P. anomala
FM58  Olive brine
D. hansenii
FM32  Olive brine
P. anomala
FM62  Olive brine
D. hansenii
FM72  Olive brine
P. anomala
FM73  Olive brine
Olive brine
P. anomala
FM74  Olive brine
K. marxianus
FM2   Olive brine
P. anomala
FM79  Olive brine
K. marxianus
FM6   Olive brine
P. guilliermondii
MP6   Fresh olives
K. marxianus
FM7   Olive brine
K. marxianus
FM11  Olive brine
S. cerevisiae
FM8   Olive brine
K. marxianus
FM12  Olive brine
S. cerevisiae
FM9   Olive brine
K. marxianus
FM19  Olive brine
S. cerevisiae
FM20  Olive brine
K. marxianus
FM24  Olive brine
S. cerevisiae
FM39  Olive brine
K. marxianus
FM26  Olive brine
S. cerevisiae
FM41  Olive brine
K. marxianus
FM27  Olive brine
S. cerevisiae
FM43  Olive brine
K. marxianus
FM69  Olive brine
S. cerevisiae
FM44  Olive brine
K. marxianus
FM78  Olive brine
S. cerevisiae
FM51  Olive brine
S. cerevisiae
FM68  Olive brine
Table 2
List of potential spoilage strains used in the survey
Species        Strain  Origin       Species       Strain  Origin
Candida                          Rhodotorula
C. albicans
FM70  Olive brine
R. glutinis
FM21  Olive brine
C. glabrata
FM50  Olive brine
R. glutinis
MP9   Fresh olives
C. parapsilosis
FM57  Olive brine
R. minuta
FM17  Olive brine
C. rugosa
FM4   Olive brine
C. rugosa
FM16  Olive brine
T. cutaneum
FM3   Olive brine
C. rugosa
FM18  Olive brine
T. cutaneum
FM13  Olive brine
Cryptococcus                      T. cutaneum
FM45  Olive brine
C. albidus
FM22  Olive brine
T. cutaneum
FM54  Olive brine
C. albidus
FM33  Olive brine
C. albidus
MP5   Fresh olives
C. albidus
MP7   Fresh olives
C. laurentii
FM60  Olive brine
C. laurentii
FM63  Olive brine
C. laurentii
FM75  Olive brine
C. laurentii
MP2   Fresh olives
C. laurentii
MP8   Fresh olives
C. laurentii
MP10  Fresh olives
A. Hernández et al. / International Journal of Food Microbiology 121 (2008) 178


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