Quartz Crystal Microbalance Genosensing of Brettanomyces bruxellensis Yeast in Wine Using a Rapid and Efficient Drop and Collect Protocol

Rodica Elena Ionescu,Marisa Manzano,Giuliocesare Casari Bariani,Lan Zhou,Rakesh Mittapalli,Simone Poggesi

doi:10.3390/cryst11050562

Abstract

A miniaturized quartz crystal microbalance (QCM) genosensor is proposed for sensitive and real-time detection of short ssDNA sequences (53 bp) or DNA extracted from Brettanomyces bruxellensis (Brett) yeast cells. The presence of Brett yeast causes a depreciation of the quality of aged fine wines, producing molecules of unpleasant odors and biogenic amines that are harmful to human health. More specifically, standard quartz crystal (S-QCM) and homemade 4 nm gold transmission electron microscopy (TEM)-grid patterned quartz (multi-TEM QCM) are herein proposed for biofunctionalization steps with different ssDNA sequences. By employing a rapid and efficient drop and collect protocol, the specific detection of 1 pg/µL ssDNA Brett of a short sequence and 100 ng/μL DNA of B. bruxellensis extracted from a wine sample (VR2008) is reported.

Highlights

Wine is an alcoholic beverage obtained by fermentation of grapes and its production is very important for the three main producers of wine: Italy, France, and Spain [1].the presence of Brettanomyces bruxellensis spoilage microorganisms during the wine production, principally in the aging phase, affect the aroma quality and may be responsible for important economic losses.Brettanomyces bruxellensis was defined as “one of the most complex and controversial issues encountered in the making of red wine” [2]
B. bruxellensis cells are perceivable at very low levels through various standard laboratory techniques: (i) plating methods, (ii) microscopic characterization and (iii) biochemical analysis
The Scanning Electron Microscope (SEM) morphology of the S-quartz crystal microbalance (QCM) gold electrode shows dense, continued and inhomogeneous granular nanoislands (Figure 3A), while after the evaporation of the gold thin film (4 nm) through transmission electron microscopy (TEM)-grids (Figure 3B) the random formation of nanostructures were observed between pre-existent larger nanoislands gold structures [24]

Summary

Introduction

Wine is an alcoholic beverage obtained by fermentation of grapes and its production is very important for the three main producers of wine: Italy, France, and Spain [1].the presence of Brettanomyces bruxellensis spoilage microorganisms during the wine production, principally in the aging phase, affect the aroma quality and may be responsible for important economic losses.Brettanomyces bruxellensis was defined as “one of the most complex and controversial issues encountered in the making of red wine” [2]. Wine is an alcoholic beverage obtained by fermentation of grapes and its production is very important for the three main producers of wine: Italy, France, and Spain [1]. The presence of Brettanomyces bruxellensis spoilage microorganisms during the wine production, principally in the aging phase, affect the aroma quality and may be responsible for important economic losses. The presence of volatile phenols, 4-ethylphenol and 4-ethylguaiacol, are responsible for the off-flavor named “Brett character” [3,4]. B. bruxellensis cells are perceivable at very low levels through various standard laboratory techniques: (i) plating methods, (ii) microscopic characterization and (iii) biochemical analysis. These techniques require trained researchers and extended analysis time. Modern molecular methods based on PCR techniques may reduce the testing time but suffer from the difficulty to discriminate between viable and non-viable Brett cells [6]

Methods

Results

Conclusion