Precursors to the potent odorant wine lactone.

Abstract

This thesis describes the synthesis, hydrolysis and analysis of menthiafolic acid, a precursor to wine lactone in wine. (R)-Menthiafolic acid was synthesised and then taken through acid hydrolyses to confirm its conversion to wine lactone under wine-like conditions and to determine the chirality of the resultant product. A Gas Chromatography/ Mass Spectrometry (GC/MS) Stable Isotope Dilution Assay (SIDA) method was developed to analyse for this compound in grapes and wine. Chiral analysis was also carried out on wine extracts to confirm which enantiomers of menthiafolic acid and wine lactone are present in real wine samples. Bioconversion of the glucose ester of menthiafolic acid utilising three different microorganisms was evaluated in order to determine if menthiafolic acid is produced and hence if this compound is an indirect precursor to wine lactone through initial degradation to menthiafolic acid. Chapter 1 comprises an introduction and literature review. Chapter 2 concerns the synthesis and acid hydrolysis of (R)-menthiafolic acid. The synthesis gave a mixture of 95% (R)-enantiomer and 5% (S)-enantiomer menthiafolic acid. Hydrolysis was carried out under mild, wine-like conditions and under harsh Simultaneous Distillation Extraction (SDE) conditions. These hydrolyses showed that this compound is, in fact, converted to wine lactone under wine-like conditions but both the ‘natural’ (-)-isomer of wine lactone and its enantiomer are produced in varying proportions depending on the hydrolytic conditions. This work has been published; Giaccio et al. Journal of Agricultural and Food Chemistry 2011, 59, 660. Chapter 3 describes the development of a SIDA method for the analysis of menthiafolic acid in grapes and wine. Extraction methods were investigated for model wine solutions and then transferred to white wine. A d₅-analogue of menthiafolic acid was prepared for use in later quantifications. Grapes and wines were analysed and menthiafolic acid was found in the wines in varying concentrations ranging from < 10 µg/L to 342 µg/L with the highest concentration found in a Lexia wine. Wines analysed showed menthiafolic acid in significant concentrations which could potentially produce wine lactone in concentrations above its aroma threshold. Grape analyses were also carried out and menthiafolic acid was observed in concentrations ranging from 16 µg/L to 235 µg/L. Gerwütztraminer grapes contained the greatest concentration of this precursor. Chiral analysis of menthiafolic acid present was also carried out on grape and wine samples. The analyses showed that the (S)-enantiomer of menthiafolic acid is the more prevalent enantiomer in these particular grape and wine samples. Chapter 4 concerns fermentation studies of the glucose ester of menthiafolic acid. The SIDA method discussed in Chapter 3 was used to analyse for menthiafolic acid in these samples in order to determine if menthiafolic acid is released from the glucose ester via fermentation with various yeast and bacteria. Approximately 15% bioconversion of the glucose ester to menthiafolic acid was observed when fermenting with Saccahormyces cerevisiae (strain AWRI 838). Bioconversion occurred to a lesser extent (approx. 5%) when fermenting with the lactic acid bacteria Oenococcus oeni (strain VP-41) and even less of the glucose ester was converted to menthiafolic acid when fermenting with spoilage yeast Dekkera bruxellensis (strain AWRI 1499). Menthiafolic acid was not observed in a concentration above the limit of quantification in D.bruxellensis fermentations. Chapter 5 details an attempt to develop a quantification method for wine lactone in model wine. Extraction of wine lactone from a white wine was also attempted. Chiral analysis of wine lactone extracted from wine by continuous liquid extraction was also conducted showing that the predominant enantiomer of wine lactone present in the wine analysed was, in fact, the (+)-enantiomer which has not previously been reported in wine. The (-)-enantiomer of wine lactone was also observed and the ratios of the two wine lactone enantiomers correlated with what was expected when taking into account the ratios of menthiafolic acid also present in the wine. Chapter 6 comprises the experimental methods, materials and instrumentation utilised in these studies.