Ochratoxin A content in red wine 245

ISSN 0325-7541
ARTÌCULO ORIGINAL Revista Argentina de Microbiología (2009) 41: 245-250

Fate of ochratoxin A content in Argentinean red wine during a

pilot scale vinification
M. L. PONSONE1, M. L. CHIOTTA1, M. COMBINA2, 3, A. M. DALCERO1, 3, S. N. CHULZE1, 3*

1
Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas, Físico, Químicas y Naturales, Universidad
Nacional de Río Cuarto, Ruta Nacional Nº 36 Km 601, (5800) Río Cuarto, Córdoba; 2Instituto Nacional de Tecnología
Agropecuaria (INTA), Luján de Cuyo, Mendoza; 3 Members of the Research Career of CONICET, Argentina
*Correspondence. E-mail: schulze@exa.unrc.edu.ar

ABSTRACT
The aim of this work was to evaluate the fate of ochratoxin A (OTA) content from must to wine during the red wine
making process in a pilot scale vinification. The study was done using musts obtained from two red grape varieties
(Bonarda and Tempranillo) artificially contaminated with two OTA levels. A duplicate set of tanks of 100 l each was
established for each must (Bonarda and Tempranillo). The fermentations were initiated by inoculation of two Saccha-
romyces spp. strains having different fermentation performance. The must from the Tempranillo variety was spiked
with 6 µg/l of OTA while that from the Bonarda variety with 0.3 µg/l of the toxin. Samples were collected at different
stages of the process. Performance of the alcoholic and malolactic fermentations was monitored. Titratable and
volatile acidity, pH, ethanol, sugar and SO2 concentrations were determined following standard methods proposed by
the Office International de la Vigne et du Vin (OIV). OTA analysis was done by HPLC. Detection and quantification
limits were 0.01 and 0.1 ng/ml, respectively. The OTA levels during the vinification trials dropped to an average of
about 86.5%. The type of Saccharomyces strains used showed no effect on toxin reduction.
Key words: ochratoxin A, wine, grapes, vinification process

RESUMEN
Evolución del contenido de ocratoxina A en vinos tintos argentinos durante el proceso de vinificación a
escala piloto. El objetivo del presente trabajo fue evaluar la evolución del contenido de ocratoxina A (OTA) en
mostos durante un proceso de vinificación a escala piloto. Se utilizaron mostos de dos variedades de uvas tintas
(Bonarda y Tempranillo) contaminados artificialmente con dos niveles distintos de OTA. El ensayo fue llevado a cabo
por duplicado en tanques de fermentación de 100 l cada uno. La fermentación se inició mediante la inoculación de
dos cepas de Saccharomyces spp. con diferentes características fermentativas. El mosto de la variedad Tempranillo
fue contaminado con 6 µg/l de OTA y el mosto de la variedad Bonarda con 0,3 µg/l de la toxina. Se colectaron
muestras durante los diferentes estadios del proceso de vinificación. Se estableció el avance de dicho proceso sobre
la base de la evolución de las fermentaciones alcohólica y maloláctica. Se determinó la acidez total y volátil, el pH y
el contenido de etanol, de azúcar y de SO2 siguiendo los protocolos estándares propuestos por la Oficina Internacio-
nal de la Vid y el Vino (OIV). El contenido de OTA se evaluó por HPLC. Los límites de detección y cuantificación
fueron 0,01 y 0,1 ng/ml, respectivamente. Los niveles de OTA disminuyeron alrededor del 86,5% al final del proceso
de vinificación. El tipo de cepa de Saccharomyces spp. utilizada no tuvo efecto sobre la reducción de OTA.
Palabras clave: ocratoxina A, vino, uvas, proceso de vinificación

INTRODUCTION toxic effects on animals (13, 14). The International

Argentina is a major producer and exporter of wine
in the world. The wine industry plays an important role
in the Argentinean economy. Also, Argentina is con-
cerned with producing wine with both good quality
standards and absence of fungal natural contaminants
such as ochratoxin A (OTA). OTA is a mycotoxin pro-
duced by several species of Penicillium and Aspergillus
in different food commodities, including grapes (12).
The toxin shows nephrotoxic, immunotoxic and neuro-
Agency for Research of Cancer (IARC) has classified
OTA as a group 2 B carcinogen based on toxicity on
rats (13).
The presence of OTA in wine was reported for the first
time in 1996 (25). Later surveys in Europe, Australia, and
South America have shown OTA occurrence in wine and
grape products (3, 6, 7, 12, 18). Studies carried out in
different countries, including Argentina have demonstrated
that Aspergillus niger aggregate and Aspergillus carbonarius
are the main prevalent species on grapes (2, 6, 19).
246
Wine has been shown to be a significant contributor
for human OTA exposure together with cereals, coffee,
beer and raisins (14). During the wine making process,
OTA needs to be considered as a possible contaminant,
since OTA levels in wine have been shown to be related
to pre-harvest grape contamination.
The European Community has fixed a maximum allowed
limit of 2.0 µg/l of OTA for wines, grape must and grape
juice intended for direct human consumption (8).
During the winemaking process, the Argentinean com-
panies use different Saccharomyces spp. strains as start-
ers. The strain selection depends on the winemaker’s ob-
jectives.
Information about OTA persistence and transforma-
tion during processing will be useful to establish quality
control systems based on hazard analysis and critical
control points (HACCP) and the implementation of cor-
rective actions during the vinification process.
Studies on the fate of OTA during the vinification proc-
ess have been conducted in different countries using
microvinification trials (9, 15, 16, 20). The aim of this
study was to evaluate the fate of OTA during vinification
at a pilot scale for the first time in Argentina, in order to
assess the scaling effect on OTA reduction, using two
red grape varieties commonly used in Argentina for red
wine production, and two types of Saccharomyces spp.
strains.
MATERIALS AND METHODS
Winemaking trials. Winemaking trials were performed dur-
ing the 2006 vintage using “Tempranillo” and ”Bonarda” red grape
varieties. The pilot - scale vinification was carried out employing
technology currently used in the wine industry in Argentina.
Chemical composition of the musts to initiate the fermentation
process is shown in table 1. Due to the fact that OTA natural
grape contamination was not observed, musts for vinification
were spiked with two OTA levels (Sigma, St Luis, USA) in order
to evaluate the effects of high and low contamination levels
during the fermentation process. Tempranillo and Bonarda musts
were artificially contaminated with 6 µg/l and 0.3 µg/l of OTA
respectively. These levels were chosen arbitrarily, considering
higher and lower levels than the maximum limit established by
EU (2 µg/l).
Table 1. Chemical composition of musts used for vinification
Musts Total Acidity Free
(g/l SO2
Tartaric (mg/l)
Acid)
Tempranillo 3.59 16.00
Bonarda 4.13 8.96
*EAN: Easily assimilable Nitrogen
Revista Argentina de Microbiología (2009) 41: 245-250
In order to evaluate the effect of the yeast strain used as
starter in the fermentation process on the ochratoxin A dynamic,
two strains of Saccharomyces, S. bayanus EC1118 and S.
cerevisiae ICV-D80, were assayed: The former shows good per-
formance for barrel fermentation, it ferments well at low tem-
peratures and flocculates well with very compact lees. Besides,
it produces a lot of SO2 (up to 30 ppm) so it can inhibit malolactic
fermentation. S. cerevisiae ICV-D80 can ferment high sugar and
polyphenol musts. Under proper nutrition conditions, aeration
and fermentation temperatures below 28 °C, the strain ferments
up to 16% alcohol. S. cerevisiae ICV-D80 also brings high fore-
mouth volume, big mid-palate mouthfeel and intense fine grain
tannin to red wines.
The fermentation trials were carried out in four 100 l tanks as
follows:
i. Must from Tempranillo grapes inoculated with a commer-
cial S. bayanus EC1118.
ii. Must from Tempranillo grapes inoculated with a commer-
cial S. cerevisiae ICV-D80.
iii. Must from Bonarda grapes inoculated with a commercial
S. bayanus EC1118.
iv. Must from Bonarda grapes inoculated with a commercial
S. cerevisiae ICV-D80.
Vinification trials were started by crushing-destemming grapes,
producing must pomaces (skins and seeds). 50 mg/l of SO2 was
added to each must, and inoculated with 2 x 106 CFU/ml of the
commercial Saccahromyces spp. strains. The temperature in
each tank was kept in the range of 24-28 °C during maceration
and alcoholic fermentation (AF) for 7-10 days. After completion
of alcoholic fermentation, a first settling and racking was carried
out to remove the pomaces from the wine. Then, malolactic
fermentation (MLF) took place spontaneously, due to lactic acid
bacteria present in the wine. After a second racking to remove
the yeast sediment, the wine was stabilized for bottling and
storage.
Sampling of must and wine during winemaking. The sam-
ples were taken from each tank, in triplicate, after the pumping-
over to obtain homogeneous samples during fermentation, at
the stages indicated in Figure 1.
OTA analysis. The OTA content was determined following
the methodology proposed by Visconti et al (1999). In brief, must
and wine were diluted with water solution containing PEG (1%)
and NaHCO3 (5%), mixed, and filtered to remove particulate
matter. Ten ml portion was taken and added to an immunoaffinity
column (OchraTestTM; Vicam, Digen Ltd, Oxford, UK). The col-
umn was washed with 10 ml PBS containing 1% Tween 20 and
then with 10 ml double distilled water. OTA was eluted from the
column with 1.5 ml of methanol (HPLC grade), at a flow rate of
1-2 drops per second.
The HPLC apparatus system was a Hewlett-Packard
(Waldbronn, Germany) chromatograph with a loop of 50 ml,
Total Brix Bé pH EAN*
SO 2 T (mg/l)
(mg/l)
54.40 24.70 13.90 3.50 169.4
44.80 21.50 12.45 3.50 126.0
Ochratoxin A content in red wine
equipped with a spectrofluorescence detector (excitation, 333
nm; emission, 460 nm) and a C18 column (150 x 4.6 mm, 5 µm
particle size; Supelcosil LC-ABZ, Supelco, Bellefonte, PA, USA),
connected to a precolumn (20 x 4.6 mm, 5 µm particle size;
Supelguard LC-ABZ, Supelco). The mobile phase was pumped
Figure 1. Red winemaking flow-chart, showing the sampling
stages chosen for OTA analysis during the vinification process.
Figure 2. Evolution of L-Malic Acid content through a pilot scale vinification process (n=3), at each time analyzed (T: Tempranillo; B:
Bonarda; EC11 18: S. bayanus EC1118; D80: S. cerevisiae ICV-D80).
247
Table 2. Recoveries from blank musts fortified with ochratoxin
A at different levels (n = 3)
Spicking level Mean Recovery (%) ± SD*1 R.S.Dr (%)*2
ng/ml
2 107.6 ± 4.0 3.7
5 100.6 ± 3.0 2.9
10 100.5 ± 4.7 4.6
Mean of means 104.7 ± 3.9 3.73
*1SD = Standard Deviation
*2RSDr (%) = Relative standard deviation intra-laboratory.
at 1.0 ml/min, and consisted of an isocratic system as follows:
99:99:2 acetonitrile, water and acetic acid respectively. OTA was
quantified on the basis of HPLC fluorometric response compared
with OTA standard (purity > 99%; Sigma Aldrich Co., St Louis,
MO, USA). The lowest limit of detection was 0.01 ng/ml and the
quantification limit was 0.1 ng/ml.
Recovery experiments. Recovery experiments were per-
formed in triplicate by spiking OTA free samples of must with
OTA levels of 2, 5 and 10 ng/ml. (Table 2).
Physico-chemical analysis. Progress of the alcoholic fer-
mentation (AF) was monitored daily by decline in total soluble
solids using a gravimetric method as density (Bé). pH, total sugar,
ethanol (%), SO2, volatile acidity and total acidity were deter-
mined according to the standard methods of OIV (1990) (17).
MLF was followed by malic acid detection with a commercial
ELISA kit (Roche®) following the manufacturer’s protocol.
Statistical analysis. To determine the significance of OTA
reduction at the end of the process, an statistical analysis of the
OTA reduction percentage in bottled wines was carried out by
the analysis of variance (ANOVA; p<0.001), followed by a Tukey
test.
RESULTS AND DISCUSSION
During the vinification process the performance of
malolactic fermentation was monitored (Fig. 2). The
characteristics of the wines were evaluated; the results
are shown in table 3. The mean ethanol content was
14.1% (v/v) with a minimum of 13.2% (v/v) and a maxi-
mum of 15% (v/v). The levels of OTA in the wines after
248 Revista Argentina de Microbiología (2009) 41: 245-250

the vinification trial are reported in Figure 3. The mean
OTA content, reached 70 ng/l in the musts contaminated
with 0.3 µg/l, while in those contaminated with 6 mg/l the
mean final value was 903 ng/l.
This study showed the fate of OTA concentration
throughout the vinification process at a pilot scale (from
must to wine). The step that showed the first OTA level
reduction, 62% and 46% for must with high (Tempranillo)
and low (Bonarda) levels of OTA contamination, respec-
tively, occurred soon after conditioning the contaminated
musts with additional reductions upon racking from juice
and gross lees. From this point to the end of the alcoholic
fermentation process, the mean reduction of OTA reached
84 and 89% for wines obtained from Tempranillo and
Bonarda, respectively (Fig. 3 a, b). During fermentation
(either alcoholic or malolactic), the OTA content decreased
in the liquid fraction. This reduction was then further rein-
forced after the final stages of the process (Table 4). There
were highly significant differences (p < 0.001) between the
OTA content of the grape must and wines for all the
trials evaluated. Both yeast strains were able to noticeably
and significantly reduce the initial OTA content, but in the
Tempranillo grape variety no significant differences in the
behaviour between the strains used were observed. In
this particular case, the lack of differences among OTA
reduction according to the yeast strain used agrees with
data obtained by Scott et al. (21) and Caridi et al. (5),
which showed no differences in OTA reduction by sev-
eral Saccharomyces spp. strains evaluated. The differ-
ence in the behaviour between the yeast strains observed
in Bonarda musts could be explained by the low level of
OTA used at the beginning of the process.
The significant reduction of OTA during the vinification
process could be explained by the partition of the toxin
between the liquid and the solid phase, due to an exten-
sive adsorption of OTA onto the solid parts of the grapes

Table 3. Chemical composition of final wines

Wine Total Acidity Volatil Acidity Sugar Content Free SO2 Total SO2 Ethanol
g/l g/l (g/l) (mg/l) (mg/l) (% v/v)
Tartaric Acid Acetic Acid

Tempranillo
S. bayanus 5.625 0.305 1.88 40.96 64.00 14.8
ICV- EC1118
Tempranillo
S. cerevisiae D80 5.620 0.275 5.90 35.85 57.60 15.0
Bonarda
S. bayanus 6.000 0.330 1.80 34.60 57.60 13.2
ICV-EC1118
Bonarda
S. cerevisiae D80 5.770 0.310 2.18 38.40 55. 04 13.4

Figure 3. Evolution of ochratoxin A content through the pilot scale vinification process in must from Tempranillo and Bonarda grapes.
The method for evaluation of OTA contamination through the process showed a mean recovery of 101.3% evaluated in the spiking
range (n=3), at each time analyzed (T: Tempranillo; B: Bonarda; EC11 18: S. bayanus EC1118; D80: S. cerevisiae ICV-D80).
Ochratoxin A content in red wine
Table 4. Removal of OTA in wines obtained from two grape varieties using two
different commercial strains of Saccharomyces after 30 days of fermentation
(p<0.001).
Wine Range of removed
OTA (%)
Tempranillo
S. bayanus ICV- EC1118 82-84
Tempranillo
S. cerevisiae D80 85-88
Bonarda
S. bayanus 99-100
ICV-EC1118
Bonarda
S. cerevisiae D80 75-79
*SD: Standard deviation
and yeast lees as it has been demonstrated by other re-
searchers (10, 16). An adsorption mechanism onto
biomass surface could be explained by the overall nega-
tive charge in the cell walls and the acidic nature of OTA
(4). Also, Fernandes et al. (10), Gambuti et al. (11) and
Leong et al. (16) working on vinification trials reported a
decrease in wine OTA content mainly due to the removal
of OTA by adsorption onto suspended solids in musts
and wines. Solfrizzo et al. (22) and Visconti et al. (24)
reported that, on average, between 70-95% of OTA is
retained in pressed grape pomace during micro
vinification trials.
In general, all the vinification trials showed an average
86.5% of OTA reduction. Similar results were obtained
by Leong et al. (16) during micro-vinification trials of
grapes with an initial OTA concentration, ranging from 2
to 114 µg/kg. Under our experimental conditions, the OTA
reduction was dependent on the initial OTA level in the
musts.
Also, it was observed that during fermentation (either
alcoholic or malolactic) the OTA content decreased in
the liquid fraction. These data agree with Fernandes et
al. (9), who reported a decrease in the OTA content from
must to wine.
Since the risk of OTA contamination increases during
the grape ripening, a good sanitary stage of grapes at
harvest time will be essential to prevent OTA wine con-
tamination. Therefore, this stage will be one of the criti-
cal control points in the wine production chain as it was
postulated in a previous study (1).
The results showed that under the conditions simulat-
ing the vinification process in Argentina, OTA levels can
be reduced by around 86.5% during the process.
Acknowledgements: This work was supported by a grant
from ANPCyT (PICT 25522) and SECyT Secretaría de Ciencia
249
Media
percentage of removed OTA
(n=3) ± SD*
83 ± 0.82(b)
86 ± 1.25 (b)
100 ± 0.47(a)
77 ± 1.63 (c)
y Técnica, Universidad Nacional de Río Cuarto). We also thank
CONICET (L. Ponsone is a CONICET fellow).
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