ARTÍCULO ORIGINAL

Fate of ochratoxin A content in Argentinean red wine during a pilot scale vinification

M. L. Ponsone¹, M. L. Chiotta¹, M. Combina^{2, 3}, A. M. Dalcero^{1, 3}, S. N. Chulze^{1, 3*}

¹Departamento 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;
³Members 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 Saccharomyces 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 Internacional 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

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 concerned with producing wine with both good quality standards and absence of fungal natural contaminants such as ochratoxin A (OTA). OTA is a mycotoxin produced by several species of Penicillium and Aspergillus in different food commodities, including grapes ⁽¹²⁾. The toxin shows nephrotoxic, immunotoxic and neurotoxic effects on animals ^{(13, 14)}. The International 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)}.
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 companies use different Saccharomyces spp. strains as starters. The strain selection depends on the winemaker's objectives.
Information about OTA persistence and transformation during processing will be useful to establish quality control systems based on hazard analysis and critical control points (HACCP) and the implementation of corrective actions during the vinification process.
Studies on the fate of OTA during the vinification process 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 during 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

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 performance for barrel fermentation, it ferments well at low temperatures 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 foremouth 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 commercial S. bayanus EC1118.
ii. Must from Tempranillo grapes inoculated with a commercial 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 samples were taken from each tank, in triplicate, after the pumpingover to obtain homogeneous samples during fermentation, at the stages indicated in Figure 1.

Figure 1. Red winemaking flow-chart, showing the sampling stages chosen for OTA analysis during the vinification process.

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 (OchraTest^TM; Vicam, Digen Ltd, Oxford, UK). The column 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, 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 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 performed in triplicate by spiking OTA free samples of must with OTA levels of 2, 5 and 10 ng/ml. (Table 2).

Table 2. Recoveries from blank musts fortified with ochratoxin A at different levels (n = 3)

Physico-chemical analysis. Progress of the alcoholic fermentation (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 determined 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; ρ<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 maximum of 15% (v/v). The levels of OTA in the wines after 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.

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).

Table 3. Chemical composition of final wines

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).

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, respectively, 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 reinforced after the final stages of the process (Table 4). There were highly significant differences (ρ < 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 several Saccharomyces spp. strains evaluated. The difference 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.

Table 4. Removal of OTA in wines obtained from two grape varieties using two different commercial strains of Saccharomyces after 30 days of fermentation (ρ<0.001).

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 extensive adsorption of OTA onto the solid parts of the grapes and yeast lees as it has been demonstrated by other researchers ^{(10, 16)}. An adsorption mechanism onto biomass surface could be explained by the overall negative 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 contamination. Therefore, this stage will be one of the critical control points in the wine production chain as it was postulated in a previous study ⁽¹⁾.
The results showed that under the conditions simulating 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 y Técnica, Universidad Nacional de Río Cuarto). We also thank CONICET (L. Ponsone is a CONICET fellow).

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Recibido: 10/03/09
Aceptado: 22/09/09