Polyfunctional Thiols in Wine: Chirality, Precursor Stereochemistry, Winemaking Impacts, and Fate

Abstract

Polyfunctional thiols (also known as varietal thiols) with odour detection thresholds (ODTs) in the nanogram per litre range are one of the most potent volatiles present in wine and are regarded as odour-active compounds affording significant sensory contributions. These compounds have been intensively investigated for the past few decades, along with their non-volatile grape-derived precursors in more recent years, but knowledge of the analysis, biogenesis, and fate of thiols, as well as the influences of environmental and winemaking factors, is still incomplete. This thesis therefore begins by reviewing the current knowledge of polyfunctional thiols in wine and precursors in grapes (Chapter 1), and then covers the analytical approaches that have been developed to identify and quantify thiols in foods and beverages, with a particular focus on wine (Publication in Chapter 2). A number of original research studies (Publications in Chapters 3 to 6) are then presented to address the knowledge gaps related to characteristic thiols in wine and their precursors in grapes. Polyfunctional thiols 3-sulfanylhexan-1ol (3-SH) and 3-sulfanylhexyl acetate (3- SHA) are two of the most evaluated thiols in wine. 3-SH and 3-SHA are chiral molecules, which give rise to pairs of enantiomers that differ in aroma quality and ODT. However, chiral analytical methods required to study 3-SH and 3-SHA enantiomers in wine were essentially non-existent. Addressing this gap, a novel stable isotope dilution assay (SIDA) with chiral high-performance liquid chromatography with tandem mass spectrometry (HPLC–MS/MS) using a polysaccharide-based column has been developed and validated for analysing the enantiomers of 3-SH and 3-SHA, after extraction from wine as their 4- thiopyridine derivatives. Authentic derivatives were synthesised to enable chiral column screening, and method validation encompassed calibration range, linearity, accuracy, precision, limit of detection, and matrix effects. The validated method demonstrated excellent analytical performance and offers opportunities for further research around chirality of polyfunctional thiols. By applying this method, the distribution pattern of enantiomers of 3-SH and 3-SHA in a set of commercial wines has been reported (Publication in Chapter 3). The formation of 3-SH and 3-SHA during fermentation involves conjugated precursors that are present as pairs of diastereomers in grapes. However, there was no literature on thiol precursor stereochemistry in grapes in relation to thiol chirality in wine. Employing the newly developed method for analysing thiol enantiomers, the relationship between precursor diastereomers in grapes and thiol enantiomers in wine was examined for the first time through a fermentation trial using five co-located clones of Sauvignon blanc grapes (Publication in Chapter 4). No correlation was observed between precursor diastereomers in grapes analysed by SIDA HPLC–MS/MS and 3-SH or 3-SHA enantiomers in wines measured by SIDA chiral HPLC–MS/MS, but the results have provided insight that can be further explored to understand the implications of thiol biogenesis on thiol precursor stereochemistry. Tackling thiols and their precursors from a viticultural and oenological perspective, the impacts of sub-region within one geographic indication, grape clone, grape amino acids, yeast strain, commercial enzyme, fermentation nutrient, and pre-fermentation freezing have been assessed in controlled fermentation trials with Sauvignon blanc (Publications in Chapter 4 and 5). Substantial intraregional variations existed among thiol precursors and thiols in the examined grapes and wines, respectively, and clonal differences were noted at the diastereomeric and enantiomeric levels for precursors and thiols, respectively. In terms of the impact of grape metabolites on thiols and precursors, grape amino acids were revealed for the first time to have stronger correlations to 3-SH precursors in grapes (e.g., |r| > 0.73 and 0.62 for glutamic acid and glycine) than thiols in wines (|r| < 0.42), highlighting the potential interaction between grape amino acids and thiol precursor metabolism in grapes. With regard to thiol management during winemaking, significant elevations of polyfunctional thiols in wine occurred with the use of a commercial enzyme in juices (up to an approximate two-fold increase in a clone-dependent manner) or pre-fermentation freezing treatment on fresh grapes (up to an approximate 10- fold enhancement regardless of clone). These practical approaches and novel results are of potential interest for winemakers who seek to be one step closer to thiol management during winemaking. The fate of polyfunctional thiols in wine requires continued investigation to comprehend the impacts on varietal aroma profiles of wine. Based on the coexistence of 3-SH and acetaldehyde in wine, the presence of a new volatile sulfur compound (VSC) with an oxathiane structure was theorised. After the synthesis of a deuterated standard and the development of a SIDA headspace solid-phase microextraction (HS–SPME) with gas chromatography and mass spectrometry (GC–MS) method, 2-methyl-4-propyl-1,3-oxathiane was identified and quantitated in wine for the first time. Only detectable as the cis-isomer, this compound strongly correlated (r = 0.72) with the concentration of 3-SH determined by HPLC–MS/MS analysis. The ODT of this newly discovered wine volatile was determined in a neutral white wine. Concentrations of cis-2-methyl-4- propyl-1,3-oxathiane (up to 460 ng/L) determined in a range of surveyed commercial wines were below the measured ODT of 7.1 μg/L. Nonetheless, the presence of this new wine volatile still has potential implications for wine aroma, due to its direct link with the fate of important wine aroma compound 3-SH. On one hand, this may help account for 3-SH that is lost during fermentation and ageing, and on the other hand, a sizeable proportion of 3-SH could be masked as the oxathiane, thus diminishing the impact of 3-SH on wine aroma (Publication in Chapter 6). In summary, this PhD thesis has combined modern analytical methods, chemistry synthesis, fermentation trials, and sensory testing to shed light on aspects of wine aroma related to important polyfunctional thiols and their precursors. The outcomes not only contribute to a better scientific understanding of thiol chemistry but also offer opportunities for potential industrial applications.