Multi-enzyme production by pure and mixed cultures of Saccharomyces and non-Saccharomyces yeasts during wine fermentation

Abstract

Saccharomyces and non-Saccharomyces yeasts release enzymes that are able to transform neutral compounds of grape berries into active aromatic compounds, a process that enhances the sensory attributes of wines. So far, there exists only little information about enzymatic activity in mixed cultures of Saccharomyces and non-Saccharomyces during grape must fermentations. The aim of the present work was to determine the ability of yeasts to produce extracellular enzymes of enological relevance (β-glucosidases, pectinases, proteases, amylases or xylanases) in pure and mixed Saccharomyces/non-Saccharomyces cultures during fermentation. Pure and mixed cultures of Saccharomyces cerevisiae BSc562, Hanseniaspora vinae BHv438 and Torulaspora delbrueckii BTd259 were assayed: 1% S. cerevisiae/99% H. vinae, 10% S. cerevisiae/90% H. vinae, 1% S. cerevisiae/99% T. delbrueckii and 10% S. cerevisiae/90% T. delbrueckii. Microvinifications were carried out with fresh must without pressing from Vitis vinifera L. c.v. Pedro Jiménez, an autochthonous variety from Argentina. Non-Saccharomyces species survived during 15–18 days (BTd259) or until the end of the fermentation (BHv438) and influenced enzymatic profiles of mixed cultures. The results suggest that high concentrations of sugars did not affect enzymatic activity. β-Glucosidase and pectinase activities seemed to be adversely affected by an increase in ethanol: activity diminished with increasing fermentation time. Throughout the fermentation, Saccharomyces and non-Saccharomyces isolates assayed produced a broad range of enzymes of enological interest that catalyze hydrolysis of polymers present in grape juice. Vinifications carried out by a pure or mixed culture of BTd259 (99% of T. delbrueckii) showed the highest production of all enzymes assayed except for β-glucosidase. In mixed cultures, S. cerevisiae outgrew H. vinae, and T. delbrueckii was only detected until halfway the fermentation process. Nevertheless, their secreted enzymes could be detected throughout the fermentation process.

Our results may contribute to a better understanding of the microbial interactions and the influence of some enzymes on vinification environments.

Introduction

Wine is the product of complex microbial interactions. Yeasts, bacteria and filamentous fungi all contribute to the microbial ecology of wine production, although yeasts (mainly Saccharomyces species) are dominating due to their role in conducting the alcoholic fermentation (Fleet and Heard, 1993, Fleet, 2003, Fugelsang and Edwards, 2007).

Over the past decades, application of enzymes has shown an increasing interest in winemaking. Enzymatic treatments of grapes, musts and wines are nowadays carried out for multiple purposes, since they positively influence wine clarification and filtration, juice yield, color and aroma extraction as well as wine stability (Rogerson et al., 2000). The enzymes used are usually produced by bacteria or by filamentous fungi (Esteve-Zarzoso et al., 1998, Fernández et al., 2000). An alternative could be the use of indigenous starter yeasts that produce extracellular hydrolytic enzymes. Moreover, wine yeasts could be potential sources for the commercial production of enzymes used in the winemaking process (Charoenchai et al., 1997, Fernández et al., 2000, Strauss et al., 2001).

Degradation of structural grape polysaccharides by carbohydrolases (including pectinases, cellulases, hemicellulases and amylases) and haze-active proteins by proteases can improve the clarification and filtration process during winemaking (Colagrande et al., 1994, Esteve-Zarzoso et al., 1998, Haight and Gump, 1994, Jayani et al., 2005, Louw et al., 2006, van Rensburg and Pretorius, 2000). The action of these enzymes also facilitates the release of aromatic precursors from the cells of the skin, seeds and flesh of the grape berries (Bautista-Ortín et al., 2007, Fornairon-Bonnefond et al., 2002, Ganga et al., 2001, González et al., 2004, Gump and Haight, 1995, Watson et al., 1999). β-Glucosidases are hydrolases capable of breaking down the links in aryl-β-glucosides, which means they can unmask aromatic compounds from non-volatile wine constituents (Blasco et al., 2006, Charoenchai et al., 1997, Esteve-Zarzoso et al., 1998, Fernández et al., 2000).

Many traditional quantitative studies (Ciani and Maccarelli, 1998, Fleet et al., 1984, Heard and Fleet, 1985, Pardo et al., 1989) and culture-independent techniques (Andorrra et al., 2008, Hierro et al., 2006, Zott et al., 2010) on winemaking ecology showed that non-Saccharomyces genera (Candida, Debaryomyces, Hanseniaspora, Hansenula, Kloeckera, Metschnikowia, Pichia, Schizosaccharomyces, Torulaspora and Zygosaccharomyces) are able to survive during fermentation at significant levels for longer periods than previously thought (Zott et al., 2010). Therefore, they could contribute to the analytical composition of wine (Fleet, 2008).

Hanseniaspora and Torulaspora genera are reported good producers of enzymes like β-glucosidases, pectinases, proteases and those involved in xylan degradation (Charoenchai et al., 1997, Ganga and Martínez, 2004, Manzanares et al., 1999, Masoud and Jespersen, 2006, Pérez et al., 2011, Romo-Sánchez et al., 2010). However, the secretion of each enzyme is not characteristic of a particular genus or species, but depends on the yeast strain analyzed (Ganga and Martínez, 2004).

There are many reports that enzymatic activity in yeasts from enological environments are influenced and/or conditioned by different parameters like pH and temperature, as well as the presence of inhibitors such as sugars and ethanol (Barbagallo et al., 2004, Cordero Otero et al., 2003, Hernández et al., 2002, Jurado et al., 2004, Manzanares et al., 1999, McMahon et al., 1999, Rosi et al., 1994). Studies of these enzymatic activities in winemaking and fermentations are scarce (Blasco et al., 2006, Fia et al., 2005, Zamuz et al., 2004). In addition, there is not much information about the production of enzymatic activity in mixed cultures of Saccharomyces and non-Saccharomyces during grape must fermentations. Therefore, there is a lack of information about yeast–yeast interactions regarding enzymatic activity during vinification. The aim of this work was to determine the ability of 3 yeasts to produce extracellular enzymes of enological relevance (β-glucosidases, pectinases, proteases, amylases and xylanases) in pure and mixed Saccharomyces/non-Saccharomyces cultures during enological fermentations.