Study of yeast populations and their dynamics during pre-fermentative cold soak and alcoholic fermentation using cultures-independent and dependent methods

MATURANO Y.P.; MESTRE M.V.; TORO M.E.; ESTEVE-ZARZOSO B.; VAZQUEZ, F.; COMBINA M.

Mendoza

Congreso; 37° Congreso Mundial de la Vid y el Vino; 2014

Organizacion Internacional de la Vid y el Vino (OIV)

Conversion of grape must in wine is carried out by a complex microbial ecosystem, wherein yeasts play a central role. Several oenological and viticultural practices are carried out, in order to obtain high quality products. Among these practices, highlightings pre-fermentative cold maceration or cold soak (CS). It can be defined as the submission of grape must to low temperatures (0-15 °C) during a period of time, in order to allow a greater extraction of hydrosoluble compounds which contribute to color stability and aromatic complexity. These low temperatures can impact in yeasts community and, consequently, in composition and sensorial quality of the wine. The knowledge about population changes during CS allows to select the temperature according to desired product. In the last decade, new methods have been developed based on analysis of nucleic acids (DNA and RNA) that allow knowing microbial populations without a preculture. Polymerase Chain Reaction followed by Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and Real Time PCR (thereafter named qPCR for quantitative PCR) are 2 culture independent techniques mostfrequently used. The aim of this study was to compare yeast populations and their dynamics during CS and alcoholic fermentation (AF) using culture dependent and independent techniques. Methodology: two grape musts of Malbec from 2 different zones in Mendoza were used. Eighty liters of the musts were put in 100-L stainless steel tanks. Treatments: T1) Classic maceration (witness) where maceration and AF developed simultaneously, T2) CS 12- 15 °C, T3) CS 8- 10 °C and T4) CS 0- 4 °C. CS period was carried out during 7 days. Samples were taken in initial must, during CS (Day: 2, 5 and 7) and AF (onset, medium and end of fermentation). A representative percentage of each different colony was isolated, purified and identified by sequencing the D1/D2 domain of the 26S ribosomal gene. On the other hand, total DNA from samples was isolated and purified. The total DNA extracted was used for both independent culture techniques: PCR-DGGE and qPCR. Twelve species were detected in initial musts and throughout CS by dependent culture technique. This method allowed evidence of a greater diversity of species than PCR-DGGE, which has a detection limit of 103 cells /mL. Hanseniasora uvarum, Saccharomyces cerevisiae and Candida zemplinina were detected during pre-fermentative stage by PCR-DGGE and culture dependent technique. By these techniques only S. cerevisiae was detected during AF. However, C. zemplinina and H. uvarum were found in low proportions during AF using qPCR technique. The three species analyzed by qPCR were quantified during the CS and AF in different proportions depending on the CS temperature. The three used techniques showed the important presence of the species H. uvarum, S. cerevisiae and C. zemplinina. Only the qPCR method showed that the non- Saccharomyces species remained during alcoholic fermentation.