Wang, Xingchen. "Varietal Thiols and Precursors: Biogenesis, Reactivity, and Impact of Winemaking Practices." Thesis, 2022. https://hdl.handle.net/2440/136792.
Анотація:
Over one thousand volatile compounds have been identified in wine and these arise
from different chemical classes, varietal thiols are of one group which is particularly important
given their substantial sensory impact on certain wine varieties. As such, furthering the
understanding of thiol biogenesis through the analysis of known precursors and exploration of
new ones, and reactivity of thiols based on factors related to viticulture, winemaking, and wine
storage, is crucial to the manipulation of varietal thiols in wine and thus overall wine sensory
profile. Additionally, exploring of other sulfur-containing volatile compounds in wine is one of
the aims of the project, which could contribute to the appreciation of wine aroma complexity
as a result of this important class of molecules.
A review of the literature (Chapter 1) summarises the production and manipulation of
varietal thiols in wine, including 3-sulfanylhexan-1-ol (3-SH), 3-sulfanylhexyl acetate (3-SHA),
4-methyl-4-sulfanylpentan-2-one (4-MSP), and 4-methyl-4-sulfanylpentan-2-ol (4-MSPOH),
and considers the impact on non-volatile L-glutathione and L-cysteine conjugated precursors
in grapes from viticultural and winemaking practices. Specialised analytical methods for
studying such compounds are necessary given their chemical reactivity. The review also
identified research gaps, namely knowledge regarding potential new precursors and reactivity
of varietal thiols, given that the concentrations of varietal thiols in wine could not be
adequately accounted for by the utilisation of known precursors. Impacts of some winemaking
practices on the concentrations of thiol precursors in grape materials and varietal thiols in the
resultant wines were proposed. The review highlighted the possibility of sulfur-containing
volatile compounds awaiting discovery in wine, including grapefruit mercaptan and
blackcurrant mercaptan, which are key contributors to the aroma of grapefruit and
blackcurrant, respectively.
Chapter 2 (prepared in manuscript format) reports a preliminary study aiming to
identify new precursors to 4-MSP and 3-SH. 4-MSP can be released from its L-glutathionylated
or L-cysteinylated conjugate (i.e., GSH-4-MSP and Cys-4-MSP) with mesityl oxide (MO)
assumed to be their precursor but not been identified in grapes. It was hypothesised in
Chapter 1 that one route could involve MO produced by soil bacteria near grapevine roots,
with subsequent uptake and transformation of MO into 4-MSP precursors. To verify this, a
feeding experiment was conducted by applying deuterium labelled MO to grape leaves and bunches of potted grapevines. Analysis of the grape tissues by HPLC-MS/MS showed the
presence of deuterium labelled GSH-4-MSP or Cys-4-MSP, indicating that MO is the precursor
of GSH-4-MSP and Cys-4-MSP in grapevines. However, further experiments should explore the
existence of MO in vineyard soil as well as soil bacteria strains that could potentially yield MO.
Additionally, this chapter studied the presence of a potential 3-SH precursor, namely the Nmalonylcysteine
conjugated 3-SH (MalCys-3-SH), in Sauvignon blanc juice extracts. The
extracts were screened after optimising MS parameters using an authentic MalCys-3-SH
standard based on a published HPLC-MS/MS method for thiol precursors. Although MalCys-
3-SH was not identified at this stage, it cannot be concluded with the small sample set that this
or other precursors are not present in juice. A greater number of grape samples should be
analysed in the future, jointly with MS experiments that look for the loss of specific fragments
associated with L-glutathione, for example.
The research publication presented in Chapter 3 explores the reactivity of varietal thiols,
particularly 3-SH. The recent identification of cis-2-methyl-4-propyl-1,3-oxathiane (cis-2-MPO)
in wine was speculated to be the product of 3-SH reacting with acetaldehyde. In a continuation
of this work, the evolution profile of cis-2-MPO during alcoholic fermentation was studied,
revealing moderate to strong Pearson correlations with 3-SHA and acetaldehyde. Yeast strains
significantly affected cis-2-MPO production during fermentation, but resulted in similar
concentrations in the resultant wines. The instability of cis-2-MPO was illustrated by the
continuous decline in a commercial Sauvignon blanc wine spiked with cis-2-MPO which was
stored for one-year under various conditions (pH, temperature, presence of acetaldehyde or
SO2). In this case, cis-2-MPO appeared to be preserved by lower pH, acetaldehyde addition,
and 4 °C storage temperature.
The research publication in Chapter 4 was based on the hypothesis that enantiomers
of cis-2-MPO were produced from the corresponding 3-SH enantiomers upon their reaction
with acetaldehyde. To verify this, a validated stable isotope dilution assay (SIDA) using gas
chromatography coupled to mass spectrometry (GC-MS) with a chiral GC column stationary
phase was developed and utilised to analyse wine samples. Chemical formation of cis-2-MPO
from the co-spiking of 3-SH and acetaldehyde standards in a commercial wine was verified and
used to confirm enantiomer elution order. cis-2-MPO was revealed to consist of (2R,4S)-2-MPO
and (2S,4R)-2-MPO in wine, which had strong Pearson correlations with (3S)-3-SH and (3R)-
3-SH, respectively, upon analysing the thiol enantiomers. Additionally, one enantiomer of cis-
2,4,4,6-tetramethyl-1,3-oxathiane (cis-TMO), derived from the reaction of 4-MSPOH and acetaldehyde, was identified and quantified in a few white wine samples (≤ 28 ng/L). Although
presenting below the odour detection threshold (14.9 μg/L) determined in this work, its
presence demonstrated a pathway for the production of 1,3-oxathianes from acetaldehyde and
varietal thiols bearing a 1,3-sulfanylalkanol substitution in their structures.
Chapter 5 (prepared in manuscript format) reports the preliminary method
development to resolve 4-MSPOH enantiomers and identify the proposed sulfur-containing
volatile compounds in wine. Bearing a chiral centre, 4-MSPOH can conceivably consist of two
enantiomers. However, the identification of a single cis-TMO enantiomer (Chapter 3) implied
that only a single 4-MSPOH enantiomer might be present in wine. To examine this, an
assessment of two chiral stationary phases (CSPs), column temperature, eluent composition,
and mobile phase flow rate were performed to separate 4-MSPOH enantiomers by adapting a
method involving thiol derivatisation and HPLC-MS/MS analysis with a CSP. Base line
separation of 4-MSPOH enantiomers was not achieved with the columns on hand, but the
method was adequate to verify the hypothesis, and different wine samples were screened. 4-
MSPOH was not identified at this stage and further method optimisation using alternative
CSPs along with analysing additional wines with a more sensitive instrument may provide more
conclusive results. Chapter 5 also studied the potential presence of grapefruit mercaptan
(GFM) and blackcurrant mercaptan (BCM) using a published HPLC-MS/MS method after thiol
derivatisation. Calibration of BCM was undertaken and a selection of wines was analysed, but
BCM was not detected. Further work with a broader set of wines is required to provide evidence
about the presence and concentration of BCM. In contrast, GFM appears to be unstable under
light or protic conditions and the degradation product was investigated, although additional
study is required to verify its identity.
Research manuscripts in Chapter 6 and Chapter 7 highlight the effect of a novel grape
crushing technique, known as accentuated cut edges (ACE), on the release of thiol precursors
and varietal thiols in Shiraz and Sauvignon blanc during winemaking. Other practices,
including water dilution and skin contact time for Shiraz, and yeast strain and lactic acid
bacteria for Sauvignon blanc, were also evaluated. Concentrations of thiol precursors in Shiraz
grape must and varietal thiols in Shiraz wine were not significantly affected by ACE (Chapter
6, research publication). Nonetheless, as best as it can be ascertained, this was the first time
that thiol precursors were identified in Shiraz grape must. Sauvignon blanc and Pinot noir
wines made with ACE or conventional crushing on a commercial scale were also analysed,
giving preliminary insight into the potential of ACE for increasing concentrations of varietal thiols in Sauvignon blanc, but causing a decrease of 3-SH in Pinot noir. The impact of ACE on
varietal thiols may depend on grape variety was highlighted, which was pursued further with
fermentation trials involving ACE and Sauvignon blanc (Chapter 7, research publication).
Varietal thiols and their precursors in the Sauvignon blanc trials were markedly increased by
ACE. However, the potential for browning of white wine was raised given the higher amounts
of total phenolics and hydroxycinnamates in ACE treatments. The impact of crushing method,
yeast strain, and malolactic fermentation on other volatile compounds was evaluated for the
Sauvignon blanc fermentations. Interactions with other winemaking parameters were
observed and discussed in Chapter 6 and Chapter 7. The overall sensory quality of the Shiraz
and Sauvignon blanc wines was assessed with a rate-all-that-apply (RATA) methodology,
revealing that the grape crushing method and other winemaking practices could modify the
wine sensory profiles, with ACE playing a relatively minor role, as outlined in the respective
chapters.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2022