Onsite testing for Brettanomyces bruxellensis contaminations during winemaking

Abstract

World wine production is estimated at 262 million hectolitres in 2022. Long spared from the constraints of the agri-food industry due to the absence of pathogens, the oenological sector is now facing the yeast Brettanomyces bruxellensis, which has a high potential for organoleptic deviation due to the production of volatile phenols that make wines unmarketable. This problem is currently the most concerning regarding wine quality. It poses significant challenges in the current context of reduced inputs (sulfites) and climate change, which raises the pH of wines and, as a result, increases their sensitivity. One of the main difficulties is the lack of efficient tools to detect and quantify the presence of this yeast. Current tests only partially meet the criteria to enable effective prevention and management of contamination. Indeed, the assay should be fast, inexpensive, easy to use, and usable on-site while quantifying only living cells. Here, a new approach was developed based on quantifying a specific ribosomal RNA (rRNA) sequence of Brettanomyces Bruxellensis. Thanks to loop-mediated isothermal amplification (LAMP), the assay was highly sensitive and specific, allowed easy detection, exhibited a large dynamic range, and was compatible with inexpensive commercially available readers. Furthermore, using rRNA as a target allowed for the quantification of living cells only. An analytical workflow was developed to simplify the isolation of rRNA using simple laboratory tools and commercial kits. The analysis of wines took less than 2h for a small cost per test. We will describe our latest results in this project.