Abstract
The effects of common winemaking procedures on ochratoxin A (OTA) concentrations in the Portuguese wine Vinho Verde were studied. Natural contamination of grapes was not observed, so grapes for vinification were inoculated with Aspergillus carbonarius. Ochratoxin A concentration in these grapes ranged from 0.43 to 7.48 μg/kg. Vinification consistently reduced OTA concentrations in wine independent of the initial OTA concentration in grapes. Mean carry-over of OTA from grapes to wine was 8.1% (w/w) after malolactic fermentation, even without use of enological adjuvants (fining agents). Reduction in OTA was associated with removal of spent fractions during winemaking, such as wine lees after fermentation or sediment after racking. OTA concentrations were higher in these fractions than in the original grapes. Degradation by yeast or bacteria was not observed under the tested conditions.
Ochratoxin A (OTA) is a carcinogenic mycotoxin produced by various fungus species of Aspergillus and Penicillium and is a known contaminant of various foods. Aspergillus ochraceus is most associated with production within the Aspergillus genus; however, A. carbonarius is associated with wine contamination (Cabañes et al. 2002, Serra et al. 2003). Ochratoxin A in wines was first reported by Zimmerli and Dick (1995) and has been further confirmed in different countries (Burdaspal et al. 1999, Pietri et al. 2001, Sage et al. 2002). In Portuguese wines, OTA was found in Vinho Verde among others, although at lower concentrations than recommended limits (Festas et al. 2000, Ratola et al. 2004). The significance of these findings was recognized in 2005 when the European Union set a maximum permitted concentration of 2 μg/kg (Commission Regulation [EC] no. 123/2005, 26 Jan 2005).
Grape products originating from southern Europe and northern Africa (Mediterranean climates) are more affected by ochratoxin A than those from more temperate regions of central Europe (Otteneder and Majerus 2000, Battilani and Pietri 2002). Higher concentrations of OTA in red than white wines were interpreted as a consequence of differences in wine processing. Furthermore, a greater abundance of OTA-producing fungi in southern European grapes is one possible cause of the higher incidence of OTA in these regions (Varga and Kozakiewicz 2006), although a direct correlation has not yet been established (Serra et al. 2006).
It has been hypothesized that grape processing may reduce the concentration of OTA (Fernandes et al. 2003), although this subject has not been studied extensively. However, such reductions have been demonstrated for other undesirable compounds in wine such as pesticides (Cabras and Angioni 2000). Several physical, chemical, and microbiological methods are proposed for the removal/reduction of mycotoxins from foods and feeds, but few have a practical application. Detoxification of OTA from wines has also been attempted. Removal of mycotoxins by adsorption with fining agents has been studied most frequently (Dumeau and Trioné 2000, Castellari et al. 2001), and most fining agents had little effect on the removal of OTA at the dosages currently used in wine production. Active charcoal was most effective, but only at relatively high dosages that cause severe damage to wine characteristics. Microbiological methods were proposed (Abrunhosa et al. 2002, Bejaoui et al. 2004, 2005), but applicability has not yet been established. The effect of other vinification steps has not been studied sufficiently (Gambuti et al. 2005, Ratola et al. 2005).
The Vinho Verde region of northern Portugal (wine-growing zone CIa) is well known for wine production. The region has a sub-Mediterranean climate that is a variant of the temperate Mediterranean climate and with greater humidity because of Atlantic Ocean influences. Wines produced in this region have low sugar and alcohol (<0.3 g/L sugar and 9% alcohol strength) and are slightly more acidic than common table wines. We assessed changes in OTA throughout vinification of Vinho Verde by inoculating grapes with an OTA-producing A. carbonarius and measuring OTA concentrations at each step to determine critical control points in the winemaking process.
Materials and Methods
Grapes.
Grapes from the Vinho Verde region were collected in Estação Vitivinícola Amândio Galhano (EVAG), a research and experimentation centre of Comissão de Viticultura da Região dos Vinhos Verdes (wine board). The two most representative varieties of grapes were collected: Vinhão (red), which was used in all experiments, and Loureiro (white), which was only used for experiments where fining agents were used to aid clarification.
Grape contamination.
Grapes were spiked with an A. carbonarius (strain MUM 03.59) spore suspension (103 spores/mL) that had been isolated from grape must and preserved in the MUM culture collection (www.micoteca.deb.uminho.pt). Treatments were incubated in controlled temperature chambers at 25°C for 3 to 6 days to achieve different degrees of contamination. Four lots of red grapes were prepared (A to D) with different incubation times to obtain different concentrations of OTA. Humidity control was not attempted. Lots A and B each contained 20 kg of grapes and were used in two independent vinification trials. Lots C and D each consisted of 60 kg of grapes and were divided into three sublots. Each sublot was used for vinification as an independent experiment.
Vinification experiments.
Vinification trials were performed according to various technologies used for commercial production of red Vinho Verde with the Vinhão variety. Four trials were prepared (A to D), with trials C and D performed in triplicate, as noted above.
Red wine vinification had six main steps: grape crushing, alcoholic fermentation (AF), pressing, natural wine clarification, malolactic fermentation (MLF) with natural wine settling, and clarification of wine aided by enological adjuvants (i.e., the fining agents). The red grapes were crushed, yielding a mixture of a liquid (the must) and solids (pomace: skins and seeds). Crushed grapes were transferred to a stainless-steel vat, where a sulfur dioxide-generating agent was added (72 mg SO2/kg grapes). Samples were withdrawn in triplicate after crushing.
Alcoholic fermentation was started immediately by adding Saccharomyces cerevisiae (strain QA23). Each vat was inoculated with 20 g/hL of lyophilized yeast, and fermentation took place with temperature control (18 ± 1°C). After alcoholic fermentation, the mixture was racked to remove wine lees (pomace and biomass) from fermented must (i.e., wine), the must was further clarified by natural settling in a controlled temperature chamber (two weeks at 4°C). After racking and clarification, samples were withdrawn in triplicate. The weight of wine and wine lees after alcoholic fermentation and the weight of wine and sediment after clarification were measured to determine mass balance.
Following clarification, Oenoccus oeni (strain Enoferm Alpha, Proenol, Portugal) was added for MLF (1 g/hL of lyophilized bacteria). This fermentation stopped when all malic acid was converted to lactic acid, after which the wine was allowed to clarify by natural settling in a controlled temperature chamber (two weeks at 4°C). Racking was performed after clarification to separate the wine lees from wine, before fining agents were used. At this stage, samples were withdrawn in triplicate, and the weight of wine and wine lees after MLF was measured.
Red wines were combined, and clarification with fining agents was performed as a “parallel” experiment. A white wine (Loureiro variety) containing OTA (again obtained after spiking grapes with A. carbonarius) was also used in this experiment. Commercial samples of common fining agents for Vinho Verde production were used at recommended commercial concentrations: bentonite, potassium caseinate (casein), and polyvinylpolypyrrolidone (PVPP) for white wines, and gelatin and egg albumin for red wines. For these experiments, 200 mL of wine was placed in a glass tube, and the adjuvant was added at the appropriate doses. After settling, a sample of the clarified wine was taken from the top for analysis. The settled layer with the fining agent on the bottom of the glass tube was also collected for analysis. As a control, duplicates of the same wine sample were allowed to clarify without any adjuvant. Several samples were withdrawn throughout each vinification trial, with samples collected in triplicate before and after each step. These samples were either liquid or a mixture of liquid and solids.
Ochratoxin A analysis.
Ochratoxin A concentrations of solid samples were determined differently from liquid samples. Clarified musts or wines were analyzed according to the method of Visconti et al. (1999). Solid samples such as whole grapes or wine lees were analyzed according to Serra et al. (2004). Both methods used OchraTest immunoaffinity columns (VICAM, Boston, MA) and a Visiprep SPE vacuum manifold (Supelco, Bellefonte, PA) to control the flow rate.
The samples were analyzed using reversed-phase HPLC equipped with a FP-920 fluorescence detector (330 nm excitation wavelength; 460 nm emission wavelength) (Jasco, Tokyo, Japan). Chromatographic separations were performed on a C18 column (Spherisorb ODS2, 4.6 mm x 250 mm, 5 μm) (Waters, Milford, MA) fitted with a precolumn with the same stationary phase. The mobile phase consisted of acetonitrile:water:acetic acid (99:99:2, v/v), with a flowrate of 1.0 mL/min and injection volume of 100 μL.
The OTA standard was supplied by Sigma Chemical Co. (St. Louis, MO). Samples were assumed to contain OTA if they yielded a peak at a retention time similar to the OTA standard peak (~12 min). The limit of detection was 0.004 μg/kg (Serra et al. 2004). The OTA peak had been derivatized in a previous experiment and the reaction product determined to confirm its identity (Serra et al. 2004). In addition, the sample was spiked with a standard to confirm the mycotoxin peak.
Results
The natural concentration of OTA in grapes was below the detection limit of the analytical system. Grapes were inoculated with an ochratoxigenic strain of A. carbonarius, and OTA was allowed to accumulate for three to six days. Four vinification experiments were prepared on grapes with an initial OTA concentration of 0.43 μg/kg (trial A), 0.96 μg/kg (trial B), 4.12 μg/kg (trial C), and 7.48 μg/kg (trial D). A clear reduction in wine OTA concentration was observed during vinification (Figure 1⇓). The mean overall carry-over of OTA from grapes to the wine after alcoholic fermentation, clarification/natural settling, and MLF was 31.8%, 10.9%, and 8.1%, respectively, of the initial OTA in grapes (Table 1⇓).
Recovery of OTA (%) throughout four vinification trials: after alcoholic fermentation [AF], after wine clarification, and after malolactic fermentation [MLF], compared with the initial concentration in grapes.
Evolution of OTA concentration in red wine production, from whole grapes to clarified wine after malolactic fermentation (MLF). Initial OTA concentration for trials A, B, C, and D was 0.43, 0.96, 4.12, and 7.48 μg/kg, respectively.
The concentration of OTA in vinification by-products was also determined (Figure 2⇓). By-products generated during alcoholic and malolactic fermentation (wine lees after AF and wine lees after MLF, respectively) and during natural settling (sediment after clarification) have an OTA concentration that is higher than the original concentration in grapes. Overall mass balances were performed to determine whether OTA was being removed or degraded during vinification (Table 1⇑). Compared to grapes, the solids recovered after alcoholic fermentation (wine lees) had a mean OTA content of 50.4%. After natural settling of this wine (sediment) and MLF (wine lees after MLF), OTA content was 17.6 and 3.0%, respectively.
OTA in grapes and sequestered in red vinification byproducts: wine lees after alcoholic fermentation (AF), sediment after clarification, and wine lees after malolactic fermentation (MLF). Initial OTA concentration for trials A, B, C, and D was 0.43, 0.96, 4.12, and 7.48 μg/kg, respectively.
The use of fining agents to aid clarification of wines can reduce the concentration of OTA as much as 34% (Table 2⇓). The reduction was dependent on the fining agent, with some products being totally ineffective (e.g., PVPP). The settled layer on the bottom of the glass tube contained high OTA concentrations of up to 10.8 μg/L.
Effect of different fining agents on OTA concentration after clarification in control samples, in clarified wine, and in the sediment.
Discussion
The mass balance performed in each vinification trial revealed that the decrease in wine OTA was due predominantly to the removal of OTA by adsorption onto suspended solids in musts and wines. These by-products resulted in removal of 71% of the OTA, which corroborates the findings of other authors (Gambuti et al. 2005), who reported a decrease in OTA of ~80% when wine was filtered through a 0.45-μm membrane, indicating that a significant fraction of OTA is adsorbed onto suspended solids and is removed with them. The concentration of OTA in the tested by-products was always higher than in the raw grape material.
The mass balance of the ethanol fermentation vessel demonstrated that almost all the OTA in the grapes was accounted for in the resulting wine and wine lees (82.4% ± 14.4) (Table 1⇑). The difference from a hypothetical 100% may be from error in the initial OTA determination (Ratola et al. 2004). These authors report an uncertainty of OTA determination by current methodology of about 30% when calculated according to published guidelines (EURACHEM/CITAC 2000). However, degradation of OTA by yeast cannot be discounted. Some authors report that the decrease in OTA during alcoholic fermentation may be affected by yeast strain, although no degradation products have been observed (Bejaoui et al. 2004).
The change of OTA during wine clarification and MLF clearly shows that all the reduction in OTA in wine was due to the removal of this mycotoxin by the solid fraction (Table 1⇑). The overall recovery of OTA in both of these steps was greater than 90%. This last observation indicates that the bacterium used in these experiments could not metabolize OTA, although other strains are reported to do so (Battilani et al. 2003).
The use of fining agents to aid wine clarification may also contribute to the detoxification of wines by removing OTA (Castellari et al. 2001, Gambuti et al. 2005). However, this approach may reduce the value of the final wine by removing color, aroma, flavor, or other desirable wine characteristics (Gambuti et al. 2005). In the present work, control samples showed a degree of OTA removal comparable to wines clarified with fining agents for white wines treated with casein and PVPP. The most efficient removal was in red wines treated with egg albumin and gelatin. After this last clarification step, the settled layer with the fining agent was tested for OTA concentration, which was, in some cases, much higher than in the clarified wine. A possible explanation may be that at wine pH (~3.5), these proteins have a net positive charge, which could interact with the partially dissociated carboxyl group of OTA. However, the interactions between OTA and fining agents are much more complex (Castellari et al. 2001).
Conclusions
The reduction in OTA concentration during vinification is significant. The distribution of OTA between must and pomace after grape crushing favors the pomace fraction. It is common in white wine vinification to separate pomace from must after crushing, which may explain the consistent reports that white wines are less contaminated with OTA than red. Furthermore, the reduction in OTA observed during vinification clearly explains why juices are usually more contaminated than wines and does not require OTA degradation during primary fermentation.
Data collected from these trials support the conclusion that a reduction in OTA occurs during vinification and that such a reduction is mainly due to its adsorption onto suspended solids. According to this data, no evidence of OTA degradation into other compounds due to the metabolic activity of S. cerevisiae (QA23) was observed. However, it is known that the use of selected yeast or bacteria enhances the reduction of OTA to ochratoxin alpha (a breakdown product of OTA). Of concern was the finding that by-products from vinification with high levels of OTA are generated and that use of these by-products to recover other products such as tannins should consider this risk of contamination by ochratoxin A.
Footnotes
Acknowledgments: The authors are grateful for the support of the EC Quality of Life Programme, key action 1 on Food, Nutrition, and Health; contract QLK1-CT-2001-01761, Wine-Ochra Risk, and the Instituto Nacional de Investigação Agrária e das Pescas, through the Programme AGRO, contract 255.
The authors acknowledge the English revision by Russell Paterson.
- Received May 2006.
- Revision received August 2006.
- Copyright © 2007 by the American Society for Enology and Viticulture
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