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Glycosides are sugar conjugates of aroma compounds that are found in many fruits and vegetables, and while glycosides are non-volatile, they can release flavor during eating, through enzyme hydrolysis from oral microbiota. Recently, a range of sensory phenotypes for glucoside perception have been observed, reflecting interindividual variation in response to precursors of floral and smoky flavors, geranyl glucoside and guaiacyl glucoside. To understand this variation and investigate the role of oral microbiota on in vitro hydrolysis of glucosides in saliva, metagenomic screening was conducted using individuals representing the range of sensory phenotypes for geranyl and guaiacyl glucosides. In parallel, sensory retronasal detection thresholds for geranyl glucoside, guaiacyl glucoside, and the volatile odorants geraniol and guaiacol were determined. Oral microbial communities correlated with hydrolysis of glucosides in saliva, but the relationship did not extend to sensory phenotypes. Overall, the retronasal detection threshold of the volatile odorants studied was the main factor determining sensory phenotype. Refereed/Peer-reviewed

The negative effects of smoke exposure of grapes in vineyards that are close to harvest are well documented. Volatile phenols in smoke from forest and grass fires can contaminate berries and, upon uptake, are readily converted into a range of glycosylated grape metabolites. These phenolic glycosides and corresponding volatile phenols are extracted into the must and carried through the winemaking process, leading to wines with overtly smoky aromas and flavours. As a result, smoke exposure of grapes can cause significant quality defects in wine, and may render grapes and wine unfit for sale, with substantial negative economic impacts. Until now, however, very little has been known about the impact on grape composition of smoke exposure very early in the season, when grapes are small, hard and green, as occurred with many fires in the 2019–20 Australian grapegrowing season. This research summarises the compositional consequences of cumulative bushfire smoke exposure of grapes and leaves, it establishes detailed profiles of volatile phenols and phenolic glycosides in samples from six commercial Chardonnay and Shiraz blocks throughout berry ripening and examines the observed effects in the context of vineyard location and timing of smoke exposure. In addition, we demonstrate the potential of some phenolic glycosides in leaves to serve as additional biomarkers for smoke exposure of vineyards.

β-Glycosidases enhance wine aroma by releasing volatile aglycones from non-volatile glycosides. Commercial preparations contain primarily pectinases, with β-glycosidase as a secondary activity, which limits their potential. Here, the extremophilic β-glucosidase A from Halothermothix orenii, (BglA) has been compared with Rapidase® for the production of aromatic wines and in the remediation of smoke-tainted wines. Model systems, real juices and wines have been enriched with geranyl glucoside, typical of white varieties, and guaiacyl glucoside, commonly found in red wines exposed to oak and wines made from grapes exposed to smoke. The hydrolytic capacity of BglA was evaluated by measuring the released volatiles in the gas phase with solid-phase microextraction and GC–MS. BglA, despite an apparent instability at low pH, is twice as effective in releasing volatiles in sweeter wines and in grape juices, offering an excellent alternative for the early stages of the winemaking process and in the juice industry.

Pioneering investigations into precursors of fruity and floral flavors established the importance of terpenoid and C 13 -norisoprenoid glycosides to the flavor of aromatic wines. Nowadays flavor precursors in grapes and wine are known to be structurally diverse, encompassing glycosides, amino acid conjugates, odorless volatiles, hydroxycinnamic acids, and many others. Flavor precursors mainly originate in the grape berry but also from oak or other materials involved in winemaking. Flavors are released from precursors during crushing and subsequent production steps by enzymatic and nonenzymatic transformations, via microbial glycosidases, esterases, C-S lyases, and decarboxylases, and through acid-catalyzed hydrolysis and chemical rearrangements. Flavors can also be liberated from glycosides and amino acid conjugates by oral microbiota. Hence, it is increasingly likely that flavor precursors contribute to retronasal aroma formation through in-mouth release during consumption, prompting a shift in focus from identifying aroma precursors in grapes to understanding aroma precursors present in bottled wine. Refereed/Peer-reviewed

Background and Aims:Grape glycosides are an important source of wineflavour, especially for ‘floral’ cultivars. This study tested whether phenol-free glycosides from marc could be a latent source of flavour in white wine without affecting bitterness Methods and Results:Glycosides were extracted from Gewürztraminer marc with water followed by a polymeric resin adsorption and purification step, which removed phenolic substances. The glycosides were added at single and double the concentration of that in the grapes to Riesling and Chardonnay juices prior to fermentation, and to wines at bottling. The addition of phenolic-free glycosides significantly increased the concentration of monoterpenes and monoterpene glycosides,resulting in an increase in fruity and floral aroma, flavour and aftertaste attributes, as determined by sensory descriptive analysis, while not significantly altering the bitterness or astringency. The timing of the addition had only a minor effect.Consumer liking data on a subset of the wines indicated that a double addition of glycosides was not well accepted, although a cluster of consumers liked the Riesling with a single addition of glycosides Conclusions:Phenol-free glycosides extracted from marc can increase floral and fruity flavour in Riesling and Chardonnay wines, without altering bitterness or astringency Significance of the Study:Adding phenol-free glycosides from grape marc can enhance wine flavour and persistence. The relatively simple extraction method allows utilisation of marc as a source of flavour at a production scale Refereed/Peer-reviewed

The terpenoids are a major class of natural products with a range of functions in plants. In the grape, monoterpenes, sesquiterpenes and C13-norisoprenoids are present either in a free volatile form, or bound to sugars and rendered non-volatile. Of the many terpenoids in grapes, only a relatively small number makes an important contribution to wine aroma. This review summarises new developments in understanding terpenoids, including the monoterpenes linalool, cis-rose oxide, 1,8-cineole and wine lactone; the sesquiterpene rotundone; and the C13-norisoprenoids β-damascenone, β-ionone, 1,1,6-trimethyl-1,2-dihydronaphthalene and (E)-1-(2,3,6-trimethylphenyl)buta-1,3-diene. Their origin, formation and behaviour during winemaking and ageing, and viticultural influences on their concentration, will be discussed, as well as their role in different wine styles.

This study investigated the sensory significance of monoterpene glycosides during tasting, by retronasal perception of odorant aglycones released in-mouth. Monoterpene glycosides were isolated from Gewürztraminer and Riesling juices and wines, chemically characterised and studied using sensory time-intensity methodology, together with a synthesised monoterpene glucoside. When assessed in model wine at five times wine-like concentration, Gewürztraminer glycosides and geranyl glucoside gave significant fruity flavour, although at wine-like concentrations, or in the presence of wine volatiles, the effect was not significant. Gewürztraminer glycosides, geranyl glucoside and guaiacyl glucoside were investigated using a sensory panel (n = 39), revealing large inter-individual variability, with 77% of panellists responding to at least one glycoside. The study showed for the first time that grape-derived glycosides can contribute perceptible fruity flavour, providing a means of enhancing flavour in wines, and confirms the results of previous studies that the effect is highly variable across individuals. Refereed/Peer-reviewed

Bushfires occur frequently in the vicinity of grape growing regions, resulting in smoke drifting over the vineyards. Wine made from smoked grapes is often downgraded or unfit for sale due to negative sensory characters. To manage or avoid the risk of producing smoke-affected wine, a diagnostic assay was developed for assessing the extent of smoke exposure in grapes and the resulting wines. The method relies on the quantitation of the glycosidic grape metabolites that are formed from major volatile phenols present in smoke. Using HPLC-MS/MS with APCI, a quantitation method for phenolic glycosides as smoke marker compounds was developed and validated. The method was confirmed to be of sufficient sensitivity and reliability to use as a diagnostic assay. On the basis of phenolic glycoside concentrations, grapes or wine can be assessed as smoke exposed or not, and the relative intensity of smoke exposure can be determined.

Background and Aims: Australian grape growers and winemakers have typically relied on guaiacol and 4-methylguaiacol measurements to determine smoke exposure of grapes following bushfires or prescribed burns. However, the guaiacol and 4-methylguaiacol content of grapes does not always correlate with the extent of taint in resultant wines. This study compared several methods for the analysis of smoke related phenols and their conjugates in grapes and wine, to determine their capacity as diagnostic assays for smoke exposure. Methods and Results: Smoke-affected grapes were sourced from commercial vineyards exposed to bushfire smoke and from experimental field trials involving the application of smoke to grapevines, and small-scale wines were made from a number of these samples. Several analytical methods were applied to grapes and wine to determine the concentration of smoke related phenols and their conjugates. Strong correlations were observed between the glycoconjugate content of smoke-affected grapes and the concentration of guaiacol and 4-methylguaiacol released following acid hydrolysis of juice. Conclusions: Where smoke-affected grapes contain low or non-detectable levels of guaiacol and 4-methylguaiacol, analytical methods that quantify their glycoconjugate forms (either directly or indirectly) provide a better indication of the extent of smoke exposure. Significance of the Study: This is the first study to compare different methods for assessing smoke exposure in grapes and wine, through analysis of free and bound guaiacol and 4-methylguaiacol. These methods will allow grape growers and winemakers to more reliably assess smoke exposure of grapes, enabling better informed decisions to be made with regards to harvesting and processing smoke-affected grapes.

To assess the effects of exogenous tannin addition on the properties of a red wine made from Vitis vinifera cv. Shiraz, a winemaking study was performed with pre- and post-alcoholic fermentation additions of 200 mg/L of a commercial grape seed-derived oenotannin product. The influence of the added oenotannin on wine tannin concentration, pigmented polymer concentration and colour parameters was monitored throughout the winemaking process and for up to two years of post-bottling storage. Additionally, after one year of storage, the in-mouth sensory attributes of the wines were characterised using sensory descriptive analysis. No significant differences in wine colour properties and pigment profiles were found between treatments. Tannin concentration as measured by HPLC was not significantly different at the end of the experiment, but when considered over the whole time-course it was significantly higher in the post-ferment treatment. The magnitude of the effect on astringency of the oenotannin addition at 200 mg/L during winemaking was small and the sensory response was the same regardless of addition pre- or post-fermentation. The magnitude of the sensory response to the wines was not significantly different from that of the control wine assessed after (i) 200 mg/L of the oenotannin was added immediately prior to sensory evaluation, or (ii) addition of 200 mg/L of two other commercial tannin preparations. For the type of red wine and oenotannin studied, tannin addition did not affect wine colour properties and had only a minor effect on perceived astringency.

Near infrared (NIR) spectroscopy was used to simultaneously predict the concentrations of malvidin-3-glucoside (M3G), pigmented polymers (PP) and tannins (T) in red wine. A total of 495 samples from 32 commercial scale red wine fermentations over two vintages using two grape varieties (Cabernet Sauvignon and Shiraz), and also including as additional variables two types of fermenters, two different yeasts, and three fermentation temperatures were used. Samples were scanned in transmission mode (400–2500 nm) using a monochromator instrument (NIRSystems6500). Calibration equations were developed from high performance liquid chromatography (HPLC) and NIR data using partial least squares (PLS) regression with internal cross validation. Using PLS regression, very good calibration statistics (Rcal2>0.80) were obtained for the prediction of M3G, PP and T with standard deviation (S.D.)/standard error in cross validation (SECV) ratio (residual predictive deviation, RPD)) ranging from 1.8 to 5.8. It was concluded that near infrared spectroscopy could be used as rapid alternative method for the prediction of the concentration of phenolic compounds in red wine fermentations.

The volatile phenols guaiacol, 4-methylguaiacol, syringol, 4-methylsyringol, o-, m-, and p-cresol, as well as their glycoconjugates, have previously been shown to be present in elevated concentrations in smoke-tainted wine. Sensory descriptive analysis experiments, with addition of free volatile phenols in combination with their glycosidically bound forms, were used to mimic smoke taint in red wines. The addition of volatile phenols together with glycoconjugates gave the strongest off-flavor. The hydrolysis of glycosidically bound flavor compounds in-mouth was further investigated by in vitro and in vivo experiments. The results indicate that enzymes present in human saliva are able to release the volatile aglycones from their glycoconjugates even under low pH and elevated ethanol conditions, confirming that in-mouth breakdown of monosaccharide and disaccharide glycosides is an important mechanism for smoke flavor from smoke affected wines, and that this mechanism may play an important general role in the flavor and aftertaste of wine.

The phenolic composition of red wine impacts upon the color and mouthfeel and thus quality of the wine. Both of these characteristics differ depending on the age of a wine, with the purple of young wines changing to brick red and the puckering or aggressive astringency softening in older wines. This study investigated the color parameters, tannin concentrations and tannin composition of a 50 year series of Cabernet Sauvignon wines from a commercial label as well as 30 year series of Cabernet Sauvignon and Shiraz wines from a separate commercial label to assess the impact of wine age on phenolic composition and concentration. The wine color density in wines of 40 to 50 years old was around 5 AU compared with 16 AU of wine less than 12 months old, which correlated well with the concentration of non-bleachable pigments and pigmented polymers. Conversely, the anthocyanin concentrations in 10 year old wines were substantially lower than that of recently bottled wines (around 100 mg/L compared with 627 mg/L, respectively), adding further evidence that non-bleachable pigments including pigmented polymers play a much larger role in long-term wine color than anthocyanins. No age-related trend was observed for tannin concentration, indicating that the widely noted softer astringency of older red wines cannot necessarily be directly related to lower concentrations of soluble wine tannin and is potentially a consequence of changes in tannin structure. Wine tannins from older wines were generally larger than tannins from younger wines and showed structural changes consistent with oxidation.

Guaiacol and 4-methylguaiacol are well-known as contributors to the flavor of wines made from smoke-affected grapes, but there are other volatile phenols commonly found in smoke from forest fires that are also potentially important. The relationships between the concentration of a range of volatile phenols and their glycoconjugates with the sensory characteristics of wines and model wines were investigated. Modeling of the attribute ratings from a sensory descriptive analysis of smoke-affected wines with their chemical composition indicated the concentrations of guaiacol, o-cresol, m-cresol, and p-cresol were related to smoky attributes. The best-estimate odor thresholds of these compounds were determined in red wine, together with the flavor threshold of guaiacol. Guaiacol β-D-glucoside and m-cresol β-D-glucoside in model wine were found to give rise to a smoky/ashy flavor in-mouth, and the respective free volatiles were released. The study indicated that a combination of volatile phenols and their glycosides produces an undesirable smoke flavor in affected wines. The observation of flavor generation from nonvolatile glycoconjugates in-mouth has potentially important implications.

The presence of glycosides of smoke-derived volatile phenols in smoke-affected grapes and the resulting wines of Chardonnay and Cabernet Sauvignon was investigated with the aid of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). All volatile phenols studied (phenol, p-, m-, and o-cresols, methylguaiacol, syringol, and methylsyringol) could be detected as glycosylated metabolites in smoke-affected grapes in a similar fashion to that previously reported for guaiacol. These phenolic glycosides were found in smoke-affected grapes and wines at significantly elevated levels compared to those in non-smoked control grapes and wines. The extraction of these glycosides from grapes into wine was estimated to be 78% for Chardonnay and 67% for Cabernet Sauvignon. After acid hydrolysis, a large proportion of these phenolic glycosides in grapes (50%) and wine (92%) disappeared but the concentrations of volatile phenols determined by gas chromatography-mass spectrometry (GC-MS) were lower than expected. In the case of wine, the majority of the glycosides of phenol, cresols, guaiacol, and methylguaiacol were decomposed upon acid hydrolysis without releasing their respective aglycones, while syringol and methylsyringol were more effectively released.

Background and Aims: Flavonols are present in Vitis vinifera L. wine grapes as glycosides and are found in wines in both their glycosidic and aglycone forms. Flavonols and their glycosides are important components in wine because of their impact on colour, taste and health properties. An assessment of the flavonols and flavonol glycosides present in a wide selection of Australian red and white wines (n = 121 and 44, respectively) was undertaken. Methods and Results: Analyses were performed using HPLC, with compound identification being aided by HPLC-MS. In the red wines, quercetin and myricetin dominated the flavonol profile, and quercetin-3-glucuronide was by far the most abundant glycoside present. White wines had considerably lower levels of flavonols and their glycosides, in most cases, being below the detection limit of the HPLC method. Conclusions: There appeared to be no correlation between the flavonol profile and wine variety or region in either white or red wines. Contrary to literature reports of rutin in wine, rutin was not found in any of the wines we analysed, and spiking experiments showed rutin rapidly degraded to the aglycone quercetin. Furthermore, changes in elution order for some quercetin glycoside standards were observed depending upon the acid used in the mobile phase. Significance of the Study: This is the first time the flavonol contents of an extensive cross section of Australian wines have been reported. The concentrations are consistent with those reported in wines from other countries. In light of the instability of rutin in wine, critical review should be given to reports discussing its presence.

An obscure sesquiterpene, rotundone, has been identified as a hitherto unrecognized important aroma impact compound with a strong spicy, peppercorn aroma. Excellent correlations were observed between the concentration of rotundone and the mean 'black pepper' aroma intensity rated by sensory panels for both grape and wine samples, indicating that rotundone is a major contributor to peppery characters in Shiraz grapes and wine (and to a lesser extent in wine of other varieties). Approximately 80% of a sensory panel were very sensitive to the aroma of rotundone (aroma detection threshold levels of 16 ng/L in red wine and 8 ng/L in water). Above these concentrations, these panelists described the spiked samples as more 'peppery' and 'spicy'. However, approximately 20% of panelists could not detect this compound at the highest concentration tested (4000 ng/L), even in water. Thus, the sensory experiences of two consumers enjoying the same glass of Shiraz wine might be very different. Rotundone was found in much higher amounts in other common herbs and spices, especially black and white peppercorns, where it was present at approximately 10000 times the level found in very 'peppery' wine. Rotundone is the first compound found in black or white peppercorns that has a distinctive peppery aroma. Rotundone has an odor activity value in pepper on the order of 50000-250000 and is, on this criterion, by far the most powerful aroma compound yet found in that most important spice.

'Black pepper' aroma and flavor is important to some Australian Shiraz red wine styles but the aroma compounds involved have yet to be identified, and no objective analytical method to assess 'pepper' grape aromas is available to date. Samples of potentially 'spicy'/'peppery' grapes were obtained from vineyards in South Australia and Victoria over two vintages. The important sensory attributes of the grapes, including the aroma descriptor 'pepper', were rated by a sensory panel. The sensory study revealed a strong correlation between the intensity of 'pepper' aroma and the intensity of 'pepper' flavor perceived on the palate. The grape homogenates were analyzed by static headspace GC-MS using a cool inlet system. Vectors obtained by analysis of over 13 000 individual mass spectra per grape sample were then subjected to multivariate analyses. Both principal component analysis and partial least-squares regression were used to develop multivariate models based on mass spectra and aroma descriptors to explain the intensity of the rating of the 'pepper' character. Corresponding differences in mass spectra and aroma were observed among vineyards and from the same vineyards in different years. Additional optimization of the methodology enabled selection of a single region of the GC-MS chromatogram that allowed prediction of 'pepper' aroma intensity with a correlation coefficient0.98 and led to the identification of alpha-ylangene, a tricyclic sesquiterpene. To assess the potential of alpha-ylangene as a marker for this sensory characteristic, a method for alpha-ylangene analysis of grapes and wine using HS-SPME-GC-MS was developed. Although not a significant aroma compound by itself, alpha-ylangene was a satisfactory marker for the 'pepper' aroma in grapes and wine, and its concentration showed similar discrimination between 'peppery' vineyards and vintages as that obtained using the multivariate models. Despite its presence in grapes, we could not detect alpha-ylangene in wine.

The modern wine industry needs tools for process control and quality assessment in order to better manage fermentation or bottling processes. During wine fermentation it is important to measure both substrate and product concentrations (e.g. sugars, phenolic compounds), however, the analysis of these compounds by traditional means requires sample preparation and in some cases several steps of purification are needed. The combination of visible/near-infrared (Vis/NIR) spectroscopy and chemometrics potentially provides an ideal solution to accurately and rapidly monitor physical or chemical changes in wine during processing without the need for chemical analysis. The aim of this study was to assess the possibility of combining spectral and multivariate techniques, such as principal component analysis (PCA), discriminant partial least squares (DPLS), or linear discriminant analysis (LDA), to monitor time-related changes that occur during red wine fermentation. Samples (n = 652) were collected at various times from several pilot scale fermentations with grapes from either Cabernet Sauvignon or Shiraz varieties, over three vintages (2001-2003) and scanned using a monochromator instrument (Foss-NIRSystems 6500, Silver Spring, MD) in transmission mode (400-2,500 nm). PCA was used to demonstrate consistent progressive spectral changes that occur through the time course of the fermentation. LDA using PCA scores showed that regardless of variety or vintage, samples belonging to a particular time point in fermentation could be correctly classified. This study demonstrates the potential of Vis/NIR spectroscopy combined with chemometrics, as a tool for the rapid monitoring of red wine fermentation.