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- Materials and methods
- Conflict of interest
The key role of yeasts in conducting the alcoholic fermentation has long been recognized (Fleet 1999; Mortimer and Polsinelli 1999), and many factors have been found to influence their growth and metabolism and, as a consequence, the quality of wine (Charoenchai et al. 1998; Torija et al. 2003; Mendes-Ferreira et al. 2004). The temperature is a significant parameter for the course of wine fermentation (Pizarro et al. 2008), as it can strongly impact on yeast growth (Charoenchai et al. 1998), sugar utilization and ethanol production (Torija et al. 2003). Another significant variable is the concentration of fermentable sugars in grape musts (Charoenchai et al. 1998); concentrations from 200 to 300 g l−1 hinder yeast growth and decrease both the maximum population and ethanol yield (D'Amato et al. 2006). Furthermore, nitrogen can often be a limiting factor for yeast growth and sugar attenuation (Mendes-Ferreira et al. 2004). A value <150 mg l−1 of yeast assimilable nitrogen (YAN) in the must is associated with a higher probability of fermentation problems such as sluggish or stuck fermentations (Henschke and Jiranek 1993). In these cases, the addition of diammonium phosphate (DAP) or, to a lesser extent, organic yeast nutrients to nitrogen-deficient musts is a widely used practice (Barbosa et al. 2012).
Ochratoxin A (OTA) is a potent nephrotoxic and hepatocarcinogenic mycotoxin, recognized as a possible human carcinogen by the International Agency for Research on Cancer (Cecchini et al. 2006). The presence of the toxin in musts and wines is due to fungal contamination of the grapes by Aspergillus and Penicillium genera that may develop in both pre- and postharvest or during the phases before the winemaking (Esti et al. 2012).
Decontamination methods based on physical and chemical mechanisms were proposed in the past (Patharajan et al. 2011), but biological removal is considered the best solution, because it is possible to remove mycotoxins without using harmful chemicals and without losses in nutrient value or palatability of decontaminated food (Anly and Bayram 2009). In particular, some authors reported that yeasts were able to remove OTA during winemaking (Caridi et al. 2006; Cecchini et al. 2006; Angioni et al. 2007; Meca et al. 2010; Piotrowska et al. 2013).
Although many papers focused on the role of temperature, sugar and nitrogen on the course of fermentation, few data are available on the effects of DAP, as well as on its combination with temperature and sugar, on the removal of OTA by yeasts (Petruzzi et al. 2014).
Therefore, the main aim of this research was to study the OTA-removing ability of two strains of Saccharomyces cerevisiae and its correlation with temperature, sugar and addition of DAP; the planning of the research, as well as data modelling, was based on a completely randomized design with a factorial arrangement of treatments. As an additional goal, the oenological aptitude of yeasts (fermentative performances and technological and qualitative traits) was assessed to explore the possibility of a selection programme focusing on yeast cultures acting as biological tools to remove the toxin.
- Top of page
- Materials and methods
- Conflict of interest
The level of OTA in wine is markedly lower than that found in the grapes, due to toxin removal throughout the vinification, and especially during the alcoholic fermentation (Paster 2008), as a result of yeast activity (Caridi et al. 2006; Cecchini et al. 2006; Angioni et al. 2007; Meca et al. 2010; Piotrowska et al. 2013) and/or the interaction between OTA and grape constituents (Paster 2008). The adsorption to cell wall is suggested as a mechanism for OTA removal by yeasts (Bejaoui et al. 2004), because harbouring polysaccharides (glucan, mannan), proteins and lipids exhibits numerous different and easy accessible adsorption sites (Cecchini et al. 2006). Recently, Petruzzi et al. (2014) studied the effect of the fermentative conditions on the removal of OTA mediated by two commercial yeasts as well as by a potential starter strain (S. cerevisiae W13). This research focused on the influence of the most important environmental parameter (i.e. temperature) (Pizarro et al. 2008), as well as on the effect of two levels of sugar (200 and 250 g l−1), and the addition of one rate of DAP (300 mg l−1) on OTA-removing ability of two wild S. cerevisiae strains (W28 and W46) using a completely randomized design with factorial arrangements of the treatments to find a possible general trend on the toxin removal. With this scope, a semi-synthetic medium was used to avoid the interference of must components towards OTA.
After 3 days, the strains W28 and W46 were able to remove OTA by c. 70%; this result confirmed the ability of S. cerevisiae to reduce OTA content in different matrices, including synthetic and natural grape juice (Bejaoui et al. 2004), white and red must (Caridi et al. 2006; Cecchini et al. 2006) and model wine (Nunez et al. 2008). Cecchini et al. (2006) found that yeasts removed OTA by c. 50–70% in a red grape must after 36 days of fermentation. Caridi et al. (2006) obtained similar results, as they observed that yeasts reduced OTA by c. 70–80% after 90 days of fermentation. However, both these papers were in vivo studies, whilst our research was performed in vitro, and OTA removal in wine could be attributed to a synergistic effect between yeasts and phenolic compounds, as suggested by the cited authors. On the other hand, Bejaoui et al. (2004) and Nunez et al. (2008) reported higher removal indices (c. 90%), but they used heat-treated cells. Heating may cause changes in the surface properties of cells, for example denaturation of proteins or formation of Maillard reaction products. These products could harbour higher adsorption sites than viable cells with an enhancement of OTA removal (Piotrowska et al. 2013).
The ability of the wild strains W28 and W46 to remove OTA relied upon some important parameters, namely temperature, sugar and addition of DAP, with a mathematical positive effect. Concerning the temperature, our results were in agreement with other findings. In particular, a temperature-dependent mechanism was observed for the aflatoxin B1 (AFB1) removal by Lactobacillus rhamnosus GG and LC-705 (El-Nezami et al. 1998) and for the OTA removal by Metschnikowia pulcherrima MACH1, Pichia guilliermondii M8 and Rhodococcus erythropolis AR14 (Patharajan et al. 2011). Some preliminary experiments performed showed that sugar content did not affect the removal ability of some wild strains (L. Petruzzi, unpublished results), whilst its effect was found positive for a potential starter strain of S. cerevisiae (Petruzzi et al. 2014), thus suggesting a kind of strain-specific variability. Concerning DAP supplementation, the effect of this factor was found to be not significant in S. cerevisiae W13 (Petruzzi et al. 2014), whilst it affected significantly the removal mediated by the wild strains W28 and W46 as a single term, but not in interaction with sugar and/or temperature. This different result could be due to a strong strain variability in the surface properties affecting the binding of OTA to cell wall and probably connected with the oenological performances of the strains. A future trend in this sense could be the use of a robust design (e.g. central composite design) to study the effects of different levels of DAP on both OTA removal and cell surface properties and build a general model able to predict yeast trend under different conditions.
Moreover, this research showed that OTA could be released back into the medium; this kind of phenomenon was found previously under laboratory conditions (Petruzzi et al. 2012) and for the promising functional strain S. cerevisiae W13 (Petruzzi et al. 2014); the extent of OTA release was highly variable, and in some combinations, the phenomenon was completely reversible, with a lower efficiency than that found previously (Petruzzi et al. 2012, 2014).
The release of toxins after their removal was recovered for Lactobacillus amylovorus and Lact. rhamnosus towards AFB1, due to the fact that the bonds between lactobacilli/AFB1 complexes involve weak noncovalent interactions (Peltonen et al. 2001). A similar phenomenon could be associated with the release of OTA by S. cerevisiae because hydrogen bonding, ionic or hydrophobic interaction seems to be related to OTA adsorption mechanism (Cecchini et al. 2006).
Yeasts were also studied in relation to their fermentative performances under the different nutritional and environmental conditions and for their technological and qualitative traits. In particular, the strain W46 showed a higher ethanol and glycerol production and a major ability to achieve dryness than the strain W28. As expected, a high sugar concentration has a detrimental effect on ethanol production (Bely et al. 2008), whereas ethanol yield increased as the temperatures increased; this result was supported by some related findings (Fakruddin et al. 2012). For both the strains, sugars were fully consumed after 10 days of fermentation, whilst the addition of DAP determined a faster consumption of sugars, as reported by Mendes-Ferreira et al. (2004). With regard to glycerol, only the strain W28 confirmed the hypothesis of how yeast responds to increased external sugar concentration by enhanced production and intracellular accumulation of glycerol to counterbalance the osmotic pressure (Bely et al. 2008).
Finally, both the strains showed a high resistance to single and combined stressors typically encountered during alcoholic fermentation. This result was relevant because the ability to carry out vinification is largely influenced by the response of yeast cells towards stress conditions (Carrasco et al. 2001). Moreover, yeasts produced medium amounts of hydrogen sulfide, responsible for the sensorial descriptor of rotten eggs (Tristezza et al. 2012), showed a medium ability to adsorb anthocyanins, thus preserving wine colour (Caridi et al. 2002) and β-d-glucosidase, pectinase and xylanase activities, described as three of the most important extracellular enzymes of S. cerevisiae (Strauss et al. 2001); finally, they did not produce biogenic amines under in vitro conditions, but involved in toxic effects on the human organism and sensory defects in wine (Koutsoumanis et al. 2010).
In conclusion, in this article, two wild strains of S. cerevisiae (W28 and W46) were studied for their OTA-removing ability under different fermentative conditions. Both the strains were able to decrease the toxin amount by up to 70%, with the highest removing effect observed after 3 days at 30°C in the presence of 250 g l−1 sugar and with DAP supplementation (300 mg l−1). However, OTA was released back after 10 days, thus suggesting that the selection of suitable OTA-removing strains should take into account both the extent of the removed toxin and the amount of OTA released back into the medium.