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TRANSCRIPT
SMOKE TAINT: Impacts on the Chemical and Microbiological Profile of Grapes and Wine
by
Kerry Anita Pinchbeck
B.Sc., Flinders University
B.Sc. (Hons) The University of Adelaide
A thesis submitted in fulfilment of the requirements for
the degree of Doctor of Philosophy
The University of Adelaide
School of Agriculture, Food and Wine
April, 2011
TABLE OF CONTENTS
ABSTRACT....................................................................................................................
i
DECLARATION..............................................................................................................
iii
STATEMENT OF THE CONTRIBUTIONS OF JOINTLY AUTHORED PAPERS..........
iv
ACKNOWLEDGEMENTS.............................................................................................. viii
CHAPTER 1: INTRODUCTION..............................................................
1
1.1 Introduction to the Australian wine industry..........................
2
1.2 History of vineyard exposure to smoke.................................
3
1.3 The effects of smoke on nature............................................
8
1.4 Composition of smoke..........................................................
10
1.5 Smoke taint in grapes and wine............................................ 13
1.6 Research aims...................................................................... 19
CHAPTER 2: SYNTHESIS OF GUAIACOL GLUCOSIDES...................
21
2.1 Introduction to glycosides in grapes and wine......................
22
2.2 Introduction to guaiacol β-D-glucopyranoside....................... 24
2.3 Introduction to glycosylation.................................................. 25
2.4 Glycosylation of guaiacol...................................................... 26
2.5 Results and discussion........................................................
27
2.5.1 Preparation of guaiacol β-D-glucopyranoside.............. 27
2.2.5.1 Preparation of guaiacol glucoside using 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (Method 1) ..................................................................
27
2.5.1.2 Preparation of guaiacol glucoside using 2,3,4,6-tetra-O-pivaloyl-α-D-glucopyranosyl bromide. (Method 2)....................................................................
27
2.5.2 Preparation of deuterated guaiacol β-D-glucopyranoside.............................................................
28
2.6 Materials and methods.........................................................
30
2.6.1 Solvents and reagents.............................................
30
2.6.2 Chromatography.....................................................
30
2.6.3 Nuclear magnetic resonance (NMR) spectroscopy.
30
2.6.4 Ultra violet/visible spectroscopy and fluorescence spectroscopy....................................................................
31
2.6.5 Microwave synthesis...............................................
31
2.6.6 Gas chromatography-mass spectrometry (GC-MS)
31
2.6.7 High performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS).................................
32
2.6.8 Synthesis.................................................................
33
2.7 Conclusion............................................................................
42
CHAPTER 3: PROVENENCE OF GUAIACOL GLUCOSIDE IN SMOKE AFFECTED FRUIT..............................................................…..
43
Paper 1: Identification of a β-D-glucopyranoside precursor to guaiacol in grape juice following grapevine exposure to smoke.
45
CHAPTER 4: QUANTIFICATION OF GUAIACOL GLYCOSIDES IN SMOKE AFFECTED FRUIT……………………………………………….
51
Paper 2: Quantitative analysis of glycoconjugate precursors of guaiacol in smoke-affected grapes using liquid chromatography-tandem mass spectrometry based stable isotope dilution analysis…………………………………………….
53
CHAPTER 5: QUANTIFICATION OF GUAIACOL GLYCOCONJUGATES IN GRAPES AND WINE...................................
59
5.1. Introduction..........................................................................
60
5.2. Results and discussion........................................................
61
5.2.1 Method development...............................................
61
5.2.1.1Calibration function for guaiacol β-D-glucopyranoside....................................................
61
5.2.1.2 Mass transitions used for HPLC-SRM analysis........................................................................
62
5.2.2 Method validation.................................................... 63
5.2.2.1Instrument repeatability..................................... 63
5.2.2.2 Reproducibility.................................................. 63
5.2.2.3 Recovery.......................................................... 63
5.2.3 Application of wine based SIDA method to winemaking trials..............................................................
66
5.2.3.1Hydrolysis of guaiacol glycoconjugates during fermentation.................................................................
66
5.2.3.2 Influence of winemaking techniques on the glycoconjugate content of wine....................................
69
5.2.3.3 Glycoconjugate content of wine and potential for smoke taint to intensify with bottle age...................
72
5.2.3.4 Potential for carryover of glycoconjugates between growing seasons............................................
74
5.3 Materials and methods.......................................................... 76
5.3.1 Method development............................................... 76
5.3.1.1 Preparation of wine samples for HPLC-MS/MS analysis................................................
76
5.3.1.2 Calibration function for guaiacol β-D-glucopyranoside....................................................
77
5.3.1.3 Instrumental analysis.......................................
77
5.3.1.4 High performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS)................
77
5.3.2 Application of the quantitative guaiacol glycoconjugate method to winemaking trials....................
79
5.3.2.1Smoke affected grapes..................................... 79
5.3.2.2 Winemaking..................................................... 80
5.3.3 Statistical analysis................................................... 81
5.4. Conclusion...........................................................................
82
CHAPTER 6: THE EFFECT OF WINEMAKING TECHNIQUES ON THE INTENSITY OF SMOKE TAINT IN WINE.......................................
83
Paper 3: The effect of winemaking techniques on the intensity of smoke taint in wine.................................................................
85
CHAPTER 7: IMPACT OF SMOKE ON GRAPE BERRY MICROFLORA AND YEAST FERMENTATION.....................................
97
Paper 4: Impact of smoke on grape berry microflora and yeast fermentation................................................................................
99
CHAPTER 8: SUMMARY........................................................................ 104
APPENDIX .................................................................................................................. 109
REFERENCES.............................................................................................................. 117
i
ABSTRACT
Guaiacol β-D-glucopyranoside was prepared via a modified Koenigs-Knorr
glycosylation method as a reference compound to confirm its presence in smoke
affected grapes, using high performance liquid chromatography-tandem mass
spectrometry (HPLC-MS/MS) analysis. The β-D-glucopyranoside of guaiacol was
identified in extracts of Sangiovese grapes exposed to bushfire smoke and
Chardonnay grapes exposed to smoke under experimental conditions. Only trace
levels of the glucoside were identified in the corresponding control (i.e. unsmoked)
Chardonnay grapes, indicating glycosylation of smoke-derived guaiacol occurred in
response to smoke exposure. The reference compound and the glucoside present in
smoked juice samples remained largely unaffected following strong acid hydrolysis
but were highly susceptible to β-glucosidase enzyme hydrolysis, providing a plausible
explanation for the release of guaiacol during fermentation of smoke affected grapes.
Following the identification of additional guaiacol glycoconjugate precursors, the
d4-labelled analogue of guaiacol β-D-glucopyranoside was synthesised for use as an
internal standard in the development of a quantitative stable isotope dilution analysis
(SIDA) method, using HPLC-MS/MS. This method was subsequently applied to the
analysis of several grape varieties exposed to either experimental or bushfire smoke,
to investigate the accumulation of guaiacol glycoconjugates following grapevine
smoke exposure. Experimentally smoked grapes contained glycoconjugate
concentrations up to 300 µg/kg; whereas grapes affected by bushfire smoke
contained up to 2,000 µg/kg glycoconjugates, attributed to the different durations of
smoke exposure.
Analysis of separated berry components indicated that the majority of guaiacol
glycoconjugates were present in skin and pulp fractions, although approximately 6.7
times higher concentrations were found in the skins by mass. As such,
ii
glycoconjugate extraction from berry homogenate was considered to be more
efficient than from juice. To investigate the potential for smoke taint carry over to
subsequent growing seasons, grapes were collected from control and smoked Merlot
and Viognier grapevines in the season following smoke exposure. Subsequent
analysis showed no evidence to suggest grapevine sequestration of glycoconjugates.
The HPLC-MS/MS based SIDA method was adapted for the quantification of
guaiacol glycoconjugates in wine and the method applied to several winemaking
trials to investigate glycoconjugate metabolism during fermentation. Reduced skin
contact achieved using a ‘cold soak’ winemaking technique, yielded wines with
significantly lower concentrations of guaiacol glycoconjugates, compared with
traditional red winemaking practices involving extended skin contact at ambient
temperature. This suggests winemaking processes which limit precursor extraction
might offer opportunities for winemakers to ameliorate the impact of smoke taint in
wine. A yeast selection trial demonstrated only partial metabolism of glycoconjugates
during fermentation, with glycoconjugate concentrations of finished wines not
significantly influenced by choice of yeast strain. The fact that a considerable portion
of the glycoconjugate pool remained after fermentation has important implications for
industry; i.e. hydrolysis of glycoconjugates after bottling could result in enhanced
smoke characters with ageing.
The effect of grapevine smoke exposure on grape berry microflora and the
performance of several winemaking yeast in the presence of smoke-derived volatiles
were also investigated. The growth of indigenous and winemaking yeast on yeast
media agar plates spiked with guaiacol, 4-methylguaiacol or a liquid smoke
preparation was investigated to further determine the impact of smoke-derived
volatile compounds on yeast performance.
viii
ACKNOWLEDGEMENTS
Throughout the last three years I have been fortunate enough to have a great team
of people behind me supporting and helping me the whole way through my study.
Firstly many thanks to my principal supervisor Dr. Kerry Wilkinson who convinced me
to do a PhD, your guidance and support was crucial to the success of this project and
I am grateful to have had such a supporting supervisor. Thankyou also to my
supervisors Prof. Dennis Taylor for his help and advice throughout the last three
years and Dr. Yoji Hayasaka from the Australian Wine Research Institute, who has
enabled all the HPLC-MS analysis for this work, he is truly the master of the HPLC-
MS and without his expertise this project would have been a lot more difficult to
achieve.
Without valuable funding from the GWRDC this work would not have been conducted
so I thank them for their financial support. Also to the Department of Agriculture,
Fisheries and Forestry for awarding me the 2009 Science and Innovation award for
young people in Agriculture, Fisheries and Forestry and the associated funding that
supported the microbiological research to be conducted. The University of Adelaide
provided laboratories and facilities for this research for which I am grateful.
I have been fortunate enough to build some important collaborations during my
research. Firstly I would like to thank Dr. Alan Pollnitz for his helpful discussions and
guidance. Also to Mrs Gayle Baldock from the Australian Wine Research Institute
who helped with GC-MS and HPLC-MS analysis.
There are so many people who have helped me with this research, but I have to start
by thanking all the members of the Wilkinson group especially Anthea and Renata for
their assistance with the challenging field work and great friendship as well. Crista
ix
Burbidge provided me with microbiological technical assistance, without which I
would have been lost. Various other people have been essential in helping me with
my research and being there as friends, including all the guys and girls in the Taylor
group.
Finally and most importantly I need to thank my friends and family for always
supporting me with everything I have done. To my parents who helped support me
throughout my study, I am so grateful for having those opportunities. My husband
Chris you have been there for me when I was stressed and unsure about where I
was going, you have pushed me to succeed and I will love you always.
Chapter 1 1
CHAPTER 1
INTRODUCTION.
Chapter 1 2
CHAPTER 1: INTRODUCTION.
1.1 Introduction to the Australian wine industry.
The Australian wine industry contributes significantly to the Australian economy, with
in excess of $2.0 billion in domestic sales and $2.5 billion in export sales reported in
2007/08.1 In 2006, Australia became the 4th largest exporter of wine in the world,
behind France, Italy and Spain; with 776.6 million litres of wine exported, primarily to
the UK, USA, Canada, China, Germany and New Zealand.2 In any given year, the
success of the Australian industry is determined by both the yield and the quality of
grapes produced. In 2009, 1.73 million tonnes of grapes were crushed to produce
1.16 billion litres of wine, but in 2010 the winegrape crush decreased by almost 12%,
to 1.53 million tonnes.3
Over the past 10 years, annual grape yields have fluctuated from approximately
1.4 M tonne (in 2003 and 2007) to approximately 1.9 M tonne (in 2004 and 2006).4
Reduced yields have been attributed to atypical environmental conditions, in
particular drought and frost, as well as several major bushfires.2,5 The main issue
arising from bushfires is not fire damage to vineyards and wineries, although in some
cases this has occurred, but rather grapevine exposure to smoke (Figure 1), which
can result in objectionable ‘smoke’ characters being observed in subsequent wines.6
Given the incidence of vineyard exposure to smoke is likely to increase due to the
prolonged warm, dry conditions associated with climate change,7 together with the
potential for significant financial losses, ‘smoke taint’ has become an issue of
increasing concern for grapegrowers and winemakers. To ensure the continued
demand for Australian wine in both domestic and export markets, industry needs to
gain a better understanding of the impacts of grapevine smoke exposure.
Chapter 1 3
Figure 1: A bushfire occurring in close proximity to a vineyard, resulting in grapevine
smoke exposure.8
1.2 History of vineyard exposure to smoke.
In recent years, vineyard exposure to smoke has been reported in wine regions
throughout the world, including Canada (Okanagan Valley), USA (California), South
Africa and Australia.9 The first incidence of vineyard smoke exposure in Australia was
reported in mid-January 2003, following bushfires in Victoria and Canberra.
Numerous fires broke out in the Kosciuszko and Namadgi national parks, as a result
of extreme weather conditions, such as strong wind, lightning and high
temperatures.10,11 Escalation of the fires occurred rapidly, burning many outer
suburbs of Canberra and casting thick smoke over the King and Alpine Valley wine
NOTE: This figure is included on page 3 of the print copy of the thesis held in the University of Adelaide Library.
Chapter 1 4
regions. As a consequence, smoke affected juice and wine submitted to the
Australian Wine Research Institute (AWRI) from these regions, were described as
exhibiting objectionable ‘smoky’, ‘burnt’, ‘ash’, ‘ashtray’ and ‘smoked salmon’ aromas,
with an ‘excessively drying’ back-palate and a retro-nasal ‘ash’ character.7 Smoke
affected fruit became a significant concern for winemakers, in particular
determination of the extent of smoke exposure and consequences for wine quality.
Financial losses to grapegrowers and winemakers were subsequently estimated at
$4 million.6
During the 2003 vintage, the AWRI, reported an inundation of samples for ‘smoke
taint’ analysis and enquiries from concerned grapegrowers and winemakers. In
response to the 2003 bushfires, AWRI conducted a series of preliminary
investigations which were published in their annual report.7 The major outcomes
resulting from this work included:
Identification of the volatile phenols, guaiacol and 4-methylguaiacol, as the
major contributors to smoke taint; the concentration of these phenols was
found to be strongly correlated with the intensity of perceived taint, but AWRI
acknowledged other smoke-derived compounds were likely to be present also.
Detection of guaiacol and 4-methylguaiacol in skin rather than pulp fractions of
smoke affected grapes.
The discovery that increased maceration times or maceration with leaf
material gave increased guaiacol concentrations in resultant wines.
Subsequent to the above findings, AWRI conducted several vineyard and winery
trials in an attempt to identify potential amelioration methods for reducing smoke taint
in grapes and wine.7 A ‘vineyard washing’ trial was conducted, which involved the
Chapter 1 5
application of cold water, cold water plus a wetting agent, warm water, cold water
plus 5% ethanol and milk treatments to smoke affected grapevines. However, none
of these treatments gave significantly reduced juice guaiacol concentrations. The
washing liquids were found to contain some particulate matter, but guaiacol and 4-
methylguaiacol were not detected in any of these samples, indicating vineyard
washing did not effectively reduce volatile phenol concentrations.7 However, the
vineyard water wash was still considered to be beneficial, as it removed up to 90% of
smoke-derived ash and particulate matter which could potentially have contained
other smoke components capable of contributing undesirable sensory attributes.7 A
fining trial was also conducted and the capacity of various fining agents to remove
guaiacol from smoke affected wine was investigated. Of the fining agents trialled,
only activated carbon was found to remove guaiacol; however the 5% reduction
achieved was minor, and therefore not especially beneficial to winemakers.7
Based on these findings, the AWRI made a number of recommendations to the
Australian wine industry to assist grapegrowers and winemakers to minimise the
effects of smoke on grapes and wine.7 They recommended leaf plucking followed by
a cold water vineyard wash, hand picking and whole bunch pressing fruit would most
likely minimise the intensity of smoke taint in resultant wines. AWRI also suggested
that reduced maceration times would limit the extraction of guaiacol during
fermentation.7 The recommendations provided by AWRI were based on the
outcomes of their trials, but these trials lacked detailed experimental design, in
particular replication. As such these findings cannot be considered conclusive and
further research in this area is warranted.
Following their preliminary trials, AWRI reported ‘there is a possibility that smoke taint
might become a sporadic but more common occurrence in the future’. This prediction
Chapter 1 6
proved accurate and subsequent bushfires indeed occurred, with a series of major
fires occurring in north eastern Victoria between the 1st December 2006 and the 7th
February 2007.6 As a consequence, smoke taint was identified by grapegrowers and
winemakers in the King and Ovens Valleys, Milawa, Beechworth and Glenrowan
regions; i.e. not only wine regions in close proximity to fires but also more distant
regions, due to wind patterns which caused smoke to drift.12 Direct financial losses of
up to $20 million were estimated in 2007, being the cost associated with discarding
smoke affected fruit. However, total losses were estimated to be closer to $90 million,
being the value of expected profits from wine sales, together with subsequent loss of
shelf space and impact on reputation of brands.9
Severe bushfires occurred again in February 2009, in areas surrounding the Yarra
Valley wine region.13 While many vineyards reported financial losses associated with
fire damage to vineyards, drifting smoke plumes also resulted in smoke exposure of
fruit in a more widespread area of the Yarra Valley and Victoria (Figure 2). The Yarra
Valley Winegrowers Association reported fire damage or destruction of 29 vineyards,
with some individual growers losing up to 40 hectares of vineyards.13
Chapter 1 7
Figure 2: Satellite image of smoke from the Black Saturday bushfire, taken on
February 7th 2009; the Yarra Valley wine region is circled in red.14
Although bushfires have been the major cause of smoke taint in wines, prescribed
burns conducted in the vicinity of wine regions, have also resulted in smoke affected
grapes and wine.15 Winemakers in Western Australia sought millions of dollars in
compensation from the WA Department of Environment and Conservation following
vineyard exposure to smoke as a result of prescribed burns conducted during the
2004 growing season.15 Forestry Tasmania also fielded complaints from
grapegrowers regarding the detrimental impact of smoke from prescribed burns on
their vineyards.16 In response, some government agencies responsible for prescribed
burning have introduced new guidelines detailing the schedule of prescribed burns in
efforts to work more closely with growers and to identify more compatible burn times
which minimise the likelihood of smoke damage to vineyards.16 While improved
communication between government agencies and the wine industry will reduce the
NOTE: This figure is included on page 7 of the print copy of the thesis held in the University of Adelaide Library.
Chapter 1 8
impact of smoke from prescribed burns, the occurrence of bushfires is expected to
increase as a consequence of the hot and dry conditions caused by climate change.
As such, further research concerning the impact of smoke on grape and wine
production is warranted.
1.3 The effects of smoke on nature.
Prior to 2003, the effects of smoke on grape and wine composition and quality had
not been considered. However, considerable research has been undertaken to
investigate the role of smoke in seed germination. The application of smoke to seeds
from a variety of plants has been shown to stimulate seed germination, in some
cases prompting dormancy to be broken in seeds of threatened species.17 Although
smoke application doesn’t positively influence the seed germination rate of all
species, it has enabled the regeneration and conservation of many plant species;
Brown and van Staden17 reported improved germination of seeds from 45 of 94
native Western Australian plants following smoke exposure.
Flematti and coworkers18 attempted to identify the smoke constituents responsible for
seed germination by isolating an active fraction of an aqueous smoke extract using a
combination of solvent partitioning, acid-base separation, column chromatography
and semi-preparative high performance liquid chromatography (HPLC). The active
smoke fraction obtained was then applied to seeds of three plant species: Lactuca
sativa L. Grand Rapids, Conostylis aculeate R. Br. and Stylidium affine Sonder,
which improved seed germination rates by more than 100%.18 Subsequent HPLC-MS
analysis of this active fraction revealed the presence of 10 different compounds, each
of which was separated by collecting 1 minute elution fractions from the HPLC-MS.18
The seed germination potential of each individual compound was investigated using
Chapter 1 9
Grand Rapids seeds, and the active compound, with an elution time of 25 to 27 mins
and a quasi-molecular ion of m/z 151 was tentatively identified.18 The active
constituent was later confirmed to be a butenolide, following comparison with a
synthetic reference.19
While the role of smoke in seed germination has been extensively studied and
documented in the literature, there is little research concerning the effect of smoke on
plant physiology. Gilbert and Ripley investigated the photosynthetic response of
Chrysanthemoides monilifera (more commonly known as Boneseed in Australasia)
following smoke exposure, being the only physiological study conducted to date.20
Greenhouse grown plants, of no less than seven months in age, were exposed to
grass derived smoke using a commercial bee smoker, for a duration of one minute.20
Smoke exposure resulted in a significant decrease in carbon dioxide assimilation
rates, stomatal conductance and internal carbon dioxide concentrations.20 Complete
recovery of photosynthetic gas exchange rates was reported 24 hours post-smoke
exposure. Plants subjected to five consecutive days of one minute smoke treatments
showed no physiological response to smoke application on the fifth day, which
suggested the plants had developed resistance to smoke exposure.20 Longer periods
of smoke exposure, i.e. 5 minutes, had a more pronounced effect on plant health
and resulted in leaf necrosis and shoot death.20 This indicated that extended periods
of smoke exposure might damage plant tissue.
The anti-microbial effects of smoke have also been investigated. Although smoke
can have a detrimental effect on the health of living plants, it has long been used for
the preservation of food products, such as fish, cheese and meat.21 Specifically
smoke inhibits the growth of micro-organisms in food and kills bacteria known to
cause disease,22 for example, Listeria monocytogenes, is a micro-organism present
Chapter 1 10
in soft cheese, milk, meat and fish which is known to cause illness and food
poisoning when ingested.22 Niedziela et al. reported the inhibition of Listeria
monocytogenes in salmon following treatment with smoke.22 Similarly, Faith et al.
demonstrated the anti-listerial properties of liquid smoke preparations.23 In addition to
the anti-microbial nature of smoke, a range of smoke components isolated from liquid
smoke preparations, for example lignin dimers, have been shown to exhibit anti-
oxidant and organoleptic properties.24 Smoke preservation techniques therefore offer
potential benefits in addition to the inhibition of harmful micro-organisms.
Aside from preservation properties, the unique flavour and aroma imparted by smoke
has become an important characteristic of foods prepared using smoke preservation
techniques.21 The number and nature of chemicals which contribute to the aroma and
flavour of smoke has been the subject of considerable research. Of the compounds
identified to date, the volatile phenols are considered by many researchers to be the
major contributors of ‘wood smoke’ aroma.21,25-28 The volatile phenolic fraction of
smoke is also thought to be the major contributor of the ‘smoky’ aroma and flavour of
smoked food products.21 As such, the composition of smoked food has been well
investigated and guaiacol and 4-methylguaiacol have been identified as two of the
major volatile organic compounds to which the ‘smoky’ aroma in foods has been
attributed.29
1.4 Composition of smoke.
Smoke is a highly complex matrix and the precise composition of smoke depends on
the nature and moisture content of the fuel source, the temperature of combustion
and the availability of oxygen.28 More than 400 volatile organic compounds have
been identified in smoke and liquid smoke preparations.27,28,30 These compounds
Chapter 1 11
include: acids; alcohols; aldehydes; ketones; esters; furan and pyran derivatives;
lactones; phenols; ethers; hydrocarbons; and nitrogenated derivatives.26,28 Although
the combination of these compounds provide smoke with its unique flavour profile,
the volatile phenols have been identified as the major contributors.21,25,26,28,30-33
Smoke is produced by the thermal combustion of a fuel source such as wood or plant
material. The volatile phenol fraction of smoke is principally derived from the
pyrolytic degradation of lignin to give ferulic acid, which has been shown to undergo
further thermal decomposition to produce a series of volatile phenols.31
Volatile phenols comprise an aromatic ring with one or more hydroxyl groups, as well
as other functional groups such as aldehydes, ketones, acids and esters.28 Guaiacol,
4-methylguaiacol, 4-ethylguaiacol, 4-ethylphenol and eugenol are the more abundant
volatile phenols identified in smoke; their chemical structure and sensory descriptors
are shown below (Figure 3).21,28
OH
24-methylguaiacol 'sweet', 'smoky',
'toasted', 'ash'
34-ethylguaiacol
'sweet', 'smoky', 'spicy'
4eugenol
'clove', 'woody'
OH
54-ethylphenol
'pungent', 'horsey', 'barnyard'
OCH3
OH
OCH3
OH
OCH3OCH3
OH
1guaiacol 'sweet', 'smoky', 'pungent'
Figure 3: Volatile phenols identified in wood smoke and liquid smoke preparations,
and their sensory descriptors. 27,28,34
Chapter 1 12
Smoke-derived volatile phenols are important to the flavour and aroma qualities of
smoke and not surprisingly they have been identified in commercial liquid smoke
preparations used to artificially flavour foods.25,26,35 Liquid smoke preparations are
commonly applied to foods such as meat, fish and cheese to impart desirable
‘smoke’ attributes without necessitating the use of specialised smoke equipment.26
Liquid smoke flavourings are prepared by introducing smoke into a liquid matrix,
often distilled water.25,26 Smoke flavourings can differ in viscosity, colour and odour,
depending on the matrix used to retain smoke-derived volatiles, and the
concentration and ratio of individual components within the matrix; which are
influenced by fuel source and parameters used for combustion.24 Commercial smoke
preparations have generally been found to contain different ratios of carbonyl and
phenolic compounds, with those containing a higher proportion of carbonyl
compounds (for example 2-propanone, 2,3-dimethyl-2-cyclopenten-1-one and 2-
ethyl-2,5-dimethylcyclopenten-2-one), considered to best reflect the sensory
characteristics of smoke.24 Not surprisingly, many of the volatile organic compounds
present in smoke and liquid smoke flavourings are also identified in smoked food
products, and some of these volatiles could potentially be responsible for smoke taint
in grapes and wine.
The volatile phenols guaiacol and 4-methylguaiacol have not only been identified in
wine as a result of smoke, but are typically attributed to oak maturation.36-38 Wine is
traditionally aged in oak barrels to enhance aroma, flavour and complexity. During
barrel maturation, oak-derived volatile compounds including guaiacol and 4-
methylguaiacol can be extracted from the oak wood into the wine.36,37,39 Oak aged
wines typically contain between 10 and 100 µg/L of guaiacol and between 1 and 20
µg/L of 4-methylguaiacol39, and at concentrations exceeding their detection
thresholds, (Table 1) are considered to contribute desirable sensory characters.39
Chapter 1 13
However, in smoke tainted wines these phenols may contribute to the objectionable
‘smoky’, ‘burnt’, ‘ashtray’, ‘smoked salmon’ characters7, which anecdotally are
thought to be more apparent in white wine varieties.
Table 1: Aroma detection thresholds and wine concentrations for guaiacol and 4-
methylguaiacol.
Compound Aroma detection threshold (µg/L) wine concentration
water white juice red wine
guaiacol 0.4840 <641 9.542 0-10039
4-methylguaiacol 1034 6534 6534 0-2039
1.5 Smoke taint in grapes and wine.
Until recently there was no scientific literature concerning smoke taint in grapes and
wine. However, the recurrence of bushfires in close proximity to wine regions
prompted several research groups to investigate the effects of smoke on grape and
wine production.
The first scientific literature concerning smoke taint comprised a series of papers by
Kennison and collegues.43-45 Their first paper aimed to demonstrate the link between
smoke exposure of grapes and smoke taint in wine, by comparing the composition
and sensory attributes of smoke affected and control wines.45 Verdelho bunches
were exposed to straw-derived smoke post-harvest for 1 hour. Control (i.e. no smoke
exposure) and smoked grapes were then fermented according to two different
winemaking protocols: one involving juice clarification and primary fermentation, i.e.
reflecting commercial white winemaking; and one involving oxidative primary
Chapter 1 14
fermentation with skin contact, followed by malolactic fermentation, i.e. reflecting
commercial red winemaking.
Wines were then subjected to chemical and sensory analyses to determine the effect
of smoke exposure and winemaking treatments.45 The concentrations of a range of
volatile phenols including guaiacol, 4-methylguaiacol, 4-ethylguaiacol, 4-ethylphenol
and eugenol were determined by gas chromatography-mass spectroscopy (GC-MS).
Irrespective of the winemaking treatment employed, the volatile phenols were not
detected in wines made from control (unsmoked) grapes, but were detected in wines
made from smoked grapes (Table 2). Guaiacol and 4-methylguaiacol in particular
were reported at elevated levels; while the concentrations of 4-ethylphenol, 4-
ethylguaiacol and eugenol were within ranges previously reported in wine (Table 1).45
Table 2: Concentrations of guaiacol, 4-methylguaiacol, 4-ethylguaiacol, 4-
ethylphenol and eugenol present in smoked and unsmoked wines.45
Concentrationa (µg/L)
Smoked free run
Unsmoked free run
Smoked free run on skins
Unsmoked free run on skins
guaiacol 1470 a n.d. 969 b n.d.
4-methylguaiacol 326 a n.d. 250 b n.d.
4-ethylguaiacol 128 a n.d. 111 b n.d.
4-ethylphenol 59 a n.d. 67 b n.d.
eugenol 20 a n.d. 26 b n.d. a Values followed by a different letter within rows are significantly different. n.d.= not detected. Mean
values from three replicates. Values were in agreement to ca. 5%.
Sensory analysis confirmed a perceivable difference between smoked and control
wines (at the 99.9% confidence level).45 The sensory panel were able to differentiate
smoked wine blended with control wine, until a 98% dilution factor was achieved. On
Chapter 1 15
this basis, the authors concluded industry probably couldn’t rely on blending to
significantly diminish smoke related sensory attributes of smoke tainted wine.
Kennison and colleagues subsequently investigated the application of smoke to
grapevines in the field and the evolution of volatile phenols during fermentation.43
Merlot grapevines were exposed to repeated smoke treatments (8 x 30 mins each) at
different timepoints between veraison and harvest, using purpose built smoke tents
erected around the vines and straw-derived smoke.43 Control grapevines were also
enclosed in tents, but without smoke exposure, to eliminate any effects of the tent. At
maturity, grapes from control and smoked grapevines were fermented; primary
fermentation was conducted with skin contact followed by malolactic fermentation,
with samples collected at various stages of winemaking for analysis by GC-MS to
determine volatile phenol concentrations.43
Consistent with previous findings, Kennison et al.43 showed that volatile phenols were
either not detected or detected at only trace levels in control wines (Table 3). The
volatile phenol concentrations of smoked ferment samples increased progressively
throughout the winemaking process, with guaiacol and 4-methylguaiacol again being
the most abundant phenols (Table 3). This finding supported anecdotal evidence
from winemakers that the intensity of smoke taint increased with fermentation. The
authors considered these results could indicate the progressive release of phenols
from grape skins, except that phenol concentrations continued to increase after the
wines were pressed off the skins (Table 3). Further increases were also observed for
some phenols after 12 months bottle ageing. The authors instead suggested the
evolution of phenols after pressing and bottling might be due to the hydrolysis of
precursor forms of the phenols.
Chapter 1 16
Table 3: Concentrations of guaiacol, 4-methylguaiacol, 4-ethylguaiacol,
4-ethylphenol and eugenol during fermentation of fruit derived from smoked and
unsmoked grapevines.43
Concentrationa (µg/L)
Sample guaiacol 4-methyl guaiacol
4-ethyl guaiacol
4-ethyl phenol
eugenol
unsmoked
free run juice n.d. n.d. n.d. n.d. n.d.
after 1 day maceration tr. tr. n.d. n.d. tr.
after 3 days maceration tr. tr. n.d. n.d. tr.
after 5 days maceration tr. tr. n.d. n.d. tr.
after 7 days maceration tr. tr. n.d. n.d. tr.
after 10 days maceration 1 tr. n.d. n.d. tr.
after alcoholic fermentation 1 tr. n.d. n.d. tr.
finished wine 4 n.d. tr. tr. tr.
12 months post-bottling 3 tr. tr. tr. n.d.
smoked
free run juice 1 a tr. n.d. n.d. n.d.
after 1 day maceration 68 b 11 a 10 a 5 a 2 ab
after 3 days maceration 168 c 26 b 8 a 5 a 1 a
after 5 days maceration 203 cd 32 bc 9 a 15 b 2 a
after 7 days maceration 249 d 42 c 9 a 17 b 2 a
after alcoholic fermentation 249 d 43 c 8 b 23 c 1 a
finished wine 388 e 93 d 16 c 58 d 3 b
12 months post-bottling 371 e 124 e 29 c 94 e 4 c
aValues are the means from three replicates and were in agreement with ca. 10%. Values followed by a
different letter within columns are significantly different (P < 0.05). n.d.= not detected; tr.= trace (i.e. positive identification but < 1µg/L)
To investigate their hypothesis a series of hydrolysis studies were performed.43 Free
run juice from control and smoke affected Merlot grapes was hydrolysed under either
mildly acidic (i.e. pH=3.5), strongly acidic (i.e. pH=1) or enzymatic (β-glucosidase)
conditions. Only trace levels of phenols were detected in control hydrolysates;
whereas the concentration of phenols in smoked juice increased significantly
following either strong acid or enzyme hydrolysis (Table 4).43 This data provided
Chapter 1 17
further evidence to support the authors’ hypothesis that guaiacol might be bound
within the grape in precursor form. Furthermore, evolution of guaiacol following
treatment with β-glucosidase enzymes leads the authors to suggest these precursors
might be glycoconjugate in nature.
Table 4: Volatile phenol concentrations before and after mild acid (pH=3.5), strong
acid (pH=1) and β-glucosidase enzyme hydrolysis.43
Concentrationa (μg/L)
Sample guaiacol 4-methyl guaiacol
4-ethyl guaiacol
4-ethyl phenol
eugenol
unsmoked
free run juice n.d. n.d. n.d. n.d. n.d.
mild acid hydrolysate tr. tr. tr. tr. n.d.
strong acid hydrolysate tr. tr. tr. tr. 2
enzyme hydrolysate tr. tr. tr. tr. n.d.
smoked
free run juice 1 tr. n.d. n.d. n.d.
mild acid hydrolysate tr. tr. tr. tr. n.d.
strong acid hydrolysate 431 162 31 48 5
enzyme hydrolysate 325 82 13 27 n.d. a Values are the means from three replicates for juice samples and two replicates for hydrolysate
samples. Values were in agreement to ca. 10%. n.d.= not detected; tr.= trace (i.e. positive identification but < 1µg/L).
Kennison and co-workers then investigated the effect of timing and duration of
grapevine exposure to smoke.44 Merlot grapevines were exposed to a single smoke
treatment (for 30 min) at one of eight different time points between veraison and
harvest. A second treatment involved Merlot grapevines being exposed to eight
repeated smoke applications. In each case, treatments were taken to a wine
outcome for chemical and sensory analysis. Once again, only trace levels of phenols
were detected in control wines. For single smoke treatments, all smoked wines were
found to contain smoke-derived volatile phenols and to exhibit some degree of
Chapter 1 18
smoke related sensory characters. However, the highest phenol levels and most
apparent smoke taint was reported for wines made from grapes exposed to smoke 7
days post-veraison, suggesting at this phenological timepoint grapes are most
vulnerable to smoke.44 Considerably higher phenol levels were observed for wines
derived from repeated smoke treatments, demonstrating that prolonged or repeated
smoke exposure will result in a more intense smoke taint. A similar study
investigating the effects of timing of smoke exposure was conducted by Sheppard et
al.46 Fruit from Chardonnay, Pinot Gris and Merlot grapevines were exposed to
smoke produced from pine, burned in a modified barbeque and pumped into a box
surrounding the vines. Smoke was applied to vines at three different stages of
growth, preveraison, postveraison and maturity, and the grapes were harvested and
analysed by GC-MS to determine guaiacol and 4-methylguaiacol concentrations. The
authors of this study also concluded that the timing of grapevine smoke exposure
influenced guaiacol and 4-methylguaiacol concentration, and that grape variety might
also affect the uptake of smoke.46
Kennison’s research strongly suggests smoke-derived volatile phenols accumulate in
grapes in glycoconjugate forms (i.e. as glucose derivatives), following grapevine
exposure to smoke. Industry currently relies on quantification of guaiacol and 4-
methylguaiacol using existing GC-MS based analytical methods to assess the extent
of taint in smoke affected grapes.43 However these methods do not account for
‘bound’ or precursor forms of guaiacol and 4-methylguaiacol, so there is significant
potential for smoke taint to be under-estimated. Grapes with low or undetectable
volatile phenol levels might release significant levels of these phenols, and therefore
smoke taint, during fermentation. Therefore, the provenance of glycoside derivatives
of volatile phenols in smoke affected fruit needs to be established and analytical
methods specific to these precursors subsequently developed.
Chapter 1 19
1.6 Research aims.
Given the close proximity of many grape growing regions to bushland and forests,
and the warm, dry conditions experienced during summer, the incidence of bushfires,
and therefore smoke taint is likely to continue. A number of volatile phenols have
been identified in smoke tainted grapes and wine, but research findings to date
suggest these compounds might accumulate in smoke affected grapes in precursor
forms, i.e. as glycosides. This project therefore aimed to investigate the provenance
of glycoside precursors of guaiacol, as the most abundant of the smoke-derived
volatile phenols.
The project aimed to:
1. Synthesise the β-D-glucoside of guaiacol as a reference compound to confirm
its presence in smoke affected grapes.
2. Synthesise the deuterated β-D-glucoside of guaiacol for use as an isotopically
labelled internal standard for the development of a quantitative high
performance liquid chromatography tandem mass spectrometry (HPLC-
MS/MS) based Stable Isotope Dilution Analysis (SIDA) method.
3. Apply the HPLC-MS/MS method to the analysis of smoke affected grapes, to
investigate the accumulation and distribution of glycoconjugates.
4. Apply the HPLC-MS/MS method to the analysis of smoke affected wines to
investigate the behaviour of the glycoconjugates during fermentation and
bottle storage (ageing).
To date, the primary focus of smoke taint research conducted has concerned the
chemical composition and sensory characteristics of smoke affected grapes and
wine. However, the anti-microbial, preservative properties exhibited by smoke could
Chapter 1 20
potentially influence the growth of indigenous microflora on grapes or the
performance of winemaking yeast during fermentation. As such, this project also
aimed to:
5. Investigate the impact of smoke on grape berry microflora and fermentation
rates.
6. Investigate the growth of winemaking yeast in the presence of smoke-derived
volatile compounds.
Chapter 2 21
CHAPTER 2
SYNTHESIS OF GUAIACOL GLUCOSIDES.
Chapter 2 22
CHAPTER 2: SYNTHESIS OF GUAIACOL GLUCOSIDES.
2.1 Introduction to glycosides in grapes and wine.
Glycosides comprise an aglycone, with one or more sugar units attached. A range of
different glycosides have been identified in grapes including rutinosides,
disaccharides and glucosides.47 Glycosides are thought to play a role in the storage
and transport of hydrophobic compounds in the plant, facilitated by the increased
solubility afforded by sugar units, as well as reduced reactivity and potential toxicity
of aglycones.47
Glycosides are ubiquitous, occurring frequently in nature. For example, many fruits
including the cupuacu, anise, green vanilla beans, cape gooseberry and tomatoes
have been found to contain glycosides of volatile phenols.48-53 The glycosides within
these fruits are responsible for the containment of a portion of volatile aroma
compounds which may provide significant aroma potential for these fruits. For
example, 24 out of the 47 aglycones identified in the Amazonian fruit capuacu, were
found only in the enzyme hydrolysates of the glycoside fraction and not in the free
volatile fraction; identifying the important role glycosides play in the flavour profile of
this fruit.48 It is well known that many grape derived aroma volatiles occur in
glycosidic precursor forms.54-62 Although glycosides possess no odour or flavour
properties, they can be metabolised by yeast and enzymes during primary and
malolactic fermentation to release odour active aglycones.58,60 As such, acid and
enzyme hydrolysis have been employed in many wine flavour related studies to
release volatile compounds from glycosidic precursors, for example to enable the
identification of novel molecules.42,55,57,58,60,63,64 Many compounds have been isolated
and identified in grapes by this method including monoterpenes, alcohols, aliphatic
Chapter 2 23
alcohols and shikimates.62,65-67 The norisoprenoid, β-damascenone is an important
aroma compound present in grapes and wine, contributing to ‘stewed apple’, ‘exotic
fruit’ and ‘honey’ characters.68 β-damascenone occurs in grapes in either free or
glycosidically-bound forms; the glycoside typically being quantified by release of β-
damascenone following hydrolysis.64 Similarly, benzenoid compounds such as
vanillin and phenol have been identified in the acid and enzyme hydrolysates of
Merlot and Cabernet Sauvignon grapes.60
Glycosides are not only associated with grape-derived volatiles but also oak-derived
volatiles. Cis- and trans-oak lactone, possibly one of the most important oak-derived
volatiles, responsible for ‘coconut’, ‘citrus’ and ‘vanilla’ characters,69 can also exist in
glycosidic forms.70,71 The galloyl-β-D-glucoside of cis-oak lactone has been isolated
from oak wood and shown to liberate oak lactone under strongly acidic, enzymatic
and pyrolytic conditions.71 The toasting process of cooperage and enzyme activity
during fermentation can also release oak lactone from its glycosidic precursors.
Glycosides therefore play an important role in the liberation of aroma volatiles during
winemaking. For this reason, the accumulation of glycosides of smoke-derived
volatile phenols and their subsequent metabolism during fermentation, could explain
the results reported by Kennison et al.43 i.e. the release of volatile phenols from free-
run juice of smoke affected Merlot grapes following treatment with β-glucosidase
enzyme strongly supports Kennison’s hypothesis that precursors are glycosidic in
nature. However, further work is required to validate the provenance of the guaiacol
β-D-glucoside in smoke affected grapes and wine.
Chapter 2 24
2.2 Introduction to guaiacol β-D-glucopyranoside.
Guaiacol has been isolated in the enzyme and acid hydrolysates of a number of
plants and fruit, such as tomatoes, cape gooseberries and grapes, suggesting that it
is present as a glycosidic precursor.48,52,53,65,67 For example, hydroponically grown
tomato cultivars, Jorge, Durinta and p73, chosen for their economical importance and
usefulness for genetic transformation, were hand harvested at commercial maturity,
homogenised and hydrolysed to enable analysis of their volatile flavour
components.53 Tomato juice from each variety was analysed for free and bound
equivalents of volatile flavour compounds, by gas chromatography (GC). Glycosidic
fractions were isolated by solid phase extraction, and volatile components released
by pectinase hydrolysis.53 The concentration of free guaiacol in the tomato varieties
ranged from 503 - 945 µg/L, while bound concentrations ranged from 70 - 113 µg/L.53
Similarly fresh cape gooseberries were harvested, homogenised, selectively
concentrated by solid phase extraction, hydrolysed with a non-selective glucosidase
enzyme and analysed by capillary gas chromatography-mass spectrometry (GC-
MS).52 Guaiacol was measured at concentrations ranging from 300 - 700 µg/kg of
fruit in the hydrolysates, indicating the presence of guaiacol glycoside precursors in
the cape gooseberry.52
Interestingly, guaiacol has also been identified in grape juice hydrolysates derived
from a range of varieties, including Shiraz and Merlot.60,67 Shiraz berries subjected to
enzymatic hydrolysis contained 17 µg/kg of guaiacol, well above the detection
threshold of 9.5 µg/L in red wine.67 Enzyme and acid hydrolysates of Merlot juice
were found to contain up to 50 µg/L of guaiacol and the concentrations of guaiacol
were seen to increase when a greater quantity of enzyme was used during
Chapter 2 25
hydrolysis.60 The natural occurrence of guaiacol and its glycosidic precursors,
supports the presence of glycosidic precursors within smoke affected fruit.
2.3 Introduction to glycosylation.
Glucosides are comprised of a glucose unit attached to the hydroxyl group of an
aglycone in either an α or β-position (Figure 4). Glycosylation reactions usually
involve two synthetic steps: (i) linkage of a protected glucose moiety to the aglycone
unit, and (ii) deprotection, whereby the protecting groups on the glucose moiety are
removed. Glycosidic extracts isolated from plants show that β-glucosides occur more
commonly in nature than α-glucosides, due to the effectiveness of the β-glucosidase
enzyme in releasing volatile compounds.59,60,72 Various synthetic strategies have
been developed to direct β-glycosylation, but the most effective is considered to be
the modified Koenigs-Knorr method, which involves the use of a protected glucose
unit as the sugar donor, in the presence of silver triflate as a catalyst .73
Figure 4: i) β-guaiacol glucoside and ii) α-guaiacol glucoside
O
O
OH
OH
OH
HO
OCH3
O
H
OH
OH
OH
HO
OH
OCH3
ii)i)
Chapter 2 26
The oak lactone glucosides have been synthesised using a modified Koenigs-Knorr
method.70,71,74 A similar method was also used by Fudge et al. to synthesise
deuterium labelled cis-oak lactone; however, the glucose unit contained acetyl
protecting groups and the deprotection method used KOH and MeOH.70 Both
methods gave the desired reaction product, although Wilkinson et al.71 achieved
yields of 66% and 98% in the glucosylation and deprotection steps, respectively,
whereas Fudge et al.70 achieved significantly lower yields of 14% and 73%. These
methods are similar to those previously used to synthesise guaiacol β-D-glucoside.
2.4 Glycosylation of guaiacol.
The synthesis of the β-D-glucopyranoside of guaiacol has been reported previously
by Dignum et al. who reported an 18% yield using α-D-acetobromoglucose (Scheme
1).49 Zhou et al. reported the synthesis of an acetyl-protected guaiacol glucoside,
also using the α-D-acetobromoglucose as a reagent.75 These investigations provided
the basis for the preparation of guaiacol β-D-glucopyranoside in the current study.
OCH3
OH
guaiacol
OCH3
OGlu(OAc)4
protected guaiacol glucoside guaiacol glucoside
OCH3
OGlu
Scheme 1: Synthetic scheme for preparation of guaiacol β-D-glucopyranoside as
performed by Dignum et al. 49
α-(OAc)4Glu-Br, KOH, EtOH, CHCl3
(CH3)2CO/NaOH
Chapter 2 27
2.5 Results and discussion.
2.5.1 Preparation of guaiacol β-D-glucopyranoside.
2.5.1.1 Preparation of guaiacol glucoside using 2,3,4,6-tetra-O-acetyl-α-D-
glucopyranosyl bromide (Method 1).
The glycosylation of guaiacol was attempted using the methods reported by Dignum
et al.49 Low yields were obtained for both the glycosylation and deprotection steps,
and thin layer chromatography (TLC) indicated the presence of several by-products.
The major drawback of this method was the formation of significant quantities of the
α-isomer, confirmed by the characteristic anomeric proton signal, i.e. with a coupling
constant between 2-5 Hz. This glycosylation method involves non-selective isomeric
attack of the acetyl glucose giving a mixture of α- and β-isomers, which are difficult to
separate. Therefore, this glycosylation method was considered to be unsuitable for
preparation of guaiacol β-D-glucopyranoside.
2.5.1.2 Preparation of guaiacol glucoside using 2,3,4,6-tetra-O-pivaloyl-α-
D-glucopyranosyl bromide (Method 2).
A modified Koenigs-Knorr method71, using 2,3,4,6-tetra-O-pivaloyl-α-D-
glucopyranosyl bromide in the presence of lutidine and silver triflate was instead
employed for the glycosylation of guaiacol. The steric bulk of the pivaloyl protecting
groups inhibit nucleophilic attack from the α-face, thereby improving selectivity for β-
glycosylation.
Chapter 2 28
The guaiacol β-D-glucopyranoside was successfully synthesised using this method
with improved yields, i.e. 25% and 85% for the glycosylation and deprotection steps
respectively, compared with 21% and 16% obtained using method 1. NMR analysis
of the purified product showed no indication of the presence of the α-isomer. The
glycoside was characterised by 2D 1H and 13C NMR spectroscopy and HPLC-MS.
The anomeric proton produced a coupling constant of 6.6 Hz, characteristic of β-D-
glucopyranosides. HPLC-MS analysis of the guaiacol glucopyranoside further
confirmed purity; the glucoside eluted at 5.7 min with a dominant ion of m/z 345.5,
i.e. as an acetic acid adduct ion [M-H + CH3COOH]¯, with a minor ion of m/z 285.0
as the molecular ion [M-H]¯ in the mass spectrum.
2.5.2 Preparation of deuterated guaiacol β-D-glucopyranoside.
The current analytical quantification of guaiacol and 4-methylguaiacol in smoke
affected grapes and wine utilises stable isotope dilution analysis (SIDA) in
conjunction with GC-MS. SIDA is a technique commonly used for quantification,
generally using either GC-MS or HPLC-MS.39,69,70,76,77 GC-MS based SIDA is
typically used for volatile compounds such as guaiacol,39 whereas HPLC-MS based
SIDA is better suited to non-volatile compounds such as glycosides. SIDA employs
an internal standard in the form of an isotopically labelled analogue of the analyte to
be quantified, added at a known concentration prior to sample preparation and
analysis. The HPLC or GC peak area of the analyte and the internal standard are
compared to determine the concentration of the analyte. Any loss of the analyte that
might occur during the extraction process is accounted for, by an equal loss of the
isotopically labelled internal standard.
Chapter 2 29
GC-MS based SIDA methods have been developed for the analysis of a wide range
of compounds in wine, for example β-ionone, β-damascenone and cis- and trans-
oak lactone.39,64,78,79 SIDA methods developed for wine and oak analysis are
currently used to quantify smoke-derived volatile phenols in grapes and wine.39,76
SIDA methods have also been developed for the quantification of aroma precursors,
for example the oak lactone glucosides.80 The use of HPLC-MS based SIDA for the
quantification of oak lactone glucosides provides the basis for the development of a
method for the quantification of the guaiacol β-D-glucopyranoside.
Isotopically labelled guaiacol β-D-glucopyranoside was synthesised in the same
fashion as the unlabelled glucoside, but from deuterated guaiacol. Pollnitz et al.
reported the synthesis of d3-guaiacol from catechol (i.e. via methylation of a hydroxyl
group), albeit with only a 30% yield.39 Deuterium exchange of the aromatic ring would
enable incorporation of four deuterium atoms, improving the molecular mass
difference between the analyte and the deuterated internal standard for HPLC-MS
analysis. Pollnitz et al. reported deuterium exchange of 4-ethylphenol using
deuterium oxide and thionyl chloride, in a reaction performed over 5 days.76 In the
current study, the reaction was instead performed with guaiacol using a microwave
reactor to significantly reduce the duration of the reaction. The microwave assisted
synthesis gave d4-guaiacol in 80% yield after just 30 hours reaction time. Conversion
of guaiacol to its d4-equivalent was monitored by 1H NMR and deuterium exchange
was considered complete when aromatic 1H peaks could no longer be detected.
Deuterium exchange was confirmed by GC-MS analysis, with a molecular ion of m/z
128.2 [M+] observed.
Synthesis of the isotopically labelled guaiacol β-D-glucopyranoside was subsequently
performed, according to glycosylation method 2. Confirmation of deuterium retention
Chapter 2 30
on the aromatic ring was carried out by HPLC-MS; the d4-glucoside eluted at 5.97
min, and gave an acetic acid adduct ion of m/z 349.2 [M-H + CH3COOH]¯, and
molecular ion of m/z 289.1; i.e. 4 atomic mass units heavier than the unlabelled
glucoside, as expected.
2.6 Materials and methods.
2.6.1 Solvents and reagents.
Hexane was distilled at atmospheric pressure under nitrogen. Dichloromethane was
dried over 4 Å molecular sieves (2.5 – 5 mm). All other solvents and reagents were
used as purchased from Sigma Aldrich or Crown Scientific.
2.6.2 Chromatography.
Analytical thin layer chromatography was performed with aluminium backed silica gel
60 F254 sheets from Merck. Column chromatography was performed with silica gel 60
F254 obtained from Scharlau (230 - 400 mesh).
2.6.3 Nuclear magnetic resonance spectroscopy (NMR).
1H and 13C NMR spectra were recorded with a Varian Gemini spectrophotometer
operating at either 200 MHz, 300 MHz or 600 MHz. Spectra were recorded in either
deuterated chloroform (CDCl3) or deuterated pyridine (C5D5N). Chemical shifts are
reported in parts per million (ppm) downfield. The following abbreviations are used in
the assignment of 1H spectra: s=singlet, d=doublet, m=multiplet, dd=doublet of
doublets, ddd=doublet of doublets of doublets, t=triplet.
Chapter 2 31
2.6.4 Ultra violet/visible spectroscopy and fluorescence spectroscopy.
UV/Vis spectra were recorded with a Varian Cary 5000 UV-Vis-NIR
spectrophotometer. Methanol was used as the solvent and as the blank.
Fluorescence spectra were recorded with a Varian Cary Eclipse fluorescence
spectrophotometer with methanol as the solvent.
2.6.5 Microwave synthesis.
Microwave assisted synthesis was performed using a CEM Discover microwave
reactor.
2.6.6 Gas chromatography-mass spectrometry (GC-MS).
GC-MS analysis was performed with an Agilent 5973N mass spectrometer (MS)
coupled to an Agilent 6890 gas chromatograph (GC) equipped with a GERSTEL
MPS2 Multi Purpose Sampler (Agilent Technologies, Forest Hill, N.S.W., Australia). A
1 µL sample was injected and chromatographed on a ZB-WAX fused silica capillary
column (Phenomenex, 7H6 – 6007 – 11, 30 m x 0.25 mm, 0.25 µm film thickness).
The carrier gas used was helium with a flow rate of 1.9 mL/min. During analysis,
oven temperature was started at 40°C, held at this temperature for 4 mins, increased
to 130°C at a rate of 5°C/min and then increased to 220°C at a rate of 10°C/min and
held at this temperature for a further 5 mins. The injector was set to split mode (ratio
50:1) and set at a temperature of 250°C. The transfer line was also set at a
temperature of 250°C. Positive Ion electron impact mass spectra were recorded in
selected ion monitoring (SIM) mode over a scan range of m/z 20-210.
Chapter 2 32
2.6.7 High performance liquid chromatography tandem mass
spectrometry (HPLC-MS/MS).
Mass spectrometric analysis was performed with a 4000 Q TRAP hybrid tandem
mass spectrometer equipped with a Turbo ion source (Applied Biosystems/MDS
Sciex) and coupled to an Agilent 1200 HPLC system equipped with binary pump,
degasser, autosampler and column oven (Agilent Technologies, Santa Clara, CA,
U.S.A.). Data acquisition and processing were performed using Analyst software
(version 1.5.1, Applied Biosystems/MDS Sciex).
Chapter 2 33
2.6.8 Synthesis. Guaiacol 2’,3’,4’,6’-tetra-O-acetyl β-D-glucopyranoside (1) (Method 1).
OCH3
Oglu(Ac)4
To a solution of 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (2.5 g, 6 mmol), in
chloroform (5 mL), was added a solution of guaiacol (620 mg, 5 mmol) and
potassium hydroxide (280 mg, 5 mmol) in ethanol (5 mL) and heated at reflux for 2
hours. The reaction mixture was cooled, filtered and ice water added. The organic
phase was then concentrated in vacuo to give a pale orange solid, which was purified
by column chromatography (66% ethyl acetate in hexane). The crude product was
then recrystallised from ethanol to give 1 as colourless needles (476 mg, 21%) (m.p.:
150-151 °C).
1H NMR (CDCl3): 7.12 (1H, dd, J = 7.8, 1.8 Hz, ArH), 7.07 (1H, ddd, J = 7.8, 1.8 Hz,
ArH), 6.90 (1H, ddd, J = 7.8, 1.8 Hz, ArH), 6.87 (1H, dd, J = 7.8, 1.8 Hz, ArH), 5.29
(2H, m, H1’, 3’), 5.17 (1H, m, H4’), 4.96 (1H, dd, J = 5.4, 2.4 Hz, H2’), 4.28 (1H, dd, J =
12, 5.4 Hz, H6a’), 4.16 (1H, dd, J = 12, 2.4 Hz, H6b’), 3.82 (3H, s, OCH3), 3.76 (1H, m,
H5’), 2.08, 2.07, 2.04, 2.04 (12H, 4 x s, Ac).
13C NMR (CDCl3): 170.6, 170.3, 169.4, 169.4, 150.7, 146.1, 124.7, 120.9, 120.3,
112.8, 100.9, 72.7, 72.0, 71.3, 68.5, 62.0, 56.0, 20.7, 20.6, 20.6, 20.5.
Chapter 2 34
Guaiacol β-D-glucopyranoside (2) (Method 1).
OCH3
Oglu
1 (200 mg, 0.4 mmol) was added to a mixture of 1M sodium hydroxide solution (5
mL) and acetone (5 mL). The mixture was stirred at room temperature for 1 hour and
monitored by TLC (60% ethyl acetate in hexane). The mixture was then stirred for a
further 30 min in the presence of acidified Dowex (H+) ion exchange resin. The
reaction mixture was filtered and concentrated in vacuo, and recrystallised with
ethanol to give 2 as a white solid (20 mg, 16%).
1H NMR (C5D5N): 7.6 (1H, ArH), 6.92-7.02 (3H, m, ArH), 5.67 (1H, d, J = 6.6 Hz, H1’),
4.52 (1H, dd, J = 12, 2.4 Hz, H6a’), 4.33-4.41 (4H, m, H2’, 4’, 5’, 6b’), 4.10-4.12 (1H, m,
H3’), 3.70 (3H, s, OCH3).
13C NMR (C5D5N): 150.7, 148.6, 123.0, 121.9, 116.9, 113.7, 102.7, 79.3, 79.0, 75.4,
71.7, 62.8, 56.4.
Chapter 2 35
1,2,3,4,6-Penta-O-pivaloyl-β-D-glucopyranoside81 (3).
O
OPiv
OPiv
OPiv
PivOPivO
D-Glucose (4.0 g, 22.2 mmol) was added portionwise to a solution of pivaloyl chloride
(27 mL, 222.0 mmol) and pyridine (18.0 mL, 222.0 mmol) in chloroform (100 mL) and
heated at reflux for 72 hours. The solvent was evaporated, the residue dissolved in
water (100 mL) and extracted with ethyl acetate (5 x 70 mL). The organic extracts
were combined and washed with water (100 mL), hydrochloric acid (1M, 100 mL),
saturated sodium bicarbonate (100 mL) and saturated sodium chloride (100 mL). The
solution was then dried over magnesium sulphate and the solvent removed in vacuo,
to afford the crude product which was then recrystallised from ethanol to give 3 as a
white crystalline solid (11.9 g, 89%) (m.p.: 148-150 °C).
1H NMR (CDCl3): 5.70 (1H, d, J = 9.6, H1), 5.37 (1H, t, J = 9.3, H3), 5.22 (1H, dd, J =
9.3 and 8.4, H2), 5.16 (1H, t, J = 9.6, H4), 4.16 (1H, dd, J = 12.3, 2.7, H6a), 4.10 (1H,
dd, J = 12.3, 4.8, H6b), 3.86 (1H, ddd, J = 10.2, 4.8, 2.7, H5), 1.24, 1.20, 1.18, 1.15,
1.12 (45H, 5 x s, CH3).
Chapter 2 36
2,3,4,6-tetra-O-pivaloyl-α-D-glucopyranosyl bromide81 (4).
O
OPiv
PivO
PivOPivO
Br
Hydrobromic acid solution (33%) in acetic acid (5 mL) was added dropwise to a
solution of 3 (5.4 g, 9.0 mmol) in dichloromethane at 0°C, and the mixture was stirred
at room temperature for 16 hours. The reaction mixture was then concentrated, co-
evaporating with benzene (2 x 20 mL) and diethyl ether (2 x 20 mL). The residue
obtained was then dissolved in diethyl ether (40 mL), washed with saturated sodium
bicarbonate solution (3 x 30 mL), then water (40 mL) and finally dried over
magnesium sulphate and concentrated. The crude product was recrystallised from
ethanol to give 4 as a white crystalline solid (2.2 g, 42%).
1H NMR (CDCl3): 6.62 (1H, d, J = 3.9, H1), 5.63 (1H, t, J = 9.6, H3), 5.21 (1H, dd, J =
10.3 and 9.4, H4), 4.81 (1H, dd, J = 9.9 and 4.2, H2), 4.34-4.28 (1H, ddd, J = 10.5, 3.9
and 3.3, H5), 4.18-4.16 (2H, m, H6a,6b), 1.22, 1.19, 1.17, 1.13 (36H, 4 x s, CMe3).
Chapter 2 37
Guaiacol β-D-2’, 3’, 4’, 6’-tetrapivaloyl glucopyranoside (5) (Method 2).
OCH3
Oglu(Piv)4
4 (469 mg, 0.81 mmol) was added to a solution of guaiacol (100 mg, 0.81 mmol) in
anhydrous dichloromethane (6 mL) containing silver triflate (210 mg, 0.81 mmol) and
2,6-lutidine (100 µL, 0.81 mmol). The reaction mixture was then stirred in darkness
for 16 hours at ambient temperature. The reaction was quenched with saturated
sodium bicarbonate solution (20 mL) and extracted with dichloromethane (2 x 15
mL). The organic extracts were then combined, washed with saturated sodium
chloride solution, dried and concentrated in vacuo. The crude product was then
purified by column chromatography (20-60% ethyl acetate in hexane) to afford 5 as a
white crystalline solid (142 mg, 25%) (m.p.: 131-132 °C).
1H NMR (CDCl3): 7.10 (1H, d, J = 7.8 Hz, ArH), 7.02 (1H, t, J = 7.2 Hz, ArH), 6.82-
6.90 (2H, m, ArH), 5.41 (1H, t, J = 9 Hz, H 3’), 5.33 (1H, t, J = 8.4, H2’), 5.17 (1H, t, J =
9.6, H4’), 5.07 (1H, d, J = 7.8 Hz, H1’), 4.23 (1H, d, J = 12 Hz, H6a’), 4.05 (1H, dd, J =
12, 6.6 Hz, H6b’), 3.80 (1H, m, H5’), 3.79 (3H, s, OCH3), 1.18, 1.17, 1.16, 1.14 (36H, 4
x s, C(CH3)3).
13C NMR (CDCl3): 178.0, 177.2, 176.5, 176.2, 150.2, 146.0, 124.0, 120.7, 118.9,
112.5, 100.0, 72.5, 72.2, 71.1, 68.1, 62.1, 55.7, 38.8, 38.8, 38.8, 27.2, 27.1, 27.0,
27.0.
Chapter 2 38
Guaiacol β-D-glucopyranoside (2) (Method 2).
OCH3
Oglu
Sodium metal (36 mg, 1.56 mmol) was dissolved in methanol (5 mL) and the
resulting sodium methoxide solution added to a solution of 5 (100 mg, 0.14 mmol) in
methanol (5 mL). The reaction mixture was stirred for 16 hours at room temperature.
Acidified Dowex (H+) ion exchange resin was added to the reaction mixture and
stirred for a further 30 mins. The reaction mixture was then filtered and concentrated
in vacuo to produce 2 as a white solid (52 mg, 85%) (m.p.: 148-150 °C).
1H NMR (C5D5N): 7.6 (1H, ArH), 6.92-7.02 (3H, m, ArH), 5.67 (1H, d, J = 6.6 Hz, H1’),
4.52 (1H, dd, J = 12, 2.4 Hz, H6a’), 4.33-4.41 (4H, m, H2’, 4’, 5’, 6b’), 4.10-4.12 (1H, m,
H3’), 3.70 (3H, s, OCH3).
13C NMR (C5D5N): 150.7, 148.6, 123.0, 121.9, 116.9, 113.7, 102.7, 79.3, 79.0, 75.4,
71.7, 62.8, 56.4.
MS: [M-H]¯ = m/z 285.2 and [M+CH3COO]¯ = m/z 345.2 (APCI in negative mode).
Chapter 2 39
d4-Guaiacol (6).
OCH3
OH
D
D
D
D
Guaiacol (2 g, 16 mmol) and a solution of thionyl chloride (2 mL, 27 mmol) in
deuterium oxide (23 mL) were added to the reactor tube of a Discover SP-D
microwave apparatus (CEM, Matthews NC, USA). The tube was capped and
irradiated for 30 hours at 100°C. The reaction mixture was then neutralised with
potassium carbonate and extracted with pentane (3 x 40 mL). The combined organic
extracts were dried and concentrated to give 6 as a pale yellow liquid (1.65 g, 80%).
1H NMR (CDCl3): 3.89 (3H, s, OCH3) GC-MS: retention time of 24.35 mins (100% pure). Mass spectrum = m/z 128.2 [M+•], 113.2, 85.2
Chapter 2 40
d4-guaiacol 2’,3’,4’,6’-tetrapivaloyl β-D-glucopyranoside (7).
Oglu(Piv)4
OCH3
D
D
D
D
4 (4.7 g, mmol) was added to a solution of 6 (1.0 g, 7.81 mmol) in anhydrous
dichloromethane (10 mL) containing silver triflate (2.10 g, 7.81 mmol) and 2,6-lutidine
(1 mL, 7.81 mmol). The reaction mixture was then stirred in the darkness for 16
hours at room temperature. The reaction was quenched with saturated sodium
bicarbonate solution (30 mL) and extracted with dichloromethane (2 x 30 mL). The
organic extracts were combined, washed with saturated sodium chloride solution,
dried and concentrated in vacuo. The crude product was then purified by column
chromatography (20% ethyl acetate in hexane) to give 7 as colourless needles (142
mg, 25%) (m.p.:130-131°C).
1H NMR (CDCl3): 5.41 (1H, t, J = 9Hz , H 3’), 5.33 (1H, dd, J = 9.6, 8.4Hz, H2’), 5.17
(1H, t, J = 9.6 Hz, H4’), 5.07 (1H, d, J = 7.2 Hz, H1’), 4.23 (1H, dd, J = 12.6, 1.8 Hz,
H6a’), 4.05 (1H, dd, J = 12, 6.6 Hz, H6b’), 3.83 (1H, ddd, J = 9.6, 6, 1.8, H5’), 3.79 (3H,
s, OCH3), 1.18, 1.17, 1.16, 1.14 (36H, 4 x s, C(CH3)3).
13C NMR (CDCl3): 178.0, 177.2, 176.5, 176.5, 150.1, 146.0, 100.1, 72.5, 72.2, 71.1,
68.1, 62.1, 55.7, 38.8, 38.8, 38.7, 27.1, 27.0, 27.0.
Chapter 2 41
d4-guaiacol β-D-glucopyranoside (8).
Oglu
OCH3
D
D
D
D
Sodium metal (115 mg, 5 mmol) was dissolved in methanol (5 mL), and the resulting
sodium methoxide solution added a solution of 7 (300 mg, 0.42 mmol) in methanol
(10 mL). The reaction mixture was stirred for 16 hours at room temperature. Acidified
Dowex (H+) ion exchange resin was added to the reaction mixture and stirred for a
further 30 mins. The mixture was then filtered and the solvent removed in vacuo to
produce 8 as a white solid (133 mg, 84%) (m.p.: 150-151 °C).
1H NMR (C5D5N): 5.67 (1H, d, J = 6.6 Hz, H1’), 4.54 (1H, dd, J = 12.6, 2.4 Hz, H6a’),
4.34-4.42 (4H, m, H2’, 4’, 5’, 6b’), 4.12-4.14 (1H, m, H3’), 3.71 (3H, s, OCH3).
13C NMR (C5D5N): 150.7, 148.5, 102.7, 79.3, 79.0, 75.4, 71.7, 62.8, 56.4.
MS: [M-H]¯ = m/z 289.3 and [M+CH3COO]¯ = m/z 349.5 (APCI in negative mode)
Chapter 2 42
2.7 Conclusion.
The most efficient method for the synthesis of β-D-guaiacol glucopyranoside (2)
utilised 2,3,4,6-tetra-O-pivaloyl-α-D-glucopyranosyl bromide (4) as a reagent in the
presence of silver triflate. This method gave improved yields compared to those
methods previously reported, but most importantly the reaction selectivity favoured β-
glycosylation over α-glycosylation. The β-D-glucopyranoside was used as an
authentic reference sample to confirm the provenance of 2 in smoke affected grapes,
and therefore the glycosylation of guaiacol following grapevine exposure to smoke
(as described in chapter 3). Deuterated guaiacol was prepared using microwave-
assisted synthesis, significantly reducing deuterium exchange reaction times. d4-
Guaiacol was then glycosylated to give d4-guaiacol β-D-glucopyranoside (8), which
was subsequently used as an internal standard for the development of a quantitative
SIDA method using HPLC-MS/MS (as described in Chapter 4).
Chapter 3 43
CHAPTER 3
PROVENANCE OF GUAIACOL GLUCOSIDE IN SMOKE
AFFECTED FRUIT.
Chapter 3 44
CHAPTER 3: PROVENANCE OF GUAIACOL GLUCOSIDE IN SMOKE AFFECTED FRUIT.
Guaiacol is one of several volatile phenols considered to contribute significantly to
the unique aroma of smoke28 and has been identified as a component of both wood
smoke28,32,33 and smoke tainted wines.45 The evolution of guaiacol during the
fermentation of smoke affected Merlot grapes was attributed to the degradation of
one or more precursor compounds by Kennison et al.43 and the precursors were
thought to be glycosidic in nature, given significant levels of guaiacol were also
released following the addition of β-glucosidase enzymes to Merlot juice from the
same grapes.43 However the presence of a guaiacol β-D-glucopyranoside in smoke
affected grapes had yet to be confirmed.
This paper concerns an investigation into the provenance of guaiacol β-D-
glucopyranoside in smoke affected grapes, using HPLC-MS/MS analysis. The
guaiacol β-D-glucopyranoside previously synthesised in Chapter 2 was used as an
authentic reference compound to develop an HPLC-MS/MS method for its detection
in juice. The release of guaiacol from its β-D-glucopyranoside precursor following
treatment with acid and enzyme hydrolysis are also described.
A Hayasaka, Y., Dungey, K.A., Baldock, G.A., Kennison, K.R. & Wilkinson, K.L. (2010) Identification of a β-D-glucopyranoside precursor to guaiacol in grape juice following grapevine exposure to smoke Analytica Chimica Acta, v. 660 (1-2), pp. 143 -148
A NOTE:
This publication is included on pages 45-50 in the print copy of the thesis held in the University of Adelaide Library.
A It is also available online to authorised users at:
A http://dx.doi.org/10.1016/j.aca.2009.10.039
A
Chapter 4 51
CHAPTER 4
QUANTIFICATION OF GUAIACOL GLYCOSIDES IN SMOKE
AFFECTED FRUIT.
Chapter 4 52
CHAPTER 4: QUANTIFICATION OF GUAIACOL GLYCOSIDES IN SMOKE AFFECTED FRUIT. Following identification of guaiacol β-D-glucopyranoside as a component of smoke
affected fruit,82 Hayasaka et al.83 identified several guaiacol disaccharides using
stable isotope tracer experiments involving the application of a 50:50 mixture of
guaiacol and d3-labelled guaiacol to grapevine leaves. Subsequent HPLC-MS/MS
screenings enabled the tentative identification of seven different guaiacol conjugates;
a glucose-glucose disaccharide (glucosylglucoside), the glucoside, four glucose-
pentose disaccharides and a rutinoside.
To investigate the glycosylation of guaiacol in smoke affected grapes, a quantitative
analytical method was required for glycoconjugate determination. This paper
concerns the development and validation of an HPLC-MS/MS based SIDA method
using the d4-labelled guaiacol β-D-glucopyranoside synthesised in Chapter 2 as an
internal standard. The method was subsequently applied to the analysis of grapes
sourced from grapevines exposed to experimental smoke and from commercial
vineyards exposed to bushfire smoke. The accumulation of guaiacol glycoconjugates
within berry components was also investigated.
A Dungey, K.A., Hayasaka, Y. & Wilkinson, K.L. (2011) Quantitative analysis of glycoconjugate precursors of guaiacol in smoke-affected grapes using liquid chromatography -tandem mass spectrometry based stable isotope dilution analysis Food Chemistry, v. 126(2), pp. 801-806
A NOTE:
This publication is included on pages 53-58 in the print copy of the thesis held in the University of Adelaide Library.
A It is also available online to authorised users at:
A http://dx.doi.org/10.1016/j.foodchem.2010.11.094
A
Chapter 5 59
CHAPTER 5
QUANTIFICATION OF GUAIACOL GLYCOCONJUGATES IN GRAPES
AND WINE.
Chapter 5 60
CHAPTER 5: QUANTIFICATION OF GUAIACOL GLYCOCONJUGATES IN GRAPES AND WINE.
5.1 Introduction.
In Chapter 4, the development and validation of an HPLC-based SIDA method for the
quantification of guaiacol glycoconjugates in grapes was described. The application
of this method to various experimental field trials subsequently enabled the
glycosylation of smoke-derived guaiacol following grapevine exposure to smoke to be
determined, as well as the distribution of glycoconjugates within different berry
components. The preferential accumulation of glycoconjugates in grape skins
suggested winemaking techniques which involve reduced skin contact time might
offer potential methods of amelioration. Therefore, to investigate the behaviour of
guaiacol glycoconjugates during fermentation, the HPLC-MS/MS method was
adapted for wine analysis.
This chapter describes the development and validation of a SIDA based HPLC-
MS/MS method for quantification of guaiacol glycoconjugates in wine and its
application to various winemaking trials.
Chapter 5 61
5.2 Results and discussion.
5.2.1 Method development.
5.2.1.1 Calibration function for guaiacol β-D-glucopyranoside in wine.
A calibration function for guaiacol β-D-glucopyranoside was constructed by plotting
the peak area ratio of the target mass transition of guaiacol β-D-glucopyranoside (2)
to that of its deuterated equivalent (8), against known concentrations of the
glucoside, ranging from 10 to 100,000 µg/L, in a control rosé Grenache wine. This
wine was made with grapes known to contain negligible levels of guaiacol
glycoconjugates. A correlation coefficient of 0.998 was obtained, indicating a high
degree of linearity for the working range (0-5000 µg/L) (Figure 5). As with the SIDA
method developed for grape analysis (Chapter 4), the absence of labelled analogues
for the glucosylglucoside, glucose-pentose disaccharides and rutinoside required
their relative concentrations to be determined using the deuterated guaiacol β-D-
glucopyranoside (i.e. 8) as internal standard. Again a high degree of reproducibility in
glycoconjugate measurements was observed (Table 5) which leads to the
assumption that relative changes in glucosylglucoside, glucose-pentose
disaccharides and rutinoside concentrations can be accurately determined, but direct
comparison with glucoside levels are, at best, approximations.
Chapter 5 62
R² = 0.998
0.0
1.0
2.0
3.0
4.0
5.0
6.0
0 1000 2000 3000 4000 5000
An
aly
te p
eak a
rea/I
S p
eak a
rea
Concentration (μg/L)
Figure 5: Calibration function for guaiacol β-D-glucopyranoside in control rosé
Grenache wine.
5.2.1.2 Mass transitions used for HPLC-SRM analysis.
Using the deuterated guaiacol glucoside (8) as an internal standard, an HPLC-SRM
based SIDA method for the direct quantification of guaiacol glucoside and relative
quantification of the glucosylglucoside, glucose-pentose disaccharides and rutinoside
in smoke affected wines was developed, i.e. as an adaptation of the method
previously developed for use in extracts of smoke affected grapes (Chapter 4). The
glycoconjugates again predominately gave the respective acetic acid adduct ([M-H +
CH3COOH]¯) ions under the APCI conditions employed, therefore quantification was
carried out by HPLC-SRM, monitoring the mass transition from the respective [M-H +
CH3COOH]¯ ions to m/z 161 for the glucoside, m/z 293 for the glucose-pentose
disaccharides, m/z 307 for the rutinoside or m/z 323 for the glucosylglucoside.
Chapter 5 63
5.2.2 Method validation.
5.2.2.1 Instrument repeatability.
Instrument repeatability was tested by repeating the analysis of a heavily smoke
tainted Shiraz wine (5 replicates). Glycoconjugate concentrations were highly
consistent, with coefficients of variation of less than 4% obtained for each (Table 5).
5.2.2.2 Reproducibility.
Reproducibility of the method was evaluated by measuring the guaiacol β-D-
glucopyranoside concentration of five replicates of addition samples spiked with 50 or
1,000 μg/L of the glucoside. The method demonstrated a high level of consistency,
with coefficients of variation of 2.8 and 1.6% respectively (Table 5). Reproducibility of
glycoconjugate analysis was also evaluated by repeating the analysis of smoke
affected Shiraz (5 replicates) and Merlot (4 replicates) wines. Analysis of the
glycoconjugates demonstrated a high level of consistency with coefficients of
variation between 0.7 and 5.0% (Table 5), for the glucoside, glucose-pentose
disaccharides and rutinoside. The unusually high coefficients of variation obtained for
the glucosylglucoside (i.e. 10.0 and 28.3% for Shiraz and Merlot respectively) are
attributed to the extremely low levels present in these wines (i.e. 6 µg/L), compared
with the other glycoconjugates.
5.2.2.3 Recovery.
Glycoconjugate recovery was evaluated by comparing the glucoside content of a
control Grenache red wine, and the same wine spiked with 1,000 µg/L of the guaiacol
Chapter 5 64
β-D-glucopyranoside (i.e. the 1,000µg/L standard used for construction of the
calibration function). Recovery was calculated to be 97%, which demonstrates the
method can be applied to accurately quantify guaiacol β-D-glucopyranoside in wine
samples (Table 5).
Chapter 5 65
Table 5: Method validation for the quantification of guaiacol glycoconjugates in wine.
Sample Meana (μg/L)
CVb (%) nc
(a) Instrument repeatability
Smoke affected Shiraz wine
Glucosylglucoside 2 3.3 10
Glucoside 21 0.9 10
Glucose-pentose disaccharides 195 0.9 10
Rutinoside 45 1.4 10
(b) Reproducibility
50 μg/L additiond 64 2.8 5
1,000 μg/L additiond 982 1.6 5
Smoke affected Shiraz wine
Glucosylglucoside 6 10.0 5
Glucoside 81 1.9 5
Glucose-pentose disaccharides 581 0.8 5
Rutinoside 113 0.7 5
Smoke affected Merlot wine
Glucosylglucoside 6 28.3 4
Glucoside 45 2.5 4
Glucose-pentose disaccharides 657 4.5 4
Rutinoside 121 5.0 4
(c) Recovery
Control Grenache red wine 11 4.4 5
Control Grenache red wine with 1,000 µg/L addition (expected)
1,011
Control Grenache red wine with 1,000 µg/L addition (observed)
982 1.6 5
Recovery (%)e 97 a In wine sample b coefficient of variation c number of replicates d Control Grenache rosé wine spiked with a known amount of guaiacol glucoside. e (observed/expected) x 100
Chapter 5 66
5.2.3 Application of wine based SIDA method to winemaking trials.
5.2.3.1 Hydrolysis of guaiacol glycoconjugates during fermentation.
The concentration of guaiacol glycoconjugates was monitored throughout the
fermentation of smoke affected grapes to investigate their hydrolysis during
winemaking. Three seperate experiments were conducted using smoke affected
Grenache, Shiraz and Viognier grapes. Grenache and Viognier grapes were obtained
from grapevines exposed to experimentally produced smoke (for 20 or 30 min,
respectively, i.e. a relatively short duration of smoke exposure), whereas Shiraz
grapes were sourced from a vineyard exposed to bushfire smoke over a 5 week
period (i.e. prolonged smoke exposure). Must from crushed Grenache and Shiraz
grapes were inoculated with a commercial yeast strain (i.e. PDM), whereas the
Viognier must was fermented using indigenous (or „wild‟) yeast.
The smoke affected Grenache grapes contained 294 µg/kg total guaiacol
glycoconjugates. Assuming a 70% juice extraction rate,84 complete extraction of the
glycoconjugate pool would result in juice glycoconjugate concentrations of
approximately 420 µg/L. Instead, free run juice contained only 123 µg/L total
glycoconjugates. Glycoconjugate levels increased to 197 µg/L after 1 day maceration
and to 272 µg/L after 4 days maceration, but there was no significant change in
precursor concentrations from then on (Table 6). Smoke-derived volatile phenols,
including guaiacol, have been shown to evolve during fermentation, purportedly due
to the hydrolysis of glycoconjugate precursors extracted from smoke affected fruit.43
However, it is clear from the current study that a significant proportion of the
glycoconjugate pool remains in the wine, after fermentation has been completed
(Table 6).
Chapter 5 67
Table 6: Concentration of guaiacol glycoconjugates throughout fermentation of
smoke affected Grenache grapes, according to red style winemaking protocols.
Sample Total guaiacol
glycoconjugate concentration (µg/L)
grapesa 294.2 ± 35.7
free run juice 123 ± 36.8
red winemaking
after 1 day maceration 197 b ± 32.0
after 4 days maceration 272 c ± 39.8
end of alcoholic fermentation (i.e. post-pressing) 265 c ± 34.2
finished wine 290 c ± 37.0 a expressed as µg/kg Each value represents the mean of three replicates ± standard error. Means in columns followed by different letters are significantly different.
Similar results were obtained during the fermentation of smoke affected Shiraz
grapes. As expected, the increased duration of smoke exposure gave considerably
higher grape glycoconjugate concentrations, being 875 µg/kg. A greater proportion of
the glycoconjugate pool was extracted into the Shiraz must than occured for
Grenache; i.e. approximately 1,000 µg/L of an estimated 1,250 µg/L maximum (Table
7). Again, there was no significant difference in glycoconjugate concentration during
the first 7 days of maceration. However, a significant reduction in glycoconjugate
levels had occured by the time fermentations underwent pressing, i.e. approximately
20%, presumably due to hydrolysis by yeast and/or enzymes. That said, as with the
Grenache wines, the finished Shiraz wines still contained a large proportion of the
initial glycoconjugate pool.
Chapter 5 68
Table 7: Concentrations of guaiacol glycoconjugates throughout fermentation of
smoke affected Shiraz grapes.
Treatment Total glycoconjugates (µg/L)
grapesa 875 ± 111.5
after 3 days maceration 1027 b ± 50.4
after 4 days maceration 1112 b ± 90.8
after 7 days maceration 1025 b ± 64.0
after alcoholic fermentation (post-pressing) 832 c ± 28.4
finished wine 825 c ± 29.2 a expressed as µg/kg Each value represents the mean of three replicates ± standard error. Means in columns followed by different letters are significantly different.
Control and smoke affected Viognier grapes were fermented with indigenous yeast,
to determine the effects of wild fermentation on total guaiacol glycoconjugate
concentrations. Control Viognier grapes were found to contain a reasonable quantity
of glycoconjugates, being 116 µg/kg, but after fermentation, only 26 µg/L remained in
the resulting wine (Table 8). In contrast, smoke affected Viognier grapes contained
536 µg/kg glycoconjugates, but 197 µg/L remained after fermentation. Again, these
results are consistent with those obtained in the trials involving Grenache and Shiraz,
although much more variation was observed between the wild fermentation
replicates, than the inoculated fermentations, as indicated by the significantly higher
standard errors (Table 8). This is perhaps not unexpected, since populations of
indigenous yeast will differ in species and cell number, causing the observed
variations in fermentative ability between replicates.87 These fermentations were also
conducted on micro-scale (i.e. 250 mL) which likely gave much less controlled
winemaking conditions; in particular, temperature. Irrespective, the results clearly
demonstrate that guaiacol glycoconjugates can be hydrolysed during fermentation
with indigenous yeast, but that again only partial metabolism occurs, so that
glycoconjugates remain in the finished wine.
Chapter 5 69
Table 8: Concentration of guaiacol glycoconjugates in control and smoke affected
Viognier grapes and wine (produced by wild fermentation).
Sample Total glycoconjugates (μg/L)
Control Viognier grapesa 116 ± 9.3
Control Viognier wine 26 b ± 3.8
Smoke affected Viognier grapesa 536 ± 105.2
Smoke affected Viognier wine 197 c ± 74.4 a expressed as µg/kg Each grape value represents the mean of three replicates, while each wine value represents the mean of twelve replicates, ± standard error. Means in columns followed by different letters are significantly different.
The presence of glycoconjugates in finished wines has important implications for the
wine industry, since their hydrolysis in the bottle over time could result in liberation of
additional quantities of guaiacol, and therefore the intensification of smoke related
sensory attributes with ageing. This is considered in more detail, i.e. with a broader
sample set, below (i.e. in 5.2.3.3).
5.2.3.2 Influence of winemaking techniques on the glycoconjugate
content of wine.
To investigate the effect of skin contact on guaiacol glycoconjugate concentration, a
winemaking trial was conducted, in which traditional red and rosé winemaking
techniques were compared. Red and rosé style wines were made from smoke
affected and control grapes; with samples collected at various stages of fermentation,
including pre-inoculation, during alcoholic fermentation, post pressing and bottling,
and glycoconjugate concentrations determined using the wine based SIDA method.
Glycoconjugate concentrations were significantly higher in smoke affected red style
wines, compared to rosé style wines, although both contained elevated
Chapter 5 70
glycoconjugate levels compared to their corresponding control wines (Figure 6). The
lower glycoconjugate concentrations of rosé style wines is attributed to the reduced
skin contact time, given glycoconjugates were shown to preferentially accumulate in
the skins of grapes (Chapter 4). Wine style can therefore have a significant influence
on the extraction of glycoconjugates and thus the extent of smoke taint. As such,
winemaking practices need to be a consideration for winemakers when processing
smoke affected grapes.
0
50
100
150
200
250
300
350
smoked red style wine
control red style wine
smoked rosé style wine
control rosé style wine
Co
ncen
trati
on
(μ
g/L
)
Figure 6: Guaiacol glycoconjugate concentrations of control and smoke affected
Grenache wines made according to different winemaking techniques.
Although determination of total guaiacol glycoconjugate concentrations provides an
indication of the “bound” guaiacol content of wine, the relative concentrations of
individual glycoconjugates in Grenache grapes and wine was also investigated. For
both grapes and wines, the glycoconjugate pool largely comprised the glucose-
pentose disaccharides (55-65%); the rutinoside and glucoside were less abundant, at
20-30% and 6-10% respectively (Figure 7). The glucosylglucoside concentration
decreased from 13% in grapes, to less than 1% in wine, suggesting that of the
Chapter 5 71
various glycoconjugates, the glucosylglucoside is probably the most susceptible to
hydrolysis during fermentation. It is possible that a proportion of the disaccharide
glycoconjugates might be hydrolysed to the β-D-glucopyranoside.
*The glycoconjugate concentration of grapes was converted from µg/kg to µg/L
assuming a 70% juice extraction rate.
Figure 7: Relative concentrations of guaiacol glycoconjugates of smoke affected
Grenache grapes and resulting red and rosé style wines.
The concentration of guaiacol glycoconjugates in smoked and control Grenache
wines, fermented with eight different yeast strains, was measured using the wine
based SIDA method. Control wines contained negligible concentrations of all
guaiacol glycoconjugates. Smoke affected wines, fermented using AWRI 1176,
showed the highest concentration of glycoconjugates (being 374 µg/L), followed by
ICV GRE (being 356 µg/L); with the lowest concentration of glycoconjugates
observed in wines fermented with AWRI 1503 (264 µg/L) and BDX (271 µg/L). β-
Glucosidase enzyme, present in yeast and responsible for glycoconjugate
Chapter 5 72
metabolism85, will vary in activity between yeast strains, therefore explaining the
variation in glycoconjugate concentrations observed (Figure 8). Significant amounts
of glycoconjugates remained in wines at bottling; regardless of yeast strain used,
indicating the potential for further metabolism of glycoconjugates, and subsequent
release of guaiacol, during bottle storage.
Figure 8: Guaiacol glycoconjugate concentrations of control (C) and smoke affected
(S) Grenache wines fermented with eight different yeast strains. Columns with
different letters above them are significantly different
5.2.3.3 Glycoconjugate content of wine and potential for smoke taint to
intensify with bottle age.
Wines produced with Pinot Noir, Chardonnay and Cabernet Sauvignon grapes
harvested from grapevines exposed to bushfire smoke, demonstrated considerable
variation in glycoconjugate concentration between grapes and wine and showed the
potential for glycoconjugate metabolism during bottle storage. The guaiacol
Chapter 5 73
glycoconjugate concentration of grapes was again converted from µg/kg to µg/L,
assuming a 70% extraction of juice from whole berry homogenate. Glycoconjugate
concentration decreased during fermentation, regardless of grape variety, in
agreement with results obtained from previous trials (Figure 9). Smoke affected
Grenache grapes and wine, contained significantly lower (i.e. four times less)
glycoconjugate concentrations compared to the Shiraz, Chardonnay, Pinot Noir and
Cabernet Sauvignon grapes and wine (Figure 9). This was attributed to the duration
of grape smoke exposure; i.e. Grenache grapevines received only 20 minutes of
experimental smoke exposure, whereas other grapevine varieties were exposed to
bushfire smoke for up to five weeks. This demonstrates the importance of duration of
smoke exposure on guaiacol glycoconjugate concentration, as previously indicated
by Kennison and coworkers.44
0
500
1000
1500
2000
2500
3000
Co
ncen
trati
on
(μ
g/L
)
Figure 9: Concentration of guaiacol glycoconjugates in smoke affected grapes and
wine derived from grapevines exposed to experimental (Grenache) or bushfire
smoke (Shiraz, Chardonnay, Pinot Noir and Cabernet Sauvignon).
Chapter 5 74
Despite the variation in glycoconjugate concentrations between grapes and wine of
different varieties exposed to smoke under different conditions, significant amounts of
the glycoconjugates remained in the finished wines. Regardless of variety, wines
fermented with smoke affected grapes, can potentially release guaiacol thereby
enhancing the sensory attributes associated with smoke taint during bottle storage.
Again, this intensification of smoke taint in wines with time has clear implications for
winemakers, who risk releasing tainted wine, which could subsequently decrease the
value and reputation of their brands.
The grape and wine data contained within this section (5.2.3.3) has been accepted
for publication as part of a much larger study which compared different methods for
assessing smoke exposure in grapes and wine. This manuscript (currently in press)
is included in the Appendix.
5.2.3.4 Potential for the carryover of glycoconjugates between growing
seasons.
In 2009/2010 the fruit of Merlot and Viognier grapevines which had been exposed to
smoke under experimental conditions during the 2008/2009 growing season, was
harvested (at commercial maturity, i.e. ≈ 24°Brix), to investigate any carryover of
guaiacol glycoconjugates from the previous season. Smoke affected grapes
harvested in the same year as smoke exposure contained significantly higher
concentrations of guaiacol glycoconjugates compared to their corresponding control
grapes, irrespective of variety (Figures 10 and 11). However in the subsequent
growing season, no significant difference in glycoconjugate concentrations were
observed between grapes from smoked and control grapevines. These results
suggested guaiacol glycoconjugates were not sequestered within the vine prior to
Chapter 5 75
dormancy and provide no evidence to support the carryover of smoke taint from one
growing season to the next.
0
50
100
150
200
250
300
350
400
450
MC-2009 MS-2009 MC-2010 MS-2010
Co
ncen
trati
on
(μ
g/k
g)
Figure 10: Concentration of total guaiacol glycoconjugates present in grapes
harvested from smoked and control Merlot grapevines (MS and MC respectively), in
the growing season during which smoke exposure occured (2008/2009) and the
subsequent growing season (2009/2010).
Chapter 5 76
0
50
100
150
200
250
300
350
VC-2009 VS-2009 VC-2010 VS-2010
Co
ncen
trati
on
(μ
g/k
g)
Figure 11: Concentration of total guaiacol glycoconjugates present in grapes
harvested from smoked and control Viognier grapevines (VS and VC respectively), in
the growing season during which smoke exposure occured (2008/2009) and the
subsequent growing season (2009/2010).
5.3 Materials and methods.
5.3.1 Method development.
5.3.1.1 Preparation of wine samples for HPLC-MS/MS analysis.
Aliquots (1 mL) of wine were sub-sampled. After addition of labelled guaiacol
glucoside (8, 1 µg/mL wine), samples were filtered through a 0.45 µm GHP
membrane (Acrodisc®, PALL Life Sciences) and analysed by HPLC-MS/MS.
Chapter 5 77
5.3.1.2 Calibration function for guaiacol β-D-glucopyranoside in wine.
Wine produced from control (unsmoked) Grenache grapes was used for the
preparation of reference addition standards. A 200 µg/mL reference standard solution
was prepared by dissolving guaiacol glucoside (2) (5 mg) in control Grenache rosé
wine (25 mL). The reference solution was then diluted with the same control wine to
give concentration standards of 10, 50, 100, 500, 1,000, 5,000, 10,000, 20,000,
50,000, 100,000 µg/L, and following the addition of a constant amount of labelled
analogue (1 µg/mL wine) as internal standard, standards were filtered and analysed
(as above).
5.3.1.3 Instrumental analysis.
A 4000 Q TRAP hybrid tandem mass spectrometer equipped with a Turbo ion source
(Applied Biosystems/MDS Sciex) combined with an Agilent 1200 HPLC system
equipped with binary pump, degasser, autosampler and column oven (Agilent
Technologies, Santa Clara, CA) was used. Data acquisition and processing were
performed using Analyst software version 1.5.1 (Applied Biosystems/MDS Sciex).
5.3.1.4 High performance liquid chromatography tandem mass
spectrometry (HPLC-MS/MS).
A 10 µL aliquot of each wine sample was injected and chromatographed on a 150 x 2
mm internal diameter, 3µ Gemini C6-Phenyl 110A column (Phenomenex). The
column temperature was maintained at 25°C during the HPLC- run. A binary gradient
with mobile phases consisting of 0.1% acetic acid in water (solvent A) and
acetonitrile (solvent B), respectively, was used. The elution conditions were as
Chapter 5 78
follows: flow rate was 300 µL/min; a linear gradient from 10% to 30% of solvent B in
10 min, from 30% to 70% in 5 min, then held at 70% for 10 min (25 min run). The
effluent from the column was introduced directly to the Turbo ion interface.
Mass spectra were recorded in negative ion mode with nitrogen used as the curtain,
nebulizer, turbo and collision gases. The turbo ion source was set to APCI mode and
the parameters were set at -4500 V for ionspray voltage, -10 V for entrance potential,
-4 µA for nebulizer current, -40 V for declustering potential, 25 psi for gas 1, 50 psi for
gas 2 (turbo) and 350°C for gas 2 temperature. For HPLC-MS in scan mode, the
instrument scanned from m/z 50 to 500 with a step size of 0.1 Da and scan time of 1
s. For MS/MS mode, the collision parameters were set at -18 V for collision potential,
-5 V for collision cell exit potential and high for collision gas pressure. Product ion
spectra of m/z 345.1 were recorded in a mass range from m/z 50 to 360. HPLC-
MS/MS in selected reaction monitoring mode (HPLC-SRM) was used for
quantification. The following mass transitions were monitored with a dwell time of 50
ms: m/z 349 → m/z 289 and 161 for the deuterated guaiacol glucoside (8); m/z 345
→ m/z 285 and 161 for the glucoside (2); m/z 447 → m/z 417 and 293 for the four
glucose-pentose disaccharides; m/z 491 → m/z 431 and 307 for the rutinoside; and
m/z 507 → m/z 447 and 323 for the glucosylglucoside.
Chapter 5 79
5.3.2 Application of the quantitative guaiacol glycoconjugate method to
winemaking trials.
5.3.2.1 Smoke affected grapes.
Smoke affected fruit was sourced from either: (i) field trials involving the application
of smoke to grapevines under experimental conditions; or (ii) commercial vineyards
exposed to bushfire smoke. Various vineyard sites were used for field experiments.
Grenache vines were located at Nuriootpa, in the Barossa Valley district of South
Australia, approximately 80 km north-east of Adelaide (34o30‟S, 138o59‟E, altitude
274 m). Grapevines were enclosed in a purpose built smoke tent and exposed to
straw derived smoke (for 20 min) using experimental conditions described previously
(i.e. in Chapter 3). Smoke was applied at a phenological stage corresponding to
approximately 7 days post-veraison; i.e. at total soluble solids (TSS) concentrations
of approximately 14 ± 0.2 °Brix, determined using a digital handheld refractometer
(PAL-1, Atago, Tokyo, Japan). Control and smoke affected fruit was harvested when
TSS levels reached 23 ± 1 °Brix.
Shiraz grapes were sourced from a vineyard located in Coldstream, in the Yarra
Valley wine region of Victoria (37o42‟S, 145o30‟E, altitude 130 m). This vineyard was
exposed to smoke from a series of bushfires which occurred in the region between
February 7 and March 14, 2009. Fruit was harvested at a TSS level of more than 30
oBrix and stored at -20 oC prior to analysis and winemaking.
Viognier vines were located at the University of Adelaide, Waite campus in Urrbrae,
South Australia (34°58‟S, 138°38‟E) and exposed to smoke, (for 45 mins) under
Chapter 5 80
experimental conditions, as described in Chapter 3. Smoke was applied
approximately 7 days prior to harvest (i.e. at 20 °Brix). Control and smoke affected
Viognier grapes were harvested at TSS of 24 ± 1 °Brix and grapes were used fresh
for winemaking.
Additionally, control and smoke affected Viognier and Merlot grapes were harvested
again from vines used in the trials described in Chapter 4; i.e. in the season following
smoke exposure. Viognier grapes were harvested at TSS of 25 ± 1 °Brix and Merlot
grapes were harvested at TSS of 23 ± 2 °Brix.
Chardonnay, Cabernet Sauvignon and Pinot Noir grapes were sourced from a
number vineyards in the Goulburn Valley (37°42‟S, 145°30‟E), Victoria, which were
exposed to bushfire smoke between February 7 and March 14, 2009, and were
provided by the Australian Wine Research Institute. Chardonnay and Pinot Noir fruit
was harvested at juice TSS levels of 23 ± 1 °Brix, and Cabernet Sauvignon was
harvested at TSS of 19 ± 1 °Brix. Wines were made from these grapes using similar
methods as for the Shiraz winemaking trial above, by collaborators at AWRI.
5.3.2.2 Winemaking.
Grenache and Shiraz wines were made according to the methodology outlined in
Chapter 6, i.e. based on small scale winemaking techniques developed at the
University of Adelaide.86
Viognier grapes (approximately 1 kg for each of six field replicates) were harvested
from both smoked and control grapevines when they reached TSS of 24 ± 2 °Brix.
Grapes were then destemmed and crushed, and two must sub-samples (200 mL
Chapter 5 81
each) were placed in sterile conical flasks fitted with airlocks, i.e. to give 12 replicate
fermentations for each treatment. Must was fermented on skins at ambient
temperature (22°C), without the addition of sulphur dioxide or yeast; i.e. to simulate
„wild‟ fermentation. TSS levels were measured twice daily using a digital handheld
refractometer (PAL-1, Atago, Tokyo, Japan) to monitor fermentation rates.
Fermentation was considered complete when the residual sugar approached 0 g/L
(as determined by Clinitest® analysis).
5.3.3 Statistical analysis.
Statistical analysis was performed using Genstat 10.2 (10th Edition, VSN International
Limited, Herts, UK). The data was analysed using a one way analysis of variance
(ANOVA). Mean comparisons were performed by least significant difference (LSD)
multiple comparison tests at P < 0.05.
Chapter 5 82
5.4 Conclusion.
Guaiacol glycoconjugates have been identified and quantified in smoke affected
grapes and wine and winemaking techniques found to influence their concentrations
considerably. Reduced skin contact time during fermentation gave lower guaiacol
glycoconjugate levels in finished wines, while yeast selection influenced the extent of
glycoconjugate metabolism during fermentation to some degree. Guaiacol
glycoconjugate levels decreased during fermentation, irrespective of grape variety,
winemaking style or choice of yeast, but significant proportions remained in the
finished wine. The presence of glycoconjugates in wine is problematic, since
hydrolysis during bottle storage (i.e. ageing) could potentially release additional
amounts of guaiacol, increasing the intensity of smoke taint with time. Indeed, wines
thought to be free of smoke taint could develop „smoky‟ characters with ageing if
significant quantities of smoke-derived guaiacol glycoconjugates were present.
Chapter 6 83
CHAPTER 6
THE EFFECT OF WINEMAKING TECHNIQUES ON THE INTENSITY
OF SMOKE TAINT IN WINE.
Chapter 6 84
CHAPTER 6: THE EFFECT OF WINEMAKING TECHNIQUES ON THE INTENSITY OF SMOKE TAINT IN WINE.
The SIDA method developed for quantification of guaiacol glycoconjugates in smoke
affected grapes (Chapter 4) was adapted for the analysis of wine. This paper
describes the application of these methods to control and smoke affected grapes and
wine to identify winemaking techniques that influence the sensory impact of smoke
taint in wine. In particular, the metabolism of glycoconjugates during fermentation,
and the influence of wine style and yeast selection on guaiacol glycoconjugate
concentration of wine were investigated.
A Ristic, R., Osidacz, P., Pinchbeck, K.A., Hayasaka, Y., Fudge, A.L. & Wilkinson, K.L. (2011) The effect of winemaking techniques on the intensity of smoke taint in wine Australian Journal of Grape and Wine Research, v. 17 (2), pp. S29 -S40
A NOTE:
This publication is included on pages 85-96 in the print copy of the thesis held in the University of Adelaide Library.
A It is also available online to authorised users at:
A http://dx.doi.org/10.1111/j.1755-0238.2011.00146.x
A
Chapter 7 97
CHAPTER 7
IMPACT OF SMOKE ON GRAPE BERRY MICROFLORA AND YEAST FERMENTATION.
Chapter 7 98
CHAPTER 7: IMPACT OF SMOKE ON GRAPE BERRY MICROFLORA AND YEAST FERMENTATION.
The primary focus of smoke taint research conducted to date has concerned the
chemical composition and sensory characteristics of smoke affected grapes and
wine. However, the anti-microbial, preservative properties of smoke, could potentially
influence the growth of indigenous microflora on grapes and the performance of
winemaking yeast during fermentation. This paper describes experimental trials
conducted to investigate: (i) the impact of smoke on grape berry microflora and
fermentation rates; and (ii) the growth of 10 Saccharomyces and non-
Saccharomyces yeast strains in the presence of smoke-derived volatile compounds.
Chapter 7 99
Impact of smoke on grape berry microflora and yeast fermentation KERRY DUNGEY, Paul Grbin, Kerry Wilkinson
The University of Adelaide, School of Agriculture, Food and Wine, PMB 1, Glen Osmond, S.A. 5064, Australia; Email: [email protected] Abstract This study concerns the effect of grapevine smoke exposure on grape berry microflora and yeast fermentation. While smoke exposure did not appear to significantly influence the populations of indigenous yeast growing on Viognier grapes in the field, differences were observed in the fermentation rates of control (unsmoked) and smoked Viognier fruit. Smoked affected fruit completed fermentation between 2 and 4 days later than control fruit; attributed to inhibition of yeast, based on cell counts measured using a haemocytometer. The growth of different winemaking yeast (indigenous and commercial strains) on yeast media agar plates in the presence of smoke-derived volatile phenols, guaiacol and 4-methylguaiacol, or a liquid smoke preparation was also investigated.
Introduction Considerable research has been undertaken to investigate the effect of smoke on the chemical composition and sensory properties of grapes and wine,1-4 in response to several incidents of vineyard smoke exposure following forest fires in close proximity to wine regions in Australia, South Africa and North America. To date, the microbiological impact of grapevine smoke exposure has not been reported. Yeast selection plays an important role in winemaking and can significantly influence the aroma and flavor profile of finished wine.5 Additionally, some winemakers will exploit the natural microflora present on grapes (i.e. indigenous yeast) to further enhance wine complexity during fermentation.5 However, the anti-microbial, preservative properties of smoke6 could potentially influence the growth of indigenous microflora on grapes, or yeast during fermentation. This study therefore aimed to determine: (i) the effect of smoke exposure on grape microflora populations in the vineyard; and (ii) the potential inhibition of indigenous or inoculated yeasts during fermentation. Experimental Grapevine exposure to smoke under experimental conditions Viognier grapevines (6 replicates, 2 vines per replicate) located at the University of Adelaide’s Waite Campus (Urrbrae, South Australia) were exposed to straw-derived smoke approximately 1 week prior to harvest (i.e. at total soluble solids (TSS) of 21°Brix), using a purpose-built smoke tent and experimental conditions described previously.1 Grapevines were enclosed in the tent for the duration of smoke exposure (45 minutes). Grapes (200-300 berries) were randomly sampled from each replicate of smoked and control (unsmoked) grapevines at 14, 11, 5 and 3 days prior to smoke exposure and then daily following smoke exposure. Fruit was subsequently harvested from smoked and control grapevines when TSS reached 24±1 °Brix. Free amino nitrogen (FAN) was determined as described by Dukes and Butzke.7
Chapter 7 100
Determination of grape berry yeast populations Grape berry microflora populations were determined by two different methods: (i) as cell concentrations using a haemocytometer;8 and (ii) as oxygen consumption of grape must using a Hach luminescent dissolved oxygen probe (LBOD10101, Colorado, U.S.A.) calibrated according to methodology described by Comitini et al.9 Winemaking Grapes (approximately 1 kg) harvested from each replicate of smoked and control grapevines was destemmed and crushed, and two must sub-samples (200mL each) were placed in sterile conical flasks fitted with airlocks, to give 12 replicate ferments for each treatment. Must was fermented on skins at ambient temperature (22°C), without the addition of sulphur dioxide or yeast; i.e. to simulate ‘wild’ fermentation. TSS levels were measured twice daily using a digital handheld refractometer (PAL-1, Atago, Tokyo, Japan) to determine fermentation rates. Cell counts were performed daily using a haemocytometer.8 Fermentation was considered complete when the residual sugar approached 0 g/L (as determined by Clinitest® analysis). Yeast growth in the presence of smoke constituents YM agar (Amyl Media, Victoria, Australia) plates were spiked with guaiacol (10, 50, 100, 300 or 500 μg/L), 4-methylguaiacol (10, 50, 100 or 300 μg/L) or a liquid smoke preparation (0.5, 1.25, 2.5, or 5.0 mL/L; supplied by K. Dixon, Kings Park Botanical Gardens, Perth, W.A.). Plates were then inoculated (300 cells per plate) with one of ten different yeast strains (in triplicate): five non-Saccharomyces strains, representing genera typically found on grapes: Y-2311 (Aureobasidium pullulans),Y-1614 (Hanseniaspora uvarum), Y-7111 (Metschnikowia pulcherrima), Y-1453 (Candida famata) and Y-2026 (Pichia membranifaciens), (supplied by C. Kurtzman, NRRL, United States Department of Agriculture, Illinois, U.S.A.); and five commercial Saccharomyces strains: Enoferm BDX (S. cerevisiae), S6U (S. uvarum), AWRI Fusion (S. cerevisiae x S. cariocanus), AWRI 1503 (S. cerevisiae x S. kudriavzevii) and AWRI 1375 (S. bayanus). Plates were incubated at 25°C for 1-3 days, depending on the growth rate of the yeast. The resulting colonies were subsequently counted and compared with counts from control plates (i.e. plates prepared without the addition of guaiacol, 4-methylguaiacol or liquid smoke preparation). Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) using Genstat (10th Edition, VSN International Limited, Herts, UK). Mean comparisons were performed by least significant difference (LSD) multiple comparison tests at P < 0.05. Results and Discussion Effect of grapevine smoke exposure on grape berry microflora populations Grape berry microflora populations were monitored before and after grapevine exposure to smoke, by measuring cell concentrations with a haemocytometer or oxygen consumption of grape must with a luminescent dissolved oxygen probe. Differences in microflora populations were observed, irrespective of analytical method (data not shown). However, differences were attributed to inherent natural
Chapter 7 101
variation rather than smoke exposure, since the differences occurred both before and after smoke treatments were applied. Effect of grapevine smoke exposure on fermentation rates Fruit harvested from control grapevines had higher TSS levels than fruit from smoked grapevines (Figure 1a). While grapevine smoke exposure has previously been shown to inhibit sugar accumulation,10 in the current study, differences in TSS were observed before and after smoke treatments were applied (data not shown), so were attributed to natural variation. Control and smoked fermentations initially proceeded at similar rates, but smoked ferments showed signs of lagging after 4 to 5 days (Figure 1a). Measurement of yeast cell numbers (by haemocytometer) indicated this might be attributable to yeast inhibition in must derived from smoke-affected fruit; i.e. the rapid increase in yeast cell numbers observed in control fermentations during the first 3 days of fermentation was not observed in smoked ferments (Figure 1b). Smoked fermentations did eventually achieve dryness (i.e. residual sugar <2 g/L), but between 2 and 4 days later than control fermentations. (a)
(b)
Figure 1. (a) Fermentation curves for smoked and control Viognier must using indigenous yeast (n=12); and (b) average concentration of indigenous microflora cells present during fermentation of smoked and control Viognier must. Kennison et al. reported increased fermentation rates following smoke exposure by field-grown Merlot grapevines, which could be explained by the increased FAN content of the smoke-affected grapes.10 However, in the current study smoked grapes had significantly lower FAN levels than control grapes, 191 and 309 mg/L respectively, which is likely to have contributed to the reduced fermentation rates observed. The physiological response of different grapevine varieties to smoke exposure is the subject of ongoing research. Effect of guaiacol, 4-methylguaiacol and liquid smoke on growth of inoculated yeast Several volatile phenols including guaiacol and 4-methylguaiacol have been shown to contribute to the objectionable ‘smoky’ characters observed in wines exhibiting smoke taint.1,2 The growth of winemaking yeast (five non-Saccharomyces strains representing indigenous genera typically found on grapes and five commercial Saccharomyces strains) on yeast media agar plates in the presence of different concentrations of guaiacol, 4-methylguaiacol and a liquid smoke preparation was investigated. For most yeast strains, no significant difference in colony numbers were observed between control and spiked plates; yeast growth was neither inhibited nor stimulated. However, differences were observed in the growth of two Saccharomyces
Chapter 7 102
strains, S6U and AWRI 1375, in the presence of liquid smoke preparations. Interestingly, liquid smoke additions resulted in increased colony counts; i.e. stimulation rather than inhibition of yeast growth (Table 1). Metschnikowia pulcherrima growth was similarly stimulated in the presence of guaiacol and 4-
methylguaiacol, at concentrations above 50 g/L and 100 g/L respectively (data not shown); but not by the addition of liquid smoke. Table 1. Yeast colony counts for Saccharomyces strains S6U and AWRI 1375 grown on YM agar plates in the presence of a liquid smoke preparation.
Liquid smoke concentration
(mL/L)
Yeast colony countsa (per plate)
S6U AWRI 1375
(S. uvarum) (S. bayanus)
0 (control) 38 ± 2 a 208 ± 10 a
0.5 73 ± 5 b 197 ± 6 a
1.25 38 ± 3 a 193 ± 10 a
2.5 46 ± 3 a 249 ± 43 ab
5.0 49 ± 3 ab 272 ± 10 b aMean values from three replicates (± standard error); different letters within columns indicate values are significantly different. Conclusion Smoke exposure by Viognier grapevines did not appear to influence total grape berry microflora populations, but subsequent fermentation of smoke-affected fruit with indigenous yeast showed longer total fermentation times, compared with fermentation of control fruit. Since the growth of winemaking yeast was not inhibited by the presence of guaiacol, 4-methylguaiacol or a liquid smoke preparation, yeast performance was instead considered to be an indirect effect of smoke exposure, i.e. related to nitrogen availability of smoke-affected grapes. Acknowledgements The authors gratefully acknowledge the financial support of the Australian Federal Government (Department of Agriculture, Fisheries and Forestry) and the Grape and Wine Research and Development Corporation. References 1. Kennison, K.R., Wilkinson, K.L., Williams, H.G., Smith, J.H., Gibberd, M.R.
(2007) J. Agric. Food Chem. 55:10897-10901. 2. Kennison, K.R., Gibberd, M.R., Pollnitz, A.P., Wilkinson, K.L. (2008) J. Agric.
Food Chem. 56:7379-7383. 3. Sheppard, S.I., Dhesi, M.K., Eggers, N.J. (2009) Am. J. Enol. Vitic. 60:98-103. 4. Hayasaka, Y., Dungey, K.A., Baldock, G.A., Kennison, K.R., Wilkinson, K.L.
(2010) Anal. Chim. Acta 660:143-148. 5. Gil, J.V., Mateo, J., Jimenez, M., Pastor, A., Huerta, T. (1996) J. Food Sci.
61:1247-1250. 6. Faith, N.G., Yousef, A.E., Luchansky, J.B. (1992) J. Food Saf. 12:303-314. 7. Dukes, B.C., Butzke, C.E. (1998) Am. J. Enol. Vitic. 49:125-134.
Chapter 7 103
8. Iland, P.G., Grbin, P.R., Grinberg, M., Schmidtke, L., Soden, A. (2007) In: Microbiological analysis of grapes and wine: techniques and concepts. Patrick Iland Wine Promotions, pp 94-112.
9. Comitini, F., Stringini, M., Taccari, M., Ciani, M. (2009) Int. J. Wine Res. 1:53-58. 10. Kennison, K.R., Wilkinson, K.L., Pollnitz, A.P., Williams, H.G., Gibberd, M.R.
(2009) Aust. J. Grape Wine Res. 15:228-237.
Chapter 8 104
CHAPTER 8
SUMMARY.
Chapter 8 105
CHAPTER 8: SUMMARY
Bushfires occurring in close proximity to vineyards have caused significant problems
for winemakers, due to objectionable ‘smoky’ characters being imparted into grapes
and the resultant wine. Previously, smoke taint had been quantified via the direct
measurement of the volatile phenols guaiacol and 4-methylguaiacol. However, this
method does not account for the presence of glycoconjugate forms of these volatile
phenols. To more accurately assess the extent of smoke exposure of grapes, a SIDA
based method for glycoconjugate quantification by HPLC-MS/MS has been
developed.
A reference standard of guaiacol β-D-glucopyranoside was prepared via a modified
Koenigs-Knorr glycosylation method and confirmation of its presence in smoke
affected grapes was performed using high performance liquid chromatography-
tandem mass spectrometry (HPLC-MS/MS) analysis. The β-D-glucopyranoside of
guaiacol was identified in extracts of Sangiovese grapes exposed to bushfire smoke
and Chardonnay grapes exposed to smoke under experimental conditions. However,
only negligible concentrations of the glucoside were identified in the corresponding
control Chardonnay grapes, demonstrating glycosylation of smoke-derived guaiacol
occurred in response to smoke exposure. Following strong acid hydrolysis of smoke
affected juice samples, the guaiacol glucoside remained largely intact, but it was
highly susceptible to hydrolysis by β-glucosidase enzymes, providing the first
plausible explanation for the release of guaiacol during fermentation of smoke
affected grapes.
Chapter 8 106
Synthesis of the d4-labelled analogue of guaiacol β-D-glucopyranoside, as an internal
standard, enabled the development of a quantitative stable isotope dilution analysis
(SIDA) method using HPLC-MS/MS to determine the concentrations of a range of
guaiacol glycoconjugates identified in smoke affected grapes. The subsequent
application of this method to the analysis of several grape varieties exposed to either
experimental or bushfire smoke, revealed the extent of guaiacol glycoconjugate
accumulation in smoke affected grapes. Experimentally smoked grapes contained
glycoconjugate concentrations of up to 300 µg/kg; whereas grapes affected by
bushfire smoke contained up to 2,000 µg/kg, i.e. as much as 14-fold higher
concentrations, attributed to different durations of smoke exposure.
The majority of guaiacol glycoconjugates were found to accumulate in the skin and
pulp fractions of smoke affected grapes, although approximately 6.7 times higher
concentrations were found in the skins by mass. Consequently, glycoconjugate
extraction from berry homogenate was considered to be more efficient than from
juice, due to the partial loss of glycoconjugate precursors during sample preparation.
Grapes collected from control and smoked Merlot and Viognier grapevines (in the
season following smoke exposure), were analysed using the SIDA method, but no
evidence was found to support grapevine sequestration of glycoconjugates in
seasons prior to smoke exposure.
To investigate the metabolism of guaiacol glycoconjugates during fermentation, the
HPLC-MS/MS based SIDA method was adapted for application to smoke affected
wine. Several winemaking trials were conducted using fruit harvested from
grapevines exposed to either experimental smoke (Grenache) or bushfire smoke
(Shiraz, Chardonnay, Pinot Noir and Cabernet Sauvignon). Results from these trials
showed only partial metabolism of glycoconjugates during fermentation, i.e. a
Chapter 8 107
significant proportion of the glycoconjugate pool remained after fermentation. Wines
made using a rosé style winemaking technique, i.e. with reduced skin contact,
contained significantly lower concentrations of guaiacol glycoconjugates compared
with wines made using traditional red winemaking practices (i.e. involving extended
skin contact at ambient temperature). This suggests winemaking styles with limited
skin contact might limit precursor extraction, offering winemakers an opportunity to
ameliorate the impact of smoke taint in wine. Grenache grapes were fermented with
eight different yeast strains, which demonstrated that yeast selection can to some
extent affect the metabolism of glycoconjugates during fermentation. However, again
significant concentrations of the glycoconjugates remained in the finished wines,
regardless of yeast selection. The presence of significant levels of guaiacol
glycoconjugates at bottling highlights the potential for their metabolism with bottle
ageing, which could subsequently result in enhanced taint characters over time.
Since guaiacol is the most abundant smoke-derived volatile phenol, the primary
focus of this study was the occurrence of guaiacol glycoconjugates. However, it is
highly probable that the other volatile phenols identified in smoke, liquid smoke
extracts and smoke tainted wines, i.e. 4-methylguaiacol, 4-ethylphenol and 4-
ethylguaiacol, also accumulate in smoke affected grapes in glycoconjugate forms.
Additionally, there are likely to be other smoke-derived volatile compounds, besides
the volatile phenols, that contribute to smoke taint in grapes and wine. Future
research could therefore be undertaken to identify these compounds and to
determine their contribution to smoke affected grapes and wine.
The effect of grapevine smoke exposure on grape berry microflora and the
performance of several winemaking yeast during fermentation was also investigated.
Different fermentation rates were observed for control and smoked Viognier grapes,
Chapter 8 108
although the relative populations of indigenous yeast were not significantly affected.
The growth of indigenous and winemaking yeast on yeast media agar plates spiked
with guaiacol, 4-methylguaiacol or a liquid smoke preparation was investigated;
increased concentrations of liquid smoke appeared to stimulate the growth of two of
the ten yeast strains investigated, being S6U and AWRI 1375. Fermentation
performance was not affected for any of the other indigenous or commercial yeast
strains studied.
Appendix 109
APPENDIX
A Wilkinson, K.L., Ristic, R., Pinchbeck, K.A., Fudge, A.L., Singh, D.P., Pitt, K.M., Downey, M.O., Baldock, G.A.., Hayasaka, Y., Parker, M. & Herderich, M.J. (2011) Comparison of methods for the analysis of smoke related phenols and their conjugates in grapes and wine Australian Journal of Grape and Wine Research, v. 17 (2), pp. S22 -S28
A NOTE:
This publication is included on pages 110-116 in the print copy of the thesis held in the University of Adelaide Library.
A It is also available online to authorised users at:
A http://dx.doi.org/10.1111/j.1755-0238.2011.00147.x
A
References 117
REFERENCES (excluding those in papers)
1. Australian Wine and Brandy Corporation, Australian wine sales at a glance, Wine
Australia: 2010; 2010.
2. Australian Wine and Brandy Corporation, Australian Wine and Brandy Corporation
Annual Report 2007-2008, 2008.
3. Australian Wine and Brandy Corporation, Australian Wine and Brandy Corporation
Annual Report 2009-2010, 2010.
4. Winemakers Federation of Australia, 2010 WFA Vintage report, 2010.
5. Australian Wine and Brandy Corporation, Australian Wine and Brandy Corporation
annual report 2002-2003, 2003.
6. Krstic, M., Martin, S. and Lowe, S. In Australian Viticulture, 2007, Vol. March/April,
p 31-33.
7. Godden, P., Robinson, E., Francis, L., Lattey, K., Cowey, G. and Boehm, D.
Investigations conducted during 2003 and 2004 into the nature and amelioration
of taints in grapes and wine, caused by smoke resulting from bushfires, Industry
Services Report, The Australian Wine Research Institute, 2005,
http://www.awri.com.au/information_services/current/pdfs/Smoke_taint.pdf,
accessed on 4/4/2008.
8. Pizzini, A. Chrismat wines, King Valley, 2006.
9. Whiting, J. and Krstic, M. Understanding the sensitivity to timing and management
options to mitigate the negative impacts of bush fire smoke on grape and wine
quality, Department of Primary Industries Report, 2007.
References 118
10. Aon Re Australia, The January 2003 Canberra Bushfires, 2003,
http://www.extremeweatherheroes.org/media/25782/canberra_bushfires_jan_200
3.pdf, accessed on 4/4/2008.
11. Department of Sustainability and Environment, The Victorian alpine fires, The
state of Victoria, 2007, p 1-6, www.dse.vic.gov.au/DSE/nrenfoe.nsf, accessed on
4/4/2008.
12. Vallesi, M. and Howell, G. The ashes we didn't want-smoke taint in vintage 2007.
The Australian and New Zealand Grapegrower and Winemaker, 2007, May, 66-
67.
13. Visit Vineyards, The Yarra Valley and the Victorian bushfire tradgedy, 2009,
http://www.visitvineyards.com/victoria/yarra-valley-dandenong-
ranges/wine/vineyards-wineries/wine-food-travel-news/yarra-valley-victoria-bush-
fires-destroy-wineries-and-vineyards, accessed on 2/12/2010.
14. Brown, R. Bushfire imaging to determine global warming risks.
2009,http://www.abc.net.au/news/stories/2009/04/09/2539068.htm&usg=4KhGQ
5WtmKS5iQci83QriOYl32k=&h=546&w=840&sz=83&hl=en&start=19&um=1&itbs
=1&tbnid=U5-6roWe8FWd-
M:&tbnh=94&tbnw=145&prev=/images%3Fq%3Dblack%2Bsaturday%2Bbushfire
s%2B2009%2Bsatellite%2Bimages%26um%3D1%26hl%3Den%26sa%3DX%26
tbs%3Disch:1, accessed on 6/8/2010.
15. Prideaux, B. WA battle over smoke taint in wine, National rural news, 2008.
16. Neales, S. Moving slowly in the right direction, The Mercury, 2008.
17. Brown, N.A.C. and van Staden, J. Smoke as a germination cue: a review. Plant
Growth Regul., 1997, 22, 115-124.
References 119
18. Flematti, G.R., Ghisalberti, E.L., Dixon, K.W. and Trengove, R.D. Molecular
weight of a germination-enhancing compound in smoke. Plant soil, 2004, 263, 1-
4.
19. Flematti, G.R., Ghisalberti, E.L., Dixon, K.W. and Trengove, R.D. A compound
from smoke that promotes seed germination. Science, 2004, 305, 977.
20. Gilbert, M.E. and Ripley, B.S. The effect of smoke on the photosynthetic gas
exchange of Chrysanthemoides monilifera. S. Afr. J. Bot., 2002, 68, 525-531.
21. Chambers, D.H., Chambers, E., IV., Seitz, L.M., Sauer, D.B., Robinson, K. and
Allison, A.A. Sensory characteristics of chemical compounds potentially
associated with smoky aroma in foods. Dev. Food. Sci., 1998, 40, 187-194.
22. Niedziela, J.C., MacRae, M., Ogden, I.D. and Nesvadba, P. Control of Listeria
monocytogenes in salmon; antimicrobial effect of salting, smoking and specific
smoke compounds. Lebensm.-Wiss. u.-Technol., 1998, 31, 155-161.
23. Faith, N.G., Yousef, A.E. and Luchansky, J.B. Inhibition of Listeria
Monocytogenes by liquid liquid smoke and isoeugenol, a phenolic component
found in smoke. J. Food Safety, 1992, 12, 303-314.
24. Guillen, M.D. and Ibargoitia, M.L. New components with potential antioxidant and
organoleptic properties, detected for the first time in liquid smoke flavoring
preparations. J. Agric. Food Chem., 1998, 46, 1276-1285.
25. Guillen, M.D. and Ibargoitia, M.L. Volatile components of aqueous liquid smokes
from Vitis vinifera L. shoots and Fagus sylvatica L. wood. J. Sci. Food Agric.,
1996, 72, 104-110.
References 120
26. Guillen, M.D., Manzanos, M.J. and Ibargoitia, M.L. Carbohydrate and
nitrogenated compounds in liquid smoke flavorings. J. Agric. Food Chem., 2001,
49, 2395-2403.
27. Kim, K., Kurata, T. and Fujimaki, M. Identification of flavor constituents in
carbonyl, non-carbonyl neutral and basic fractions of aqueous smoke
condensate. Agr. Biol. Chem., 1974, 38, 53-63.
28. Maga, J.A. The flavor chemistry of wood smoke. Food. Rev. Int., 1987, 3, 139-
183.
29. Luten, J.B., Ritskes, J.M. and Weseman, J.M. Determination of phenol, guaiacol
and 4-methylguaiacol in wood smoke and smoked fish-products by gas-liquid
chromatography. Z. Lebensm. Unters. Forsch., 1979, 168, 289-292.
30. Pettet, A.E.J. and Lane, F.G. A study of the chemical composition of wood
smoke. J. Soc. Chem. Ind., 1940, 59, 114-119.
31. Fiddler, W., Parker, W.E., Wasserman, A.E. and Doerr, R.C. Thermal
decomposition of ferulic acid. J. Agric. Food Chem., 1967, 15, 757-761.
32. Fine, P.M., Cass, G.R. and Simoneit, B.R.T. Chemical characterization of fine
particle emissions from the fireplace combustion of wood types grown in the
Midwestern and Western United States. Environ. Eng. Sci., 2004, 21, 387-409.
33. Fine, P.M., Cass, R. and Simoneit, B.R.T. Chemical characterization of fine
particle emissions from the fireplace combustion of woods grown in the Southern
United States. Environ. Sci. Technol., 2002, 36, 1442-1451.
34. Boidron, J.N., Chatonnet, P. and Pons, M. Influence du bois sur certaines
substances odorantes des vins. Connaissance Vigne Vin, 1988, 22, 275-294.
References 121
35. Wittkowski, R. and Baltes, W. Analysis of liquid smoke and smoked meat
volatiles by headspace gas chromatography. Food Chem., 1990, 37, 135-144.
36. Towey, J.P. and Waterhouse, A.L. Barrel-to-barrel variation of volatile oak
extractives in barrel-fermented Chardonnay. Am. J. Enol. Vitic., 1996, 47, 17-20.
37. Towey, J.P. and Waterhouse, A.L. The extraction of volatile compounds from
French and American oak barrels in Chardonnay during three successive
vintages. Am. J. Enol. Vitic., 1996, 47.
38. Wittkowski, R., Ruther, J., Drinda, H. and Rafiei-Taghanaki, F. Formation of
smoke flavor compounds by thermal lignin degradation. Flavor Precursors, ACS
symposium series, 1992, 490, 232-243.
39. Pollnitz, A.P., Pardon, K.H., Sykes, M. and Sefton, M.A. The effects of sample
preparation and gas chromatograph injection techniques on the accuracy of
measuring guaiacol, 4-methylguaiacol and other volatile oak compounds in oak
extracts by stable isotope dilution analyses. J. Agric. Food Chem., 2004, 52,
3244-3252.
40. Eisele, T.A. and Semon, M.J. Best estimated aroma and taste detection threshold
for guaiacol in water and apple juice J. Food Sci., 2005, 70, S267-S269.
41. Hoj, P., Pretorius, I.S. and Blair, R. The Australian Wine Research Institute
Annual Report, AWRI, 2003.
42. Ferreira, V., Lopez, R. and Cacho, J.F. Quantitative determination of the odorants
of young red wines from different grape varieties. J. Sci. Food Agric., 2000, 80,
1659-1667.
References 122
43. Kennison, K.R., Gibberd, M.R., Pollnitz, A.P. and Wilkinson, K.L. Smoke-derived
taint in wine: the release of smoke-derived volatile phenols during fermentation of
Merlot juice following grapevine exposure to smoke. J. Agric. Food Chem., 2008,
56, 7379-7383.
44. Kennison, K.R., Wilkinson, K.L., Pollnitz, A.P., Williams, H.G. and Gibberd, M.R.
Effect of timing and duration of grapevine exposure to smoke on the composition
and sensory properties of wine. Aust. J. Grape. Wine. R., 2009, 15, 228-237.
45. Kennison, K.R., Wilkinson, K.L., Williams, H.G., Smith, J.H. and Gibberd, M.R.
Smoke-derived taint in wine: effect of postharvest smoke exposure of grapes on
the chemical composition and sensory characteristics of wine. J. Agric. Food
Chem., 2007, 55, 10897-10901.
46. Sheppard, S.I., Dhesi, M.K. and Eggers, N.J. Effect of pre- and postveraison
smoke exposure on guaiacol and 4-methylguaiacol concentration in mature
grapes. Am. J. Enol. Vitic., 2009, 60, 98-103.
47. de Roode, B.M., Franssen, M.C.R., van der Padt, A. and Boom, R.M.
Perspectives for the industrial enzymatic production of glycosides. Biotechnol.
Prog., 2003, 19, 1391-1402.
48. Boulanger, R. and Crouzet, J. Free and bound flavour components of Amazonian
fruits: 3-glycosidically bound components of cupuaca. Food Chem., 2000, 70,
463-470.
49. Dignum, M.J.W., van der Heijden, R., Kerler, J., Winkel, C. and Verpoorte, R.
Identification of glucosides in green beans of Vanilla plantifolia Andrews and
kinetics of Vanilla β-glucosidase. Food Chem., 2004, 85, 199-205.
References 123
50. Fujimatu, E., Ishikawa, T. and Kitajima, J. Aromatic compound glucosides, alkyl
glucoside and glucide from the fruit of anise. Phytochem., 2003, 63, 609-616.
51. Leong, G., Uzio, R. and Derbesy, M. Synthesis, identification and determination
of glucosides present in green vanilla beans (Vanilla fragrans Andrews). Flavour
Fragrance J., 1989, 4, 163-167.
52. Mayorga, H., Knapp, H., Winterhalter, P. and Duque, C. Glycosidically bound
flavor compounds of cape gooseberry (Physalis peruviana L.). J. Agric. Food
Chem., 2001, 49, 1904-1908.
53. Ortiz-Serrano, P. and Gil, J.V. Quantitation of free and glycosidically bound
volatiles in and effect of glycosidase addition on three tomato varieties (Solanum
lycopersicum L.). J. Agric. Food Chem., 2007, 55, 9170-9176.
54. Bureau, S., Razungles, A., Baumes, R. and Bayonove, C. Glycosylated flavor
precursor extraction by microwaves from grape juice and grapes. J. Food Sci.,
1996, 61, 557-560.
55. Bureau, S.M., Baumes, R.L. and Razungles, A.J. Effects of vine or bunch
shading on the glycosylated flavor precursors in grapes of Vitis vinifera L. Cv.
Syrah. J. Agric. Food Chem., 2000, 48, 1290-1297.
56. Cabrita, M.J., Freitas, A.M.C., Laureano, O. and Di Stefano, R. Glycosidic aroma
compounds of some Portuguese grape cultivars. J. Sci. Food Agric., 2006, 86,
922-931.
57. Fernandez-Gonzalez, M. and Di Stefano, R. Fractionation of glycoside aroma
precursors in neutral grapes. Hydrolysis and conversion by Saccharomyces
cerevisiae. Lebensm.-Wiss. u.-Technol., 2004, 37, 467-473.
References 124
58. Francis, I.L., Sefton, M.A. and Williams, P.J. Sensory descriptive analysis of the
aroma of hydrolysed precursor fractions from Semillon, Chardonnay and
Sauvignon Blanc grape juices. J. Sci. Food Agric., 1992, 59, 511-520.
59. Gunata, Y.Z., Bayonove, C.L., Baumes, R.L. and Cordonnier, R.E. The aroma of
grapes I. Extraction and determination of free and glycosidically bound fractions
of some grape aroma components. J. Chromatogr., 1985, 331, 83-90.
60. Sefton, M.A. Hydrolytically-released volatile secondary metabolites from a juice
sample of Vitis vinifera grape cvs Merlot and Cabernet Sauvignon. Aust. J.
Grape. Wine. Res., 1998, 4, 30-38.
61. Skouroumounis, G.K. and Sefton, M.A. Acid-catalyzed hydrolysis of alcohols and
their β-D-glucopyranosides. J. Agric. Food Chem., 2000, 48, 2033-2039.
62. Ugliano, M. and Moio, L. Free and hydrolytically released volatile compounds of
Vitis vinifera L. cv. Fiano grapes as odour-active constituents of Fiano wine. Anal.
Chim. Acta., 2008, 621, 79-85.
63. Ibarz, J., Ferreira, V., Hernandez-Orte, P., Loscos, N. and Cacho, J. Optimization
and evaluation of a procedure for the gas chromatographic-mass spectrometric
analysis of the aromas generated by fast acid hydrolysis of flavor precursors
extracted from grapes. J. Chromatogr. A., 2006, 1116, 217-229.
64. Kotseridis, Y., Baumes, R.L. and Skouroumounis, G.K. Quantitative
determination of free and hydrolytically liberated β-damascenone in red grapes
and wines using a stable isotope dilution assay. J. Chromatogr. A., 1999, 849,
245-254.
References 125
65. Souid, I., Hassene, Z., Palomo, E.S., Perez-Coello, M.S. and Ghorbel, A. Varietal
aroma compounds of Vitis Vinifera cv. Khamri grown in Tunisia. J. Food Quality,
2007, 30, 718-730.
66. Williams, P.J., Strauss, C.R., Wilson, B. and Massy-Westropp, R.A. Studies on
the hydrolysis of Vitis vinifera monoterpene precursor compounds and model
monoterpene β-D-glucosides rationalizing the monoterpene composition of
grapes. J. Agric. Food Chem., 1982, 30, 1219-1223.
67. Wirth, J., Guo, W., Baumes, R. and Gunata, Z. Volatile compounds released by
enzymatic hydrolysis of glycoconjugates of leaves and grape berries from Vitis
vinifera Muscat of Alexandria and Shiraz cultivars. J. Agric. Food Chem., 2001,
49, 2917-2923.
68. Ribereau-Gayon, P., Glories, Y., Maujean, A. and Dubourdieu, D. Handbook of
enology, The chemistry of wine stabilization and treatments; 2 ed., 2000; Vol. 2.
69. Pollnitz, A.P., Jones, G.P. and Sefton, M.A. Determination of oak lactones in
barrel-aged wines and in oak extracts by stable isotope dilution analysis. J.
Chromatogr. A, 1999, 857, 239-246.
70. Fudge, A.L., Elsey, G.M., Hayasaka, Y. and Wilkinson, K.L. Quantitative analysis
of β-D-glucopyranoside of 3-methyl-4-hydroxyoctanoic acid, a potential precursor
to cis-oak lactone, in oak extracts using liquid chromatography-tandem mass
spectrometry based stable isotope dilution analysis. J. Chromatogr. A., 2008,
1215, 51-56.
71. Wilkinson, K.L., Elsey, G.M., Prager, R.H., Tanaka, T. and Sefton, M.A.
Precursors to oak lactone. Part 2: Synthesis, separation and cleavage of several
References 126
β-D-glucopyranosides of 3-methyl-4-hydroxyoctanoic acid. Tetrahedron, 2004,
60, 6091-6100.
72. Gunata, Z., Bitteur, S., Brillouet, J., Bayonove, C. and Cordonner, R. Sequential
enzymatic hydrolysis of potentially aromatic glycosides from grape. Carbohyd.
Res., 1988, 184, 139-149.
73. Koenigs, W. and Knorr, E. Some derivatives of grape sugars and galactose.
Berichte der Deutschen Chemischen Gesellschaft, 1901, 34, 957-981.
74. Wilkinson, K.L. Doctoral Thesis, Flinders University, 2004.
75. Zhou, F.Y. and Zhong, J.H. 6-Acetoxymethyl-2-(2-methoxyphenoxy)-
tetrahydropyran-3,4,5-triyl triacetate. Acta. Crystallogr. E., 2005, E61, 2701-2703.
76. Pollnitz, A.P., Pardon, K.H. and Sefton, M.A. Quantitative analysis of 4-
ethylphenol and 4-ethylguaiacol in red wine. J. Chromatogr. A., 2000, 874, 101-
109.
77. Ugliano, M. and Moio, L. The influence of malolactic fermentation and
Oenococcus oeni strain on glycosidic aroma precursors and related volatile
compounds of red wine. J. Sci. Food Agric., 2006, 86, 2468-2476.
78. Kotseridis, Y., Baumes, R., Bertrand, A. and Skouroumounis, G.K. Quantitative
determination of β-ionone in red wines and grapes of Bordeaux using a stable
isotope dilution assay. J. Chromatogr. A., 1999, 848, 317-325.
79. Kotseridis, Y., Baumes, R. and Skouroumounis, G.K. Synthesis of labelled [2H4]-
β-damascenone, [2H2]-2-methoxy-3-isobutylpyrazine, [2H3]-α-ionone, and [2H3]-β-
ionone for quantification in grapes, juices and wines. J. Chromatogr. A., 1998,
824, 71-78.
References 127
80. Fudge, A.L. Honours Thesis, Flinders University, 2007.
81. Kunz, H. and Harreus, A. Glycoside synthesis using 2,3,4,6-tetra-O-pivaloyl-α-D-
glucopyranosyl bromide. Liebigs Annalen der Chemie 1982, 41-48.
82. Hayasaka, Y., Dungey, K.A., Baldock, G.A., Kennison, K.R. and Wilkinson, K.L.
Identification of a β-D-glucopyranoside precursor to guaiacol in grape juice
following grapevine exposure to smoke. Anal. Chim. Acta., 2010, 660, 143-148.
83. Hayasaka, Y., Baldock, G.A., Pardon, K.H., Jeffery, D.W. and Herderich, M.J.
Investigation into the formation of guaiacol conjugates in berries and leaves of
grapevine Vitis vinifera L. Cv. Cabernet Sauvignon using stable isotope tracers
combined with HPLC-MS and MS/MS analysis. J. Agric. Food Chem., 2010, 58,
2076-2081.
84. Iland, P.G., Bruer, N., Edwards, G., Weeks, S. and Wilkes, E. Chemical analysis
of grapes and wine: techniques and concepts; Patrick Iland Wine Promotions
Pty. Ltd. Adelaide, Australia, 2004.
85. Vazquez, L.C., Perez-Coello, M.S. and Cabezudo, M.D. Effects of enzyme
treatment and skin extraction on varietal volatiles in Spanish wines made from
Chardonnay, Muscat, Airen and Macabeo grapes. Anal. Chim. Acta., 2002, 458,
39-44.
86. Holt, H.E., Iland, P.G. and Ristic, R. A method for mini-lot fermentation for use in
research and commercial viticultural and winemaking trials. Australian and New
Zealand Grapegrower and winemaker, 2006, 509a, 74-81.
References 128
87. Comitini, F., Stringini, M., Taccari, M. and Ciani, M. A fast and simple method for
wild yeast flora detection in winemaking. Int. J. Wine Res., 2009, 1, 53-58.