MONITORING AND IDENTIFICATION OF BRETTANOMYCES BRUXELLENSIS IN YOUNG PORTUGUESE RED WINES

Abstract

This study focused on a key topic in the wine industry: the impact of alterative yeasts on the organoleptic quality and microbiological stability of wine. A comprehensive assessment of the presence, growth and phenolic production of Brettanomyces bruxellensis in wine, allows a deeper knowledge on wine quality and microbiological stability. The aim of the present study was to identify novel strategies to limit B. bruxellensis proliferation and mitigate the formation of volatile compounds that negatively affect wine aroma and flavour. The study objectives were pursued through the following steps: (i), collection of samples from wineries affiliated to the University of Lisbon (School of Agriculture University of Lisbon, ISA); (ii), isolation and identification of volatile phenol- producing B. bruxellensis both in young wines and in matrices that reproduce wine characteristics, such as synthetic wine medium (SWM) and sterilised young red wines; (iii), analysis of volatile organic compounds (VOCs) by gas chromatography; (iv), identification and quantification of yeasts by RT-PCR. During alcoholic fermentation, the kinetics of the yeast population were monitored. The production of 4-ethylphenol (4-EP) was frequently monitored throughout the experiment, as this compound is an index of Brettanomyces spp. contamination (Malfeito Ferreira, 2011). Among commercial red wines, three samples showed B. bruxellensis concentration at significant values that was detected only on DBDM (Dekkera/Brettanomyces Differential Medium), showing 4-EP levels above the perception threshold (481 ppb). After seven days, the viability of the yeast decreased, indicating a transition to the VBNC (vital but not cultivable) state. This transition to the VBNC state may have been an adaptive response to stressful environmental conditions, such as limited oxygen or nutrient availability. The detection of cells in the VBNC state is crucial to understand the growth kinetics of B. bruxellensis surviving cells in wines, since the presence of these cellsis not detectable by traditional culture methods, cells continue to proliferate under favourable conditions and influence the organoleptic characteristics of the wine. In the SWM growth medium, three strains showed significant growth (OD 640 nm ≥ 0.5), with an initial lag phase followed by population expansion. After 28 days, a decline phase was observed, characterised by surface film formation and increased production of acetic acid. Competition between B. bruxellensis and acetic acid bacteria, suggested by the decrease in CFU/mL.n sterilised young red wines, two samples showed the highest yeast growth, while two others showed complete inhibition. The 4-EP production exceeded the detection threshold in several samples, with high initial levels (8909.5 ppb) and further increases during growth (9186.5 ppb on day 14). RT-PCR analysis confirmed the variability of the contamination, with most samples exceeding the detection limit (>10 CFU/mL). Since recent ecological evidence has shown that Brettanomyces spp. is already present in the early stages of winemaking (Ocon et al., 2010), although it is more commonly present during the ageing of wine, it is essential, and carrier of this study, to implement early monitoring and control phases of this yeast. The approach adopted is holistic and focuses on observing the behavior and biodiversity of these yeasts in a variety of growing environments, including young red wines, wine-like mediums, and sterilized young red wines. Examining how Brettanomyces spp. adapt and multiply in these different growth environments offers new perspectives on its management, representing an innovative frontier for modern oenology.