A Review of Wine Authentication Using Spectroscopic Approaches in Combination with Chemometrics.

In a global context where trading of wines involves considerable economic value, the requirement to guarantee wine authenticity can never be underestimated. With the ever-increasing advancements in analytical platforms, research into spectroscopic methods is thriving as they offer a powerful tool for rapid wine authentication. In particular, spectroscopic techniques have been identified as a user-friendly and economical alternative to traditional analyses involving more complex instrumentation that may not readily be deployable in an industry setting. Chemometrics plays an indispensable role in the interpretation and modelling of spectral data and is frequently used in conjunction with spectroscopy for sample classification. Considering the variety of available techniques under the banner of spectroscopy, this review aims to provide an update on the most popular spectroscopic approaches and chemometric data analysis procedures that are applicable to wine authentication.

Uptake and Glycosylation of Smoke-Derived Volatile Phenols by Cabernet Sauvignon Grapes and Their Subsequent Fate during Winemaking.

Wine made from grapes exposed to bushfire smoke can exhibit unpleasant smoky, ashy characters, which have been attributed to the presence of smoke-derived volatile phenols, in free or glycosylated forms. Here we report the uptake and glycosylation of volatile phenols by grapes following exposure of Cabernet Sauvignon vines to smoke, and their fate during winemaking. A significant delay was observed in the conversion of volatile phenols to their corresponding glycoconjugates, which suggests sequestration, the presence of intermediates within the glycosylation pathway and/or other volatile phenol storage forms. This finding has implications for industry in terms of detecting smoke-affected grapes following vineyard smoke exposure. The potential for an in-canopy sprinkler system to mitigate the uptake of smoke-derived volatile phenols by grapes, by spraying grapevines with water during smoke exposure, was also evaluated. While "misting" appeared to partially mitigate the uptake of volatile phenols by grapes during grapevine exposure to smoke, it did not readily influence the concentration of volatile phenols or the sensory perception of smoke taint in wine. Commercial sensors were used to monitor the concentration of smoke particulate matter (PM) during grapevine exposure to low and high density smoke. Similar PM profiles were observed, irrespective of smoke density, such that PM concentrations did not reflect the extent of smoke exposure by grapes or risk of taint in wine. The sensors could nevertheless be used to monitor the presence of smoke in vineyards during bushfires, and hence, the need for compositional analysis of grapes to quantify smoke taint marker compounds.

Inactivating Mutations in Irc7p Are Common in Wine Yeasts, Attenuating Carbon-Sulfur ß-Lyase Activity and Volatile Sulfur Compound Production.

During alcoholic fermentation of grape sugars, wine yeasts produce a range of secondary metabolites that play an important role in the aroma profile of wines. In this study, we have explored the ability of a large number of wine yeast strains to modulate wine aroma composition, focusing on the release of the "fruity" thiols 3-mercaptohexan-1-ol (3-MH) and 4-mercapto-4-methylpentan-2-one (4-MMP) from their respective cysteinylated nonvolatile precursors. The role of the yeast gene IRC7 in thiol release has been well established, and it has been shown that a 38-bp deletion found in many wine strains cause them to express a truncated version of Irc7p that does not possess cysteine-S-conjugate ß-lyase activity. In our data, we find that IRC7 allele length alone does not fully explain the capacity of a strain to release thiols. Screening of a large number of strains coupled with analysis of genomic sequence data allowed us to identify several previously undescribed single-nucleotide polymorphisms (SNPs) in IRC7 that, when coupled with allele length, more robustly explain the ability of a particular yeast strain to release thiols from their cysteinylated precursors. We also demonstrate that allelic variation of IRC7 not only affects the release of thiols but modulates the formation of negative volatile sulfur compounds from the amino acid cysteine. The results of this study provide winemakers with an improved understanding of the genetic determinants that affect wine aroma and flavor, which can be used to guide the choice of yeast strains that are fit for purpose.IMPORTANCE Volatile sulfur compounds contribute to wine aromas that may be considered pleasant, such as "tropical," "passionfruit," and "guava," as well as aromas that are considered undesirable, such as "rotten eggs," "onions," and "sewer." During fermentation, wine yeasts release some of these compounds from odorless precursor molecules, a process that is most efficient when performed by yeasts that express active forms of the protein Irc7p. We show that most wine yeasts carry mutations that reduce activity of this protein, affecting the formation of volatile sulfur compounds that impart both pleasant and unpleasant aromas. The results provide winemakers with guidance on the choice of yeasts that can emphasize or deemphasize this particular contribution to wine quality.

Analysis of Potent Odour-Active Volatile Thiols in Foods and Beverages with a Focus on Wine.

Certain volatile thiols are some of the most potent odour-active molecules that are found in nature. Thiols play significant roles in the aroma qualities of a range of foods and beverages, including wine, with extremely low odour detection thresholds (nanogram per litre range). A fundamental understanding of their formation, fate, and impact essentially depends on the development of suitable analytical methods. The analysis of volatile thiols in foods and beverages is a challenging task when considering (1) the complexity of food and beverage matrices and (2) that thiols are highly reactive, low molecular-weight volatiles that are generally present at trace to ultra-trace concentrations. For the past three decades, the analytical evaluation of volatile thiols has been intensively performed in various foods and beverages, and many novel techniques related to derivatisation, isolation, separation, and detection have been developed, particularly by wine researchers. This review aims to provide an up-to-date overview of the major analytical methodologies that are proposed for potent volatile thiol analysis in wine, foods, and other beverages. The analytical challenges for thiol analysis in foods and beverages are outlined, and the main analytical methods and recent advances in methodology are summarised and evaluated for their strengths and limitations. The key analytical aspects reviewed include derivatisation and sample preparation techniques, chromatographic separation, mass spectrometric detection, matrix effects, and quantitative analysis. In addition, future perspectives on volatile thiol research are also suggested.

Simple quantitative determination of potent thiols at ultratrace levels in wine by derivatization and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis.

Volatile sulfur compounds contribute characteristic aromas to foods and beverages and are widely studied, because of their impact on sensory properties. Certain thiols are particularly important to the aromas of roasted coffee, cooked meat, passion fruit, grapefruit, and guava. These same thiols enhance the aroma profiles of different wine styles, imparting pleasant aromas reminiscent of citrus and tropical fruits (due to 3-mercaptohexan-1-ol, 3-mercaptohexyl acetate, 4-mercapto-4-methylpentan-2-one), roasted coffee (2-furfurylthiol), and struck flint (benzyl mercaptan), at nanogram-per-liter levels. In contrast to the usual gas chromatography (GC) approaches, a simple and unique high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for routine analysis of five wine thiols, using 4,4'-dithiodipyridine (DTDP) as a derivatizing agent and polydeuterated internal standards for maximum accuracy and precision. DTDP reacted rapidly with thiols at wine pH and provided stable derivatives, which were enriched by solid-phase extraction (SPE) prior to analysis by HPLC-MS/MS. All steps were optimized and the method was validated in different wine matrices, with method performance being comparable to a well-optimized but more cumbersome gas chromatography-mass spectrometry (GC-MS) method. A range of commercial wines was analyzed with the new method, revealing the distribution of the five thiols in white, red, rosé, and sparkling wine styles.

Unravelling glutathione conjugate catabolism in Saccharomyces cerevisiae: the role of glutathione/dipeptide transporters and vacuolar function in the release of volatile sulfur compounds 3-mercaptohexan-1-ol and 4-mercapto-4-methylpentan-2-one.

Sulfur-containing aroma compounds are key contributors to the flavour of a diverse range of foods and beverages, such as wine. The tropical fruit characters of Sauvignon Blanc wines are attributed to the presence of the aromatic thiols 3-mercaptohexan-1-ol (3-MH), its acetate ester 3-mercaptohexyl acetate (3-MHA), and 4-mercapto-4-methylpentan-2-one (4-MMP). These aromatic thiols are not detectable in grape juice to any significant extent but are released by yeast during alcoholic fermentation. While the processes involved in the release of 3-MH and 4-MMP from their cysteinylated precursors have been studied extensively, degradation pathways for glutathione S-conjugates (GSH-3-MH and GSH-4-MMP) have not. In this study, a candidate gene approach was taken, focusing on genes known to play a role in glutathione and glutathione-S-conjugate turnover in Saccharomyces cerevisiae. Our results confirm the role of Opt1p as the major transporter responsible for uptake of GSH-3-MH and GSH-4-MMP, and identify vacuolar Ecm38p as a key determinant of 3-MH release from GSH-3-MH. ECM38 was unimportant, on the other hand, for release of 4-MMP, and abolition of vacuolar biogenesis caused an increase in the amount of 4-MMP released. The alternative cytosolic glutathione degradation pathway was not involved in release of either thiol from their glutathionylated precursors. Finally, cycling of GSH-3-MH and/or its breakdown intermediates between the cytosol and the vacuole or extracellular space was implicated in modulation of 3-MH formation. Together, these results provide new targets for development of yeast strains that optimize release of these potent volatile sulfur compounds, and further our understanding of the processes involved in glutathione-S-conjugate turnover.

Impact of accentuated cut edges, yeast strain, and malolactic fermentation on chemical and sensory profiles of Sauvignon blanc wine.

This pioneering investigation involved the application of accentuated cut edges (ACE) technique to Sauvignon blanc winemaking. The concentration of varietal thiol precursors in juice was significantly higher for ACE treatment compared to conventional crushing, with two-way or three-way interactions of the experimental factors, which included yeast strain and malolactic fermentation, being determined from the wine data. ACE yielded higher concentrations of 4-methyl-4-sulfanylpentan-2-one (4-MSP) and enantiomers of 3-sulfanylhexanol (3-SH) and 3-sulfanylhexyl acetate (3-SHA) in wines that were more abundant in phenolic compounds. Compared to Sauvy yeast strain, VIN13 produced greater amounts of 3-SH and 3-SHA but less 4-MSP with wines exhibiting lower intensity 'floral' and 'fruity' notes. MLF increased 3-SH and 4-MSP concentrations and led to wines that exhibited more non-fruity sensory attributes. The study revealed the potential of ACE for increasing varietal thiol concentrations in Sauvignon blanc wine and altering overall sensory profiles, with interactions involving yeast strain and MLF.

Methoxypyrazine concentrations in the grape bunch rachis of Vitis vinifera L. Cv Shiraz: Influence of rootstock, region and light.

Vitis vinifera L. cv Shiraz appears unable to synthesise 3-alkyl-2-methoxypyrazines (MPs) in the berry, but can still produce significant concentrations in rachis. MPs are readily extracted from rachis during fermentation, producing Shiraz wines with uncharacteristic "green" flavours. Recently, rootstocks were shown to significantly alter MP concentrations in Cabernet Sauvignon rachis compared to own-rooted varieties, but whether Shiraz followed a similar trend required investigation. This study considered the effect of thirteen rootstocks on the concentrations of 3-isobutyl-2-methoxypyrazine (IBMP), 3-isopropyl-2-methoxypyrazine (IPMP), and 3-sec-butyl-2-methoxypyrazine (SBMP) in the rachis of Shiraz bunches sampled during multiple vintages across several Australian growing regions. Although IBMP was the most abundant, all measured MP concentrations were significantly affected by vintage, rootstock, and region. In addition, vine vigour showed positive correlations with IBMP, which were attributed to changes in canopy coverage impacting rachis light exposure. This hypothesis was explored with light exclusion trials, which significantly increased rachis IBMP concentrations.

Insights into the Uptake, Distribution, and Metabolism of 3-Isobutyl-2-hydroxypyrazine in Grapevine Using a Stable Isotope Tracer.

Methoxypyrazines (MPs) are potent aroma compounds that have been predominately studied in grape berries but can also be detected in other vine tissues. The synthesis of MPs in berries from hydroxypyrazines by VvOMT3 is well established, but the origin of MPs in vine tissues that have negligible VvOMT3 gene expression is unknown. This research gap was addressed through the application of stable isotope tracer 3-isobutyl-2-hydroxy-[2H2]-pyrazine (d2-IBHP) to the roots of Pinot Meunier L1 microvines and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) quantification of HPs from grapevine tissues following a novel solid-phase extraction method. Four weeks post-application, d2-IBHP and its O-methylated product 3-isobutyl-2-methoxy-[2H2]-pyrazine (d2-IBMP) were present in excised cane, berry, leaf, root, and rachis material. Translocation of d2-IBHP and d2-IBMP was investigated, but results were inconclusive. Nonetheless, knowledge that d2-IBHP, and potentially d2-IBMP, are translocated from roots to other vine organs, including the berries, could provide opportunities for controlling MP accumulation in grapevine tissues pertinent to winemaking.

Impact of accentuated cut edges (ACE) technique on volatile and sensory profiles of Shiraz wines.

Varietal thiols are important wine aroma compounds that are generally less abundant in red wines. Accentuated cut edges (ACE), known for accelerating phenolic extraction, was applied to Shiraz winemaking and compared with conventional crushing (NOACE) to examine the effects on varietal thiol precursor extraction and thiol formation. Water addition to grape must and skin contact time (SCT) during fermentation were also assessed. Although there was no difference for precursors in the must, ACE significantly decreased 3-S-glutathionylhexan-1-ol concentration during fermentation. 3-Sulfanylhexan-1-ol and ethyl esters were significantly influenced by crushing method and/or SCT, with NOACE or shorter SCT yielding higher concentrations. Acetates, higher alcohols, fatty acids, and isoprenoids differed according to the interaction of crushing method and SCT, with ACE and shorter SCT significantly enhancing all groups except acetates. Volatiles in Sauvignon blanc and Pinot noir wines produced at commercial scale with ACE were briefly evaluated, suggesting an impact of grape variety.

Consumer perspectives of wine typicity and impact of region information on the sensory perception of Cabernet Sauvignon wines.

Region of origin is used in marketing of wine and by consumers as a wine quality indicator. To better understand wine consumers' purchase decisions, sensory perception, and wine liking in connection with wine provenance, this study used regular wine consumers (n = 112) to evaluate two Cabernet Sauvignon wines from each of four wine producing regions through hedonic rating and rate-all-that-apply (RATA) testing in conjunction with pre- and post-tasting questionnaires. The majority of consumers rated the region of origin stated on the label as important for purchase intent and for deciding the price they were willing to pay for a wine. The questionnaire also revealed that consumers were familiar with the wine typicity concept, but seemed to consider it only as an extrinsic characteristic rather than an intrinsic aspect of the wine. By randomly dividing the consumers into two groups (n = 56 each), one having information on the origin of samples and the other tasting without such knowledge, it was demonstrated that origin information had a positive impact on hedonic scores. Sensory profiling revealed that origin information did not impact the sample sensory characterisation, and liking for both groups was related to 'full body', 'jammy', and 'dark fruits' attributes. Some regional profile features were apparent for the samples, such as 'minty' for Coonawarra and savoury attributes for Bordeaux. Overall, this work highlighted that consumers could differentiate wines from distinct regions on the basis of sensory characteristics.

Chemical and Sensory Profiles of Sauvignon Blanc Wine Following Protein Stabilization Using a Combined Ultrafiltration/Heat/Protease Treatment.

Ultrafiltration (UF) was evaluated as a process by which proteins can be selectively removed from white wine as an alternative approach to protein stabilization than traditional bentonite fining. Unfined Sauvignon Blanc wine (50 L) was fractionated by UF and the retentate stabilized either by heat and/or protease treatment or bentonite fining before being recombined with the permeate. The heat stability of recombined wine was significantly improved when retentate was heated following protease (Aspergillopepsin) addition and subsequently stabilized by bentonite treatment. The combined UF/heat/protease treatment removed 59% of protein and reduced the quantity of bentonite needed to achieve protein stability by 72%, relative to bentonite treatment alone. This innovative approach to protein stabilization had no significant impact on wine quality or sensory characteristics, affording industry greater confidence in adopting this technology as a novel approach to achieving protein stability.

Rootstock, Vine Vigor, and Light Mediate Methoxypyrazine Concentrations in the Grape Bunch Rachis of Vitis vinifera L. cv. Cabernet Sauvignon.

Ramsey rootstock has previously been implicated in an approximate 8-fold increase of 3-isobutyl-2-methoxypyrazine (IBMP) levels in the rachis (grape bunch stem) of Vitis vinifera L. cv. Shiraz scions over own-rooted Shiraz vines at harvest. IBMP extracted from rachis during red wine fermentation can contribute potent "green" flavors. Methoxypyrazines (MPs) are normally present in Cabernet Sauvignon grapes, rachis, and wines, but it is unknown whether rootstocks can influence the MP concentration in the rachis. This study considered the effect of eight rootstocks including Ramsey and own roots on the concentrations of IBMP, 3-isopropyl-2-methoxypyrazine (IPMP), and 3-sec-butyl-2-methoxypyrazine (SBMP) in the rachis and grapes of Cabernet Sauvignon over two seasons. IBMP predominated, and its concentration in rachis and berries at harvest was significantly affected by rootstock and growing season. In the 2020 vintage, light exclusion, vine vigor, and spatial variation in vine vigor were shown to significantly affect MP concentrations in rachis.

Impact of Lachancea thermotolerans on Chemical Composition and Sensory Profiles of Viognier Wines.

Viognier is a warm climate grape variety prone to loss of acidity and accumulation of excessive sugars. The yeast Lachancea thermotolerans can improve the stability and balance of such wines due to the partial conversion of sugars to lactic acid during alcoholic fermentation. This study compared the performance of five L. thermotolerans strains in co-inoculations and sequential inoculations with Saccharomyces cerevisiae in high sugar/pH Viognier fermentations. The results highlighted the dichotomy between the non-acidified and the bio-acidified L. thermotolerans treatments, with either comparable or up to 0.5 units lower pH relative to the S. cerevisiae control. Significant differences were detected in a range of flavour-active yeast volatile metabolites. The perceived acidity mirrored the modulations in wine pH/TA, as confirmed via "Rate-All-That-Apply" sensory analysis. Despite major variations in the volatile composition and acidity alike, the varietal aromatic expression (i.e., stone fruit aroma/flavour) remained conserved between the treatments.

Engineering Saccharomyces cerevisiae to release 3-Mercaptohexan-1-ol during fermentation through overexpression of an S. cerevisiae Gene, STR3, for improvement of wine aroma.

Sulfur-containing aroma compounds are key contributors to the flavor of a diverse range of foods and beverages. The tropical fruit characters of Vitis vinifera L. cv. Sauvignon blanc wines are attributed to the presence of the aromatic thiols 3-mercaptohexan-1-ol (3MH), 3-mercaptohexan-1-ol-acetate, and 4-mercapto-4-methylpentan-2-one (4MMP). These volatile thiols are found in small amounts in grape juice and are formed from nonvolatile cysteinylated precursors during fermentation. In this study, we overexpressed a Saccharomyces cerevisiae gene, STR3, which led to an increase in 3MH release during fermentation of a V. vinifera L. cv. Sauvignon blanc juice. Characterization of the enzymatic properties of Str3p confirmed it to be a pyridoxal-5'-phosphate-dependent cystathionine ß-lyase, and we demonstrated that this enzyme was able to cleave the cysteinylated precursors of 3MH and 4MMP to release the free thiols. These data provide direct evidence for a yeast enzyme able to release aromatic thiols in vitro that can be applied in the development of self-cloned yeast to enhance wine flavor.

Chiral analysis of cis-2-methyl-4-propyl-1,3-oxathiane and identification of cis-2,4,4,6-tetramethyl-1,3-oxathiane in wine.

cis-2-Methyl-4-propyl-1,3-oxathiane (cis-2-MPO), arising from 3-sulfanylhexan-1-ol (3-SH) and acetaldehyde, was recently identified in wine, but the enantiomeric distribution was unknown. Such information could reveal influences on wine aroma, given the impact of chirality on odorant molecules. Herein, a stable isotope dilution assay employing headspace solid-phase microextraction with chiral gas chromatography-mass spectrometry was developed, validated, and applied to a selection of wines. Studies with (3R)-3-SH revealed the elution order of the cis-2-MPO enantiomers and the concentrations of (2R,4S)-2-MPO and (2S,4R)-2-MPO in the studied wines ranged from undetected to 250 ng/L and 303 ng/L, respectively. Strong positive correlations were found between (3R)-3-SH and (2S,4R)-2-MPO (r = 0.654), and (3S)-3-SH and (2R,4S)-2-MPO (r = 0.860). Additionally, cis-2,4,4,6-tetramethyl-1,3-oxathiane, constituted from acetaldehyde and 4-methyl-4-sulfanylpentan-2-ol (4-MSPOH), was identified in wine for the first time. This new 1,3-oxathiane, which presents a novel fate for 4-MSPOH, was detected in wines as a single enantiomer at up to 28 ng/L.

Spectrofluorometric analysis combined with machine learning for geographical and varietal authentication, and prediction of phenolic compound concentrations in red wine.

Fluorescence spectroscopy is rapid, straightforward, selective, and sensitive, and can provide the molecular fingerprint of a sample based on the presence of various fluorophores. In conjunction with chemometrics, fluorescence techniques have been applied to the analysis and classification of an array of products of agricultural origin. Recognising that fluorescence spectroscopy offered a promising method for wine authentication, this study investigated the unique use of an absorbance-transmission and fluorescence excitation emission matrix (A-TEEM) technique for classification of red wines with respect to variety and geographical origin. Multi-block data analysis of A-TEEM data with extreme gradient boosting discriminant analysis yielded an unrivalled 100% and 99.7% correct class assignment for variety and region of origin, respectively. Prediction of phenolic compound concentrations with A-TEEM based on multivariate calibration models using HPLC reference data was also highly effective, and overall, the A-TEEM technique was shown to be a powerful tool for wine classification and analysis.

Authentication of the geographical origin of Australian Cabernet Sauvignon wines using spectrofluorometric and multi-element analyses with multivariate statistical modelling.

With the increased risk of wine fraud, a rapid and simple method for wine authentication has become a necessity for the global wine industry. The use of fluorescence data from an absorbance and transmission excitation-emission matrix (A-TEEM) technique for discrimination of wines according to geographical origin was investigated in comparison to inductively coupled plasma-mass spectrometry (ICP-MS). The two approaches were applied to commercial Cabernet Sauvignon wines from vintage 2015 originating from three wine regions of Australia, along with Bordeaux, France. Extreme gradient boosting discriminant analysis (XGBDA) was examined among other multivariate algorithms for classification of wines. Models were cross-validated and performance was described in terms of sensitivity, specificity, and accuracy. XGBDA classification afforded 100% correct class assignment for all tested regions using the EEM of each sample, and overall 97.7% for ICP-MS. The novel combination of A-TEEM and XGBDA was found to have great potential for accurate authentication of wines.

Impact of Lachancea thermotolerans on chemical composition and sensory profiles of Merlot wines.

Wines from warm(ing) climates often contain excessive ethanol but lack acidity. The yeast Lachancea thermotolerans can ameliorate such wines due to partial conversion of sugars to lactic acid during alcoholic fermentation. This study compared the performance of five L. thermotolerans strains in two inoculation modalities (sequential and co-inoculation) to Saccharomyces cerevisiae and un-inoculated treatments in high sugar/low acidity Merlot fermentations. The pH and ethanol levels in mixed-culture dry wines were either comparable, or significantly lower than in controls (decrease of up to 0.5 units and 0.90% v/v, respectively). The analysis of volatile compounds revealed marked differences in major flavour-active yeast metabolites, including up to a thirty-fold increase in ethyl lactate in certain L. thermotolerans modalities. The wines significantly differed in acidity perception, alongside 18 other sensory attributes. Together, these results highlight the potential of some L. thermotolerans strains to produce 'fresher' wines with lower ethanol content and improved flavour/balance.

Production of indole by wine-associated microorganisms under oenological conditions.

A high concentration of indole has been linked to 'plastic-like' off-flavour in wines, predominantly in wines produced under sluggish fermentation conditions. The purpose of this study was to determine the ability of yeast and bacteria to form indole and whether tryptophan was required for indole accumulation during winemaking. Wine-associated yeast and bacteria species (Saccharomyces cerevisiae, Saccharomyces bayanus, Candida stellata, Hanseniaspora uvarum, Kluyveromyces thermoloterans, Oenococcus oeni, Lactobacillus lindneri, Pediococcus cerevisiae and Pediococcus parvulus) were screened for their potential to generate indole during alcoholic or malolactic fermentation. Tryptophan was required for the accumulation of indole in chemically defined medium, and all yeast and bacteria fermentations were able to accumulate indole. C. stellata showed the greatest potential for indole formation (1033 microg/L) and among the bacteria, the highest concentration was generated by L. lindneri (370 microg/L). Whether primary fermentation is the principle cause of indole formation remains to be determined. We hypothesise that during an efficient fermentation, indole is removed through catabolic metabolism, but, when a sluggish fermentation arises, non-Saccharomyces species might produce excess indole that is still present by end of fermentation.