Effect of fermentation temperature on microbial population evolution using culture-independent and dependent techniques

Abstract

The population dynamics of micro-organisms during grape-must fermentation has been thoroughly studied. However, the main approach has relied on microbiological methods based on plating. This approach may overlook micro-organisms that (i) grow slowly or do not grow well on artificial media or (ii) whose population size is small enough to be detected by regular sampling. Culture-independent methods have been used and compared with the traditional plating method during wine fermentations performed at two different temperatures (13 °C and 25 °C). These methods include a qualitative technique, the DGGE; a semi-quantitative technique, the direct cloning of amplified DNA; and a quantitative technique, the QPCR. The biodiversity observed in the must and at the beginning of fermentation was much higher when DGGE or direct cloning were used. Quantification of the most frequent non-Saccharomyces yeast, Hanseniaspora uvarum and Candida zemplinina, showed that they survived throughout the fermentation process and, specifically, it revealed the quantitatively relevant presence of C. zemplinina until the end of fermentation.

Introduction

The conversion of grape-must to wine is a complex biochemical process involving interactions between yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB). The metabolism of these micro-organisms contributes to the quality of the wine by releasing metabolites which are constituents of the flavour and aroma (Swiegers, Bartowsky, & Henschke, 2005). Wine microbiota is influenced by multiple factors which can be grouped into viticultural and oenological practices (Pretorius, 1999). The temperature of fermentation is an oenological factor which influences the evolution of wine populations (Fleet, 2003): the lower the temperature of fermentation, the higher the chance of survival of the non-Saccharomyces yeasts during alcoholic fermentation (Heard and Fleet, 1988, Sharf and Margalith, 1983). Likewise, Ribereau-Gayon, Dubourdieu, Donèche, and Lonvaud (2000) reported that low temperature notably reduced the growth of acetic and lactic acid bacteria. Low-temperature fermentations (below 15 °C) are considered to improve the wine’s aromatic profile. The increase in aroma may be related to a higher retention of volatile compounds. However, Beltran, Novo, Guillamón, Mas, and Rozès (2008) observed that this increase in flavour and aroma was not only related to primary aroma retention. The evolution of yeast and bacteria species and their metabolism may also be involved in this improvement in the organoleptical characteristic of wines fermented at low temperature.

Most previous studies on wine microbial ecology have invariably been conducted after the culture of the different micro-organisms in different media. Today, new culture-independent methods allow to identify and enumerate micro-organisms, avoiding the biases associated with traditional culture-dependent methods (Rantsiou et al., 2005). The presence of viable but non-culturable micro-organisms in wine samples has been described (Divol and Lonvaud-Funel, 2005, Millet and Lonvaud-Funel, 2000). These micro-organisms are unable to grow on standard solid media within the laboratory but may justify the differences reported by various authors between isolated and naturally occurring species in wine samples (Cocolin and Mills, 2003, Hierro et al., 2006, Mills et al., 2002).

The aim of this study was to analyse the evolution of wine microbial population during the fermentation of the same grape-must at low (13 °C) and optimum temperature for wine yeasts during fermentation (25 °C). Microbial populations were evaluated by using three culture-independent techniques: a qualitative technique (DGGE), a semi-quantitative technique (the direct cloning of amplified DNA) and a quantitative technique (the QPCR). DGGE and QPCR are two of the most widely used techniques for culture-independent microbial analysis. In a previous study (Andorrà, Landi, Mas, Guillamón, & Esteve-Zarzoso, 2008), we enumerated the main wine microbial groups (yeast, lactic acid bacteria and acetic acid bacteria) using QPCR. In addition, we employed specific primers for the enumeration of two of the main yeast genera, Saccharomyces and Hanseniaspora. In the present study, we have also designed a new pair of primers for the enumeration of what is probably the third main wine yeast Candida stellata, or its current classification as Candida zemplinina (Sipiczki, Ciani, & Csoma, 2005). Moreover, in parallel to the analysis of species diversity by DGGE, we have evaluated the richness in yeast species through a direct amplification of DNA purified from wine samples and further cloning and identification of the amplicons. This technique has the additional advantage of making it possible to detect the relative abundance of the different species. To our knowledge, this is the first time that yeast diversity has been analysed using this strategy, thus avoiding some of the problems of cultivability of wine micro-organisms.