Dynamics and diversity of non-Saccharomyces yeasts during the early stages in winemaking

Abstract

This detailed study observed the yeasts present in the ecological niche of “wine must”. The dynamics and identity of non-Saccharomyces yeasts during the cold maceration and alcoholic fermentation of grape must were investigated under real production conditions in the Bordeaux region. Furthermore, we studied the impact of two oenological parameters on the development and diversity of non-Saccharomyces yeasts during cold maceration: temperature management and the timing of dried yeast addition.

The non-Saccharomyces community underwent constant changes throughout cold maceration and alcoholic fermentation. The highly diverse non-Saccharomyces microflora was present at 10⁴–10⁵ CFU/mL during cold maceration. The population increased to a maximum of 10⁶–10⁷ CFU/mL at the beginning of alcoholic fermentation, then declined again at the end. The population at this point, evaluated at around 10³–10⁴ CFU/mL, was shown to be dependent on the timing of yeast inoculation. The choice of temperature was the key factor for controlling the total yeast population growth, as well as the species present at the end of cold maceration. Hanseniaspora uvarum was a major species present in 2005 and 2006, while Candida zemplinina was very abundant in 2006. A total of 19 species were isolated.

Introduction

Yeasts live on berry surfaces throughout grape development. The population varies constantly in response to climatic conditions and the stage of ripening also plays a major role (Raspor et al., 2006). At harvest time, the yeast population is quite complex and the major fermenting yeast, Saccharomyces cerevisiae, is not very abundant. Indeed, research has demonstrated that only one grape per 1000 is S. cerevisiae positive (Mortimer and Polsinelli, 1999). Therefore, the non-Saccharomyces (NS) population is expected to be dominant in the early stages of grape must processing.

Numerous studies have evaluated the NS species present in the wine ecosystem and recent research has demonstrated the impact of grape conditions on NS populations (Fernandez et al., 1999, Raspor et al., 2006, González et al., 2007). Botrytis has been shown to alter species heterogeneity and succession (Greece) (Nisiotou et al., 2007) and the influence of ripeness on yeast dynamics and diversity has also been investigated (Hierro et al., 2006). A highly diverse yeast population, including Hanseniaspora (anamorph Kloeckera), Candida, Pichia, Metschnikowia, Kluyveromyces, and Issatchenkia is often described in the initial stages of winemaking (Mills et al., 2002, Di Maro et al., 2007).

Recently, there has been increasing interest in the industrial application of NS yeasts. Several studies have shown their capacities to contribute positive on wine flavour (Fleet, 2003, Esteve-Zarazoso et al., 1998) or evaluated the biotechnological interest of their enzymatic activities (Fernandez et al., 1999, Charoenchai et al., 1997). Mixed culture fermentations have also been assessed (Bely et al., 2008).

The impact of the oenological techniques applied on yeast development is assumed, but rarely described in the literature. Cold maceration (CM) of grape must is used in red wine production worldwide. It consists of keeping the must at a low temperature (not defined) for a certain time (not defined). CM has been shown to contribute positively to wine colour and produce more saturated wines (Gómez-Míguez et al., 2007). Colour enhancement has been reported for several red grape varieties (Marais, 2003, Villimburgo, 2003, Schödl, 2002). Higher monoterpene concentrations were also found in rosé wines following cold maceration (Salinas et al., 2003).

The microbial community present at the beginning of CM is thought to originate essentially from grapes (Ribereau-Gayon et al., 2006) but is assumed to be modified and influenced by the yeast community present on cellar surfaces (Mercado et al., 2007).

This study examines the yeast community in the ecological niche of wine, analysing the dynamics and diversity of the whole population during CM. Furthermore, the impact of the time of yeast addition and CM temperature on the yeast ecosystem was investigated. Species diversity was evaluated by PCR-ITS-RFLP and combined with the sequencing of specific regions to identify the species isolated.