Dispersive Liquid-Liquid Microextraction for the Quantitation of Terpenes in Wine.

To study the contribution of yeasts to the formation of terpene derivatives during winemaking, a dispersive liquid-liquid microextraction gas chromatography mass spectrometry method was developed for the quantitation of terpenes in white wines, synthetic wine, and a fermented synthetic medium. A mixture of acetone (disperser solvent) and dichloromethane (extraction solvent) was added to 5 mL of sample. The proposed method showed no matrix effect, good linearity in the enological range (from 10 to 200 µg/L), good recovery, and satisfactory inter- and intraday reproducibilities (below 20 and 15% of the relative standard deviation). This sample preparation technique is very interesting for high-throughput studies and economic and environmental reasons because it is fast and easy to operate with high enrichment and consumes a low volume of organic solvents. This method was applied to explore the capacities of 40 yeast strains to produce terpene compounds during fermentation of Chardonnay and Ugni Blanc musts as well as in a synthetic medium. Interestingly, most of the studied compounds were detected and quantified in the resulting wines. This study shows that yeast strains can intrinsically produce terpene derivatives under enological conditions and also highlights the differences between the de novo biosynthesis of terpenes and their precursor-linked production.

Dynamics and quantitative analysis of the synthesis of fermentative aromas by an evolved wine strain of Saccharomyces cerevisiae.

We performed a dynamic and quantitative analysis of the synthesis of fermentative aromas by an aromatic wine yeast, ECA5, obtained by adaptive evolution. During fermentation at pilot scale on synthetic and natural musts, ECA5 produced volatile compounds (higher alcohols and their acetates, ethyl esters) at higher rates than the ancestral strain, with the exception of propanol. Marked differences in the chronology of synthesis of several compounds were observed between the two strains. Overproduction of phenyl ethanol occurred mainly during the growth phase for ECA5, consistent with its higher flux through the pentose phosphate pathway, which plays a key role in biosynthetic processes. The kinetics of production of isobutanol and isoamyl alcohol were differently affected by different media (synthetic or natural must) and, in particular, according to the nature of the sterols in the media (ergosterol or phytosterols). We also observed differences in the chronology of synthesis of ethyl acetate and isoamyl acetate or ethyl esters, suggesting that the regulation of the synthesis of these compounds in the evolved strain differs from that in the ancestral strain. This study shows that a dynamic analysis of volatile compounds, using high acquisition frequency online gas chromatography, can provide novel insights into the synthesis and regulation of aromas and is thus a potentially powerful tool for strain characterization.