The Effects of Co-Fermentation with Saccharomyces and Non-Saccharomyces Yeasts

Key Words: Saccharomyces, Non-Saccharomyces, Sauvignon blanc, Aroma Authors: Utku Gundogdu, Kaitlyn Sulenski, Xinyi Zhang, Houry Zournajian Introduction Winemaking has evolved over the years in many ways. One specific aspect is the usage of yeasts; traditionally, wine was made with native yeasts on the grapes or in the cellar (Moreira et al., 2011). More recently, pure yeasts have been cultured and sold in mass quantities to wineries. The most common yeast culture is Saccharomyces cerevisiae (Sadoudi et al., 2012). A new development of coinoculations of non-Saccharomyces with Saccharomyces yeasts has become of interest in the winemaking world (Medina et al., 2012). Some believe that the commercialization of Saccharomyces yeast is leading to less diverse and complex wines (Sadoudi et al., 2012). To diversify wines, people want to use non-saccharomyces yeasts (Ciani et al., 2006). The issue with nonSaccharomyces yeast is their tolerance to wine conditions. Some yeast do not have the proper alcohol tolerance, which can lead to stuck fermentations. Therefore, with the help of a Saccharomyces yeast strain, a non-Saccharomyces co-fermentation can go to completion with positive results. There are some complications with cofermentations including nutrient availability and yeast-yeast interactions. In co-fermentations, the rate of fermentation is notably slower than a Saccharomyces cerevisiae monoculture (Andorr et al., 2010; Ciani et al., 2006). This slower rate can be related to a competition for nitrogen compounds (Ciani et al., 2006; Medina et al., 2012) and vitamins (Medina et al., 2012). This can be avoided through additions of nitrogen compounds or vitamins (Medina et al., 2012). There are also negative yeast-yeast interactions through killer characteristics (Sadoudi et al., 2012; Medina et al., 2012). Non-Saccharomyces Yeasts Non-Saccharomyces species have great potential to introduce appealing characteristics to wine that may improve its organoleptic quality and play relevant roles in the metabolic impact and aroma complexity of the final product. Mixed inoculation of non-Saccharomyces yeasts and S. cerevisiae is of interest to the wine industry for technological and sensory reasons. The major non-Saccharomyces yeasts present during alcoholic fermentation are of genera Hanseniaspora (previously known as Kloeckera), Pichia, Candida, Metschinikowia, Kluyveromyces, Debaruomyces, Saccharomyces, Zygosaccharomyces, Saccharomycodes, Torulaspora, Dekkera, Lachancea and Schizosaccharomyces, which originate from the microbial communities of the grape berry and the microbial communities of the winery environment (Fleet, 2008). Hanseniaspora, Candida, Pichia and Metschinikowia initiate spontaneous alcoholic fermentation of the juice, but are very soon overtaken by the growth of S. cerevisiae, which dominates the mid to final stages of the process (Fleet, 2008). Many studies in various wine regions of the world have now confirmed the important contribution that non-Saccharomyces species make to the overall kinetics of yeast growth during both spontaneous and S. cerevisiae inoculated wine fermentations (Fleet, 2008). However, they also contribute some negative enological characteristics, such as the production of high amounts of volatile acidity and acetic acid. These compounds can be reduced when in a mixed fermentation with S. cerevisiae. Some non-Saccharomyces yeast species have the ability to improve the fermentation behavior of Saccharomyces yeast and the chemical composition of wine, which can lead to a more complex aroma. They are able to positively contribute to overall sensory properties of wine by showing a significant influence on terpenols, esters, higher alcohols, acids, thiols and monoterpene production due to their enzyme activity (Andorr et al., 2010; Anfang et al., 2009; Medina et al., 2012). Investigations of poly-galacturonase and -D-xylosidase

produced by non-Saccharomyces yeasts during fermentation showed that these activities are widely dispersed throughout these yeasts and can be used to enhance wine quality (Ciani et al., 2010). In addition to -D-xylosidase, -glucosidase has also been found in several non-Saccharomyces species (Ciani et al., 2010). -Glucosidase hydrolyzes terpenyl-glycosides, and can enhance the wine aroma. In contrast to grape glucosidase, -glucosidase produced by yeast is not inhibited by glucose, and is involved in the release of terpenols during fermentation. Pectinases, proteases and glycosidases are other enzymes secreted by various non-saccharomyces species, which can improve the clarification, stabilization and aroma of wines (Andorr et al., 2010). Some nonSaccharomyces varieties, like Pichia kluyveri and Candida zemplinina, has a decreased ability of converting 3-Mercaptohexan-1-ol (3MH) to 3Mercaptohexyl acetate (3MHA). These compounds are shown in Table 1 (Coetzee et al., 2012). On the contrary, S. cerevisiae has a higher 3MH and 3MHA production, and different 4-Mercapto-4methylpentan-2-one (4MMP) releasing ability, which are the main volatile compounds responsible for the varietal aroma of Sauvignon blanc (Anfang et al., 2009). Multistarter fermentation in winemaking. In the last few years, there has been increased interest in the role of non-Saccharomyces yeast during the alcoholic fermentation of grape juice. These studies have shown that non-Saccharomyces species could have an impact on the chemical composition of the wine and that the contribution of these yeasts to wine character is significant

(Sadoudi et al., 2012). Co-fermentation of Saccharomyces and non-Saccharomyces yeasts lead to a faster fructose consumption, less acetic acid, and more higher alcohol production (Barrajn et al., 2011; King et al., 2010; Sadoudi et al., 2012). Unfortunately, fermentation conducted with natural flora brings diminished predictability of the process and inconsistencies in wine quality. For these reasons, an alternative process has gained interest, in particular the use of nonSaccharomyces starter cultures together with Saccharomyces starters. Multistarters containing non-Saccharomyces and Saccharomyces strains have been shown to mimic natural fermentation, avoiding the risk of stuck fermentations (Sadoudi et al., 2012). A few studies have investigated the use of controlled mixed cultures to reduce the volatile acidity and enhance the organoleptic profiles of wines (Ciani et al., 2010). Recently, the impact of mixed and sequential T. delbrueckiiS. cerevisiae cultures in high sugar fermentation was evaluated to determine whether it can improve the quality of wines and reduce the acetic acid content (Ciani et al., 2010). Mixed T. delbrueckiiS. cerevisiae cultures at a 20 : 1 ratio produced 53% and 60% reductions in the volatile acidity and acetaldehyde, respectively, while sequential cultures showed lower effects on the reduction of these metabolites. The combination of Torulaspora delbrueckii/ S. cerevisiae as co-cultures has also been shown to produce less volatile acidity and acetaldehyde in high sugar fermentations (Ciani et al., 2010). Mixed fermentations have also been proposed to enhance specific volatile compounds to improve the wine aroma. Ciani et al., (2010) proposed the

use of a mixed culture of Debaryomyces vanriji and S. cerevisiae to increase volatile compounds, particularly geraniol, in Muscat wine. Moreover the co-fermentation with S. cerevisiae and Pichia kluyveri was proposed to increase varietal thiol concentrations in Sauvignon Blanc (Anfang et al., 2009). This study showed that a 1:9 starting ratio of S. cerevisiae: P. kluyveri enhanced the 3-mercaptohexyl acetate concentrations in Sauvignon Blanc. Aromatic Compounds Co-fermentation of Saccharomyces and nonSaccharomyces yeasts promote the volatile aroma substances production, like higher alcohols and thiols (Anfang et al., 2009; King et al., 2010; Sadoudi et al., 2012). Co-fermentation of Metschnikowia pulcherrima and S. cerevisiae produces more fatty acids, ethyl esters, acetates, hotrienol, 4-terpineol and linalool. Hanseniaspora guilliermondii and S. cerevisiae produce more esters that are noticed by the flowery and fruity aroma and less ethyl acetate (Andorr et al., 2010). But, if too much Hanseniaspora pretenses in alcoholic fermentation, increased volatile acidity production has been and resulted in the synthesis of acetic acid and ethyl acetate (Andorr et al., 2010). The effect of co-fermentation on aroma is also influenced by different fermentation times and yeast strains (Sadoudi et al., 2012). Sauvignon blanc. Sauvignon blanc is a versatile grape variety that can deliver different interesting aromatic compounds (Coetzee et al., 2011). Its aromatic expression can vary with the viticultural and oenological practices employed. The main volatile thiols impacting on Sauvignon blanc flavor are 3MH, 3MHA and 4MMP. They play a critical role in the flavor of Sauvignon blanc, due to their extremely low perception threshold. The precursors of volatile thiols may be affected differently in a co-fermented wine versus single culture fermentation. A co-fermentation of P. kluyveri and VL3 (a strain of S. cerevisiae) at the ratio of 9:1 has shown a significant boost of 3MHA production compared to single ferments, which is a good method to improve the Sauvignon blanc style without applying genetic modification (Anfang et al., 2008).

However, not all of the co-inoculated combinations can improve thiols production. King et al. (2008) demonsted that co-fermentation of Vin7/QA23 can improve the production of 4MMP, 3MH and 3MHA in Sauvignon blanc while Vin7/Vin13 produced very little 4MMP and 3MHA. Therefore, only certain co-fermentation combinations can contribute to an increased volatile thiol production. They are not simply co-existing, but rather interact with each other, and generate a different wine aromatic profile, which cannot be regenerated by blending (Barrajn et al., 2011; King et al., 2008). Sensory Analysis Co-inoculated wines can be analyzed chemically, but more importantly, the wines should to be evaluated sensorily to test the consumer preferences. Co-inoculation in wines have been a place of interest in the recent years. They not only have an affect of the wine chemical structure, but also on the sensory profile. Studies have shown that there are significant sensory changes that consumers either prefer or dont prefer. In King et al. (2010), it was shown that co-inoculated Sauvignon blanc wines had different sensory profiles than single strain Sauvignon Blanc wines. Figure 1, from King et al, (2010), shows the variance between yeast inocula. This study also conducted a consumer acceptance tasting with 4 of their different wines. Their results showed that the co-inoculated yeast sample was the least preferred out of all the other strains. This result may be due to the wines strong estery and floral aroma and other sensory aspects (as seen in Fig 1). This does not mean that coinoculations are negative for sensory preferences of wine, but there needs to be a balance in flavors in the wines instead of an overpowering aroma and flavor. Another sensory analysis conducted by Parapouli et al. (2010) sensory analysis on cofermented Debina wine, a white Greek varietal. Co-fermentations were done a few years in a row with M. pulcherrima and S. cerevisiae yeast. The cofermented wines showed a higher concentration of fruity aromas compared to the mono fermentation. Jolly et al. (2003) did co-fermentations of S. cerevisiae and C. pulcherrima on Chenin Blanc wine

Fig. 1 This shows the different sensory perceptions of wines fermented by single strains and mixed strains in South Africa for 3 consecutive years. Their sensory analysis results between years varied. 2 out of the 3 years, the co-fermentation was detected at the better quality. Therefore, cofermentations do have a sensorial impact on wines, but without consistency. More studies have to be conducted with more strains and different combinations. Conclusion Co-fermententation shows great potential for wine quality. Yeast-derived aroma compounds can also have a significant effect on the aroma bouquet of different varieties, like Sauvignon blanc wine (Coetzee et al., 2011). They can produce sensorially important volatile metabolites such as esters, which mostly contribute to a pleasant smell in wine. The issues with the co-fermentations are the variance in results from different studies. Generally, cofermentation can improve the production of volatile aromatic compounds and reduce volatile acidity. This can also improve the wine quality of Sauvignon blanc. These results may be from the interactions between different yeast, but the mechanisms are not well known and still need to be studied more. Co-fermentations have also been shown to balance wine sensory profiles so that there is no over powering particular flavor over another (King et al., 2010), but the opposite effect was also shown in King et al. (2010). There have been mixed results from different studies, which means there is a lot more to research. There also has to be more sensory analysis done on trial wines. There is a lot of chemical analysis on co-fermented wines, but there is not a lot of sensory analysis by panels or consumers. This may be conducted when there is more knowledge of yeast interactions.

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