Formation of vinylphenolic pyranoanthocyanins by Saccharomyces cerevisiae and Pichia guillermondii in red wines produced following different fermentation strategies
Introduction
The traditional role of wine yeasts as transformers of grape sugars into ethanol has been significantly widened since the establishment of modern oenological microbiology. Louis Pasteur (1866) indicated long ago that the types of yeast used in the wineries of a particular region led to that region’s wines having similar organoleptic characteristics. Thus, the role of yeasts in winemaking is not purely fermentative. The metabolic activities of a particular wine yeast in a must may lead to the formation of metabolites and to the transformation of grape molecules that may sensorially enrich a wine (Pretorius, 2000). Thus, in recent years, the yeast selection has included the development of techniques for detecting strains that might improve wines in terms of their colour, aroma and structure (Suárez-Lepe & Iñigo, 2004).
Related to colour, some of the metabolites produced by yeasts during the glycolytic stage of fermentation may condense with grape anthocyanins to produce highly stable pyranoanthocyanin adducts, such as vitisin A and B (Fig. 1) (Bakker and Timberlake, 1997, Morata et al., 2007, Morata et al., 2003, Romero and Bakker, 2000). Yeasts with hydroxycinnamate decarboxylase (HCDC) activity can also be used to decarboxylate hydroxycinnamic acids and form vinylphenols that condense with grape anthocyanins to produce vinylphenolic pyranoanthocyanin adducts – molecules that show great colour stability (Fig. 1) (Morata et al., 2006, Morata et al., 2007). Pigments of this type have been detected in wines using LC–MS techniques, and characterised by mass spectrometry (Hayasaka & Asenstorfer, 2002). With respect to colour improvement, a number of Saccharomyces cerevisiae strains show desirable HCDC activity (Shinohara, Kubodera, & Yanagida, 2000), although to very varied degrees. While some show very little activity, others may transform up to 15% of the substrate into vinylphenols (Benito, Palomero, Morata, Calderón, & Suárez-Lepe, 2009). Vanbeneden, Gils, Delvaux, and Delvaux (2008) showed that of the 75 S. cerevisiae strains they examined, over 70% were capable of decarboxylating ferulic acid. However, only a few Saccharomyces strains showed high conversion rates.
HCDC activity has been described in several yeast, bacterial and fungal species (Chatonnet et al., 1993, Degrassi et al., 1995, Dias et al., 2003, Edlin et al., 1998, Suezawa, 1995, Suezawa et al., 1998). Some species, such as Pichia guillermondii show high HCDC activity. However, P. guillermondii is unable to completely ferment a normal must with a sugar content of 220 g/l, as its normal fermentation limit is 30 g/l; therefore it can only be used alongside S. cerevisiae (high fermentative power) in a mixed or sequential culture for wine fermentation to be completed. A mixed culture would involve inoculating the must with a mixture of P. guillermondii and S. cerevisiae; a sequential culture would require initial inoculation with P. guillermondii to favour the formation of vinylphenols, followed by inoculation with S. cerevisiae to ensure the fermentation is properly completed.
In this work, the HCDC activity of different wine Saccharomyces strains was compared with a non-Saccharomyces species, P. guillermondii, with the latter showing the highest activity. Therefore, this works aim was to use this non-Saccharomyces yeast either together (mixed fermentation) or following (sequential fermentation) the Saccharomyces strain, in order to increase the amount of vinylphenolic pyranoanthocyanins, due to Pichia metabolism, and complete the fermentation due to the fermentative metabolism of Saccharomyces.
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Yeast strains
The S. cerevisiae strains examined in this work included CTPL14(2), 17VAA, 1EV, 1VA, 2EV, 3EV3, 3VA, 4CV, 52(1), 52(4), 7EV, 7VA, 7VL, CTPL5(2) and G2T4, isolated and selected for red winemaking by the Laboratorio de Enología, Dept. Tecnología de Alimentos, Universidad Politécnica de Madrid (Madrid, Spain). The P. guillermondii strain 513, isolated at the same laboratory, was also used. The yeast strain 87 (S. cerevisiae) from the IFI collection (CSIC), plus the commercial strain S6U (S. uvarum
Hydroxycinnamate decarboxylase activity in different yeast strains
The HCDC activities of the different Saccharomyces strains and P. guillermondii 513 were compared by determining the content of the p-coumaric acid precursor in the must before and after fermentation, and by determining the post-fermentation concentration of 4-vinylphenol. The initial p-coumaric acid content of the must was fixed at 115 mg/l. Two strains (S. cerevisiae 87 and the commercial strain S. uvarum S6U), which show no HCDC activity, were used as controls; the initial and
Discussion
P. guillermondii 513 yeast strain has been specially selected because of its HCDC activity, which was much greater than that of other S. cerevisiae HCDC+ strains. This strain was used in mixed and sequential fermentations in order to maximise the production of high stable vinylphenolic pyranoanthocyanins. Simultaneously, the concentrations of 4-ethylphenol precursors are likely to be reduced, giving stability to wine (Benito, Palomero, Morata, Uthurry, & Suárez-Lepe, 2009).
Fermentation implies
Conclusions
Sequential fermentation involving P. guillermondii 513, a non-Saccharomyces yeast with high HCDC activity, and S. cerevisiae, allows the formation of highly stable vinylphenolic pyranoanthocyanins to be maximised while reducing the presence of p-coumaric acid (the precursor of 4-ethyphenol). The use of P. guillermondii 513 in mixed or sequential fermentations is associated with no significant deviation of the fermentation metabolite profile; unwanted organoleptic variations are therefore not a
Acknowledgements
This work was supported by the Ministerio de Ciencia e Innovación (MCeI) (Project AGL-2008-05603-C02-01/AGR). The authors would like to thank S. Somolinos and J. A. Sánchez (Dept. Tecnología de Alimentos, ETSI Agrónomos, UPM) for their excellent technical assistance.
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