Viable and culturable populations of Saccharomyces cerevisiae, Hanseniaspora uvarum and Starmerella bacillaris (synonym Candida zemplinina) during Barbera must fermentation

Abstract

The present study analyzed the viable and/or culturable populations of Saccharomyces cerevisiae, Hanseniaspora uvarum and Starmerella bacillaris (synonym Candida zemplinina) during laboratory grape must fermentation, in order to investigate the interaction between the three species considered. Firstly, population dynamics during wine fermentation were followed by culture-dependent techniques, and non-Saccharomyces yeast became non-culturable at late stages of fermentation when S. cerevisiae dominated. Four different culture-independent techniques were further applied to detect viable yeast cells at the late stage of fermentation. Both quantitative PCR techniques applied, namely ethidium monoazide bromide (EMA)-qPCR and Reverse Transcription (RT)-qPCR, detected H. uvarum and Starm. bacillaris at a concentration of 10⁵ to 10⁶cells/mL. These non-culturable cells had membranes impermeable to EMA and stable rRNA. The background signals from dead cells did not interfere with the quantification of viable cells in wine samples by EMA-qPCR technique. As a qualitative culture-independent technique, DGGE technique was coupled with EMA treatment (EMA-PCR-DGGE) or with RT (RT-PCR-DGGE). With EMA-PCR-DGGE non-Saccharomyces species during fermentation were detected although it was limited by the predominance of S. cerevisiae.

Keywords: Culture-independent technique; DGGE; Ethidium monoazide bromide (EMA); Non-Saccharomyces; Quantitative PCR (qPCR); Wine.