Development of a New Method for the Quantitative Analysis of Aroma Compounds Potentially Related to the Fruity Aroma of Red Wines.

To determine the concentrations of aroma compounds involved in the fruity aroma of red wines, an analytical method was developed and optimized using liquid-liquid extraction and gas chromatography coupled to mass spectrometry (GC/MS). The aim was to reduce sample preparation and analysis time, with a single sample preparation and a single injection being needed to quantify 43 compounds. 19 esters, 13 monoterpenes, 5 C13-norisoprenoids, and 6 C6-aldehyde and alcohol compounds were quantified in 14 red wines made from different grape varieties grown in the Mediterranean basin. Samples were selected based on typical varietal aroma by a panel of experts, who produced 7 olfactory descriptors linked to desirable or non-desirable wine aromas. The instrumental analysis showed variations in concentrations of the quantified compounds among the wines. The wines described using positive fruity descriptors had higher mean total concentrations of esters, C6-alcohols, monoterpenes, and C13-norisoprenoids. Some non-ester compounds were positively correlated with the fruity descriptors. Sensory profile results obtained by a panel of 16 trained judges revealed that the addition of non-ester compounds (including 2 cyclic esters) to a red wine initially described as having cooked fruit aromas had a positive contribution to some fresh fruity notes. This study opens up new avenues for research on the potential involvement of non-ester compounds in the fruity expression of red wines.

Color Characterization of Bordeaux Red Wines Produced without Added Sulfites.

Nowadays, the development of naturalness as a concept is illustrated in the oenological field by the development of wine produced with lower inputs, sometimes even without any addition of SO2 throughout the winemaking process, up to the bottling stage. Despite the increase in the offer of these wines, they remain poorly explored in the literature and require characterization. This study was developed to evaluate the color of Bordeaux red wines without SO2 addition using colorimetric and polymeric pigments analysis. From a batch of commercial Bordeaux red wines with and without SO2 addition, and experimental wines produced from homogenous grapes according to different winemaking processes, colorimetric analyses (CIELab and color intensity (CI)) revealed a large difference in wine color depending on the presence or absence of SO2. Indeed, wines without SO2 were significantly darker and presented with a deeper purplish color. According to these observations, polymeric pigments were quantified using UPLC-DAD/ESI QTof, and a higher concentration of polymeric pigments bound by the ethylidene bridge was observed in wines without SO2. This correlated with differences observed for CIELab and CI. Finally, a comparison with polymeric tannins bound by ethylidene bridge was made and revealed that no differences were observed between wines with and without added SO2. This underlines the affinity difference between tannins and anthocyanins to react with acetaldehyde to form ethylidene bridges.

Sensory Specificities Involving Acetaldehyde and Diacetyl in Wines Produced without Added Sulfur Dioxide.

Nowadays, the development of naturality concept is illustrated in the oenological field by the development of wine produced without the addition of SO2. Among its chemical properties, SO2 is able to react with carbonyl compounds to form carbonyl bisulfites. Acetaldehyde and diacetyl are the main carbonyl compounds of red wines, which could influence product perception. The goal of this paper was to evaluate their chemical and sensory impact in red wines produced without any addition of SO2. A first quantification approach revealed a lower concentration of these compounds in wines without added SO2 than in those produced with SO2. A sensory approach involving aromatic reconstitutions in wines in the presence or absence of SO2 revealed that analytical differences observed for acetaldehyde and diacetyl were able to impact wine freshness, with diacetyl being, moreover, involved in wine fruity aroma changes.

Impact of Grape Maturity on Ester Composition and Sensory Properties of Merlot and Tempranillo Wines.

The goal of this study was to evaluate how grape composition modifications linked to maturity level could affect the wine ester composition and aromatic expression. An experimental design has been developed from grapes of Vitis vinifera cv Merlot and cv Tempranillo. On each vine plot, grapes have been harvested at two maturity levels and have been fermented using a commercial yeast strain under standardized conditions, specifically after having the sugar and nitrogen concentrations adjusted to the same target values. Tempranillo wine ester content was not impacted by the maturity level, whereas Merlot wines from the highest maturity level showed lower concentrations for fatty acid ethyl esters and higher alcohol acetates but higher concentrations for substituted ethyl esters. Sensory analysis corroborated these analytical results: when Merlot maturity increased, wine fruity aromatic expression decreased (particularly its global intensity and the fresh, red-berry, and fermentative fruit characters). In addition, aromatic reconstitution experiments showed that esters were not, alone, responsible for the sensory differences linked to grapes' maturity. Globally, our results highlight the role of esters in the overall wine fruity aromatic expression associated to Merlot ripeness and show that their levels are impacted by other parameters than the grape content in sugars and amino acids, well known as being their precursors.

Methyl Salicylate, an Odor-Active Compound in Bordeaux Red Wines Produced without Sulfites Addition.

Red wines produced without the addition of any SO2 are currently the source of a new consumer trend. The first characterization approaches regarding these specific wines were devoted to sensory studies that highlighted differences according to the use of SO2 during winemaking. The goal of this paper is to extend our knowledge of such aromatic specificities. Examining experimental wines produced with and without the addition of SO2, aroma fractionation, gas chromatography coupled with olfactometry, and mass spectrometry were applied to identify compounds at the origin of the specificities of these wines. Thus, we identified methyl salicylate as being impacted by the use of SO2 during the winemaking process. Studying a large number of commercial Bordeaux red wines, methyl salicylate was significantly quantified at a higher content in wines without added SO2. A sensory approach revealed a significant impact of methyl salicylate on wines without the sulfite aroma, particularly concerning fruity aromas and wine freshness.

Toward a Better Understanding of Perceptive Interactions between Volatile and Nonvolatile Compounds: The Case of Proanthocyanidic Tannins and Red Wine Fruity Esters-Methodological, Sensory, and Physicochemical Approaches.

The impact of commercial proanthocyanidic tannins on fruity pool of esters, representing the fruitiness of Bordeaux red wines, was assessed in model solutions. It was shown that the presence of tannins in the matrix significantly attenuated perception of fruity notes (p = 0.013). Physicochemical analysis demonstrated that the presence of proanthocyanidic tannins in dilute alcohol solution resulted in a decrease in ester partition coefficients and thus in a decrease in ester contents in the headspace (p < 0.05). This fact highlighted the changes that may occur in wines at a pre-sensory level, prior to sensory evaluation. Finally, a new sensory tool was developed, consisting in an ISO glass containing two compartments separated by a glass wall, providing a way to compare perceived odors according to whether or not the components of the odor mixtures were actually mixed in solution (p < 0.001). This new tool was used to demonstrate the impact and the only implication of pre-sensory level in the consequences of physical mixture between proanthocyanidic tannins and esters on their odor perception.

The Sensory Space of Wines: From Concept to Evaluation and Description. A Review.

The concept of sensory space was first formulated over 25 years ago and has been widely adopted in oenology for around the last 15 years. It is based on both the common organoleptic characteristics of products and the mental representations built by specific groups of people. Exploring this concept involves first assessing whether it already exists for tasters, and, when this is the case, conducting perceptual evaluations to verify its effectiveness before potentially highlighting the associated sensory properties. The goal of this review, which focuses on applications linked to the field of oenology, is to study how these three steps are carried out, how the corresponding tasks and tests are performed and managed, and the type of results that can be obtained.

Metabolic, Organoleptic and Transcriptomic Impact of Saccharomyces cerevisiae Genes Involved in the Biosynthesis of Linear and Substituted Esters.

Esters constitute a broad family of volatile compounds impacting the organoleptic properties of many beverages, including wine and beer. They can be classified according to their chemical structure. Higher alcohol acetates differ from fatty acid ethyl esters, whereas a third group, substituted ethyl esters, contributes to the fruitiness of red wines. Derived from yeast metabolism, the biosynthesis of higher alcohol acetates and fatty acid ethyl esters has been widely investigated at the enzymatic and genetic levels. As previously reported, two pairs of esterases, respectively encoded by the paralogue genes ATF1 and ATF2, and EEB1 and EHT1, are mostly involved in the biosynthesis of higher alcohol acetates and fatty acid ethyl esters. These esterases have a moderate effect on the biosynthesis of substituted ethyl esters, which depend on mono-acyl lipases encoded by MGL2 and YJU3. The functional characterization of such genes helps to improve our understanding of substituted ester metabolism in the context of wine alcohol fermentation. In order to evaluate the overall sensorial impact of esters, we attempted to produce young red wines without esters by generating a multiple esterase-free strain (Δatf1, Δatf2, Δeeb1, and Δeht1). Surprisingly, it was not possible to obtain the deletion of MGL2 in the Δatf1/Δatf2/Δeeb1/Δeht1 background, highlighting unsuspected genetic incompatibilities between ATF1 and MGL2. A preliminary RNA-seq analysis depicted the overall effect of the Δatf1/Δatf2/Δeeb1/Δeht1 genotype that triggers the expression shift of 1124 genes involved in nitrogen and lipid metabolism, but also chromatin organization and histone acetylation. These findings reveal unsuspected regulatory roles of ester metabolism in genome expression for the first time.

Impact of Whisky Lactone Diastereoisomers on Red Wine Fruity Aromatic Expression in Model Solution.

The impact of whisky lactone diastereoisomers on the typical fruity expression of red Bordeaux wines was evaluated by sensory analysis. The detection thresholds of cis- and trans-whisky lactone in a dilute alcohol solution (12% v/v) were 20 and 130 µg/L, respectively. Consequently, considering their average concentrations found in oak-aged red wines, cis-whisky lactone was present at supra threshold levels, whereas trans-whisky lactone was below its detection threshold. Adding these diastereoisomers to a red wine fruity aromatic reconstitution at these average concentrations led to a decrease in the perception of this last one, highlighting a masking effect. Sensory profiles of cis- and trans-whisky lactone confirmed that these compounds modified the perception of fruity aromas, decreasing the intensity of red berry fruit notes and increasing that of blackberry fruit and spicy descriptors.

Influence of must yeast-assimilable nitrogen content on fruity aroma variation during malolactic fermentation in red wine.

This study assessed the impact of must yeast-assimilable nitrogen (YAN) content and lactic acid bacteria (LAB) strains used for malolactic fermentation (MLF) on the formation of substituted esters, as well as the corresponding precursors (substituted acids), to investigate the modulation of fruity expression in red wines. In microvinification experiments, a Merlot must was fermented with an initial YAN content of 111 mg/L, or supplemented up to 165 and 220 mg/L. Two Oenococcus oeni LAB strains were used for MLF. Analytical methods were used to quantify substituted esters, as well as the corresponding acids, including, any enantiomeric forms. YAN supplementation of the must significantly increased concentrations of substituted esters of short- and branched-chain alkyl fatty acids produced during alcoholic fermentation (AF) (up to 67% in samples with the highest nitrogen content) and substituted esters of hydroxycarboxylic acids generated during MLF (up to 58% in samples with the highest nitrogen content). YAN supplementation in the must did not affect substituted acid formation during AF. After MLF, short- and branched-chain alkyl fatty acid levels increased in wines made from musts with the highest nitrogen content (up to 56% in samples with the highest nitrogen content), whereas concentrations of hydroxycarboxylic acids increased (up to 55%) independently of the initial YAN content, highlighting the important role of MLF. (2S)-2-hydroxy-4-methylpentanoic acid was only found in wines after malolactic fermentation, suggesting different pathways for each enantiomer and opening up new prospects for the study of bacterial metabolisms. Moreover, sensory profiles revealed a significant increase in black-berry- and jammy-fruit aromas during MLF and a strong positive correlation between these aromas and the production of substituted esters following must nitrogen supplementation and MLF. Aromatic reconstitutions revealed that variations in the concentrations of substituted esters after MLF impacted the fruity aroma of red wines.

Oak Wood Volatiles Impact on Red Wine Fruity Aroma Perception in Various Matrices.

This research examined the impact of oak wood volatile compounds on the perception of red wine fruity aroma in several matrices. Several aromatic reconstitutions were prepared, consisting of 13 esters, representing the fruity pool of red wine, and 14 oak wood compounds at the various concentrations corresponding to the levels released by light, medium, and heavy toasting of barrels. These reconstitutions were prepared in dilute alcohol solution, dearomatized red wine, and commercial red wine. Sensory analysis revealed the impact of the addition or omission of some oak wood compounds. The "detection threshold" of the fruity pool was then evaluated. The presence of 2-furanmethanethiol individually and the oak wood compound mixture, at concentrations representing various toasting levels, had a significant masking effect on the fruity pool, whereas vanillin had a significant enhancing effect in model solution. Sensory profiles highlighted changes in the perception of fruity nuances in the presence of the oak wood compound mixture. The addition of compounds at concentrations representing different toasting levels led to a predictable increase in the perception of the oak wood descriptors (spicy, smoky, and toasty) in all of the matrices tested. The perception of fruity notes also varied depending upon the toasting level and the complexity of the matrix. In dilute alcohol solution and dearomatized red wine, light toasting preserved or intensified the fruity notes. Generally, in all matrices tested, fresh-fruit and red-berry-fruit notes decreased with the addition of wood at medium and heavy toasting levels.

Static Headspace Analysis Using Low-Pressure Gas Chromatography and Mass Spectrometry, Application to Determining Multiple Partition Coefficients: A Practical Tool for Understanding Red Wine Fruity Volatile Perception and the Sensory Impact of Higher Alcohols.

To evaluate the partition coefficients of volatiles between the liquid and gas phases, an analytical method was developed and optimized using static headspace analysis and low-pressure injection gas chromatography coupled to mass spectrometry (SHS-LP-GC/MS). Two different types of analytical columns were coupled for low-pressure chromatography injection: a narrow restriction microbore column on the inlet side and a mega-bore column on the mass spectrometer side. Coupling these two columns and static headspace analysis to gas chromatography and mass spectrometry resulted in a simple, fast, sensitive, and accurate approach. Several points have been optimized: time to reach the thermodynamic equilibrium in the gas phase, syringe filling rate, gas injection rate, and volume ratio between the gas and liquid phases. This new method was used to determine partition coefficients between the liquid and gas phases and study multicomponent mixtures for which particular perceptive interactions had previously been highlighted. The partition coefficients of 9 esters and 5 higher alcohols were determined in dilute alcohol solution (12% v/v) and dearomatized red wine. These partition coefficients revealed modifications in ester headspace release in the presence of higher alcohols for the first time in this type of matrix. The correlation of these results with sensory data highlighted the role of physicochemical, presensory effects on sensory modifications for the first time, suggesting that this type of interaction may partly modulate qualitative and quantitative fruity perception.

The complexity of wine: clarifying the role of microorganisms.

The concept of wine complexity has gained considerable interest in recent years, both for wine consumers and wine scientists. As a consequence, some research programs concentrate on the factors that could improve the perceived complexity of a wine. Notably, the possible influence of microbiological factors is particularly investigated. However, wine complexity is a multicomponent concept not easily defined. In this review, we first describe the actual knowledge regarding wine complexity, its perception, and wine chemical composition. In particular, we emphasize that, contrary to expectations, the perception of wine complexity is not related to wine chemical complexity. Then, we review the impact of wine microorganisms on wine complexity, with a specific focus on publications including sensory analyses. While microorganisms definitively can impact wine complexity, the underlying mechanisms and molecules are far from being deciphered. Finally, we discuss some prospective research fields that will help improving our understanding of wine complexity, including perceptive interactions, microbial interactions, and other challenging phenomena.

Wine yeast phenomics: A standardized fermentation method for assessing quantitative traits of Saccharomyces cerevisiae strains in enological conditions.

This work describes the set up of a small scale fermentation methodology for measuring quantitative traits of hundreds of samples in an enological context. By using standardized screw cap vessels, the alcoholic fermentation kinetics of Saccharomyces cerevisiae strains were measured by following their weight loss over the time. This dispositive was coupled with robotized enzymatic assays for measuring metabolites of enological interest in natural grape juices. Despite the small volume used, kinetic parameters and fermentation end products measured are similar with those observed in larger scale vats. The vessel used also offers the possibility to assay 32 volatiles compounds using a headspace solid-phase micro-extraction coupled to gas chromatography and mass spectrometry. The vessel shaking applied strongly impacted most of the phenotypes investigated due to oxygen transfer occuring in the first hours of the alcoholic fermentation. The impact of grape must and micro-oxygenation was investigated illustrating some relevant genetic x environmental interactions. By phenotyping a wide panel of commercial wine starters in five grape juices, broad phenotypic correlations between kinetics and metabolic end products were evidentiated. Moreover, a multivariate analysis illustrates that some grape musts are more able than others to discriminate commercial strains since some are less robust to environmental changes.

2-Methylbutyl acetate in wines: Enantiomeric distribution and sensory impact on red wine fruity aroma.

Enantiomers of 2-methylbutyl acetate were assayed in red and white commercial wines from various vintages and origins, using chiral gas chromatography (γ-cyclodextrin), revealing the exclusive presence of the S-enantiomeric form. Results also confirmed that (S)-2-methylbutyl acetate levels were generally higher in red than white wines of the same age, and that acetate levels increased gradually during ageing. Olfactory threshold of (S)-2-methylbutyl acetate was evaluated at 313µg/L in dilute alcohol solution (12% v/v) and 1083µg/L in a fruity aromatic reconstitution, reflecting its presence in wines at subthreshold concentrations. At concentrations considerably lower than its olfactory threshold, 2-methylbutyl acetate was associated with blackberry-fruit and banana notes. It was also revealed that, even at subthreshold concentrations, this compound had a modification on the perception of fruity aromas in the matrices studied. Sensory profiles highlighted, for the first time, its specific contribution to black-, fresh-, and jammy-fruit notes, despite its subthreshold concentrations.

Study of Substituted Ester Formation in Red Wine by the Development of a New Method for Quantitative Determination and Enantiomeric Separation of Their Corresponding Acids.

A new method was developed for quantifying substituted acids including, where applicable, their various unexplored enantiomeric forms. A new step was added to acids' usual quantification methods, consisting of extraction, derivatization to methyl esters, and gas chromatography analysis: preliminary extraction was performed at basic pH to eliminate ethyl esters, thus avoiding their transesterification during derivatization. Quantitation and enantiomeric distribution of some substituted esters and their corresponding acids were established in 31 commercial Bordeaux red wines from 0 to 20 years old. A strong positive correlation was observed between the age of wine and levels of ethyl 2-methylpropanoate, ethyl 3-methylbutanoate, ethyl 2-methylbutanoate, ethyl (3R)-3-hydroxybutanoate, both enantiomeric forms of ethyl 2-hydroxy-3-methylbutanoate, and ethyl (2S)-2-hydroxy-4-methylpentanoate, but not ethyl (3S)-3-hydroxybutanoate. However, the standard deviations of average concentrations for the corresponding substituted acids were so large that only few correlations between concentrations and age were observed. Concentrations of (2S)-2-hydroxy-3-methylbutanoic acid and (2S)-2-hydroxy-4-methylpentanoic acid increased slightly over time, while (2R)-2-hydroxy-4-methylpentanoic acid levels decreased slightly with the age. Variations in the ratio of substituted ethyl esters to their corresponding acids over time detected thanks to these analytical advances suggested that, in general, acids were continuously esterified during aging.

Identification of Piperitone as an Aroma Compound Contributing to the Positive Mint Nuances Perceived in Aged Red Bordeaux Wines.

Although a sensory definition of the aging bouquet of red Bordeaux wines was recently established, its chemical transcription has only partially been elucidated. A multiple-step approach, combining sensory evaluations of red Bordeaux wines and aromatic reconstitutions of wine extract fractions, was used to identify the molecular markers involved. One wine with a high aging-bouquet score and a mint nuance has received particular attention. Various reconstitution and omission tests highlighted the contribution of two specific fractions to the intensity of the perception of mint aroma. Gas chromatography coupled to olfactometry and mass spectrometry was applied to the targeted fractions to identify molecular marker(s) responsible for the mint nuance in fine red Bordeaux wines. A similar analytical process was applied to selected fractions of essential oils presenting mint odors to characterize them and interpret the mass spectrometry data. This approach resulted in the detection of piperitone, a monoterpene ketone that, to the best of our knowledge, was reported for the first time as a contributor to the positive mint aroma of aged red Bordeaux wines.

How do esters and dimethyl sulphide concentrations affect fruity aroma perception of red wine? Demonstration by dynamic sensory profile evaluation.

Our study focused on variations in wine aroma perception and molecular composition during tasting over a period of 30min. In parallel, dynamic analytical and sensory methods were applied to study changes in the wines' molecular and aromatic evolution. Dynamic sensory profile evaluations clearly confirmed the evolution of the wine's fruity notes during sensory analysis, highlighting significant differences for red-berry and fresh fruit as well as black berry and jammy fruit, after 5 and 15min, respectively. Dynamic analytical methods revealed a decrease in ester and dimethyl sulphide (DMS) concentrations in the first few minutes. Sensory profiles of aromatic reconstitutions demonstrated that the aromatic modulation of fruity notes observed during wine tasting was explained by changes in ester and DMS concentrations. These results revealed that variations in concentrations of DMS and esters during wine tasting had a qualitative impact, by modulating fruity aromas in red wine.

Distribution and Organoleptic Impact of Ethyl 3-Hydroxybutanoate Enantiomers in Wine.

Enantiomers of ethyl 3-hydroxybutanoate were assayed in 87 commercial wines from various vintages and origins, using chiral gas chromatography (ß-cyclodextrin). Generally, ethyl 3-hydroxybutanoate levels were higher in red than in white wines of the same age. The average S/R enantiomeric ratio of this compound in red wine was approximately 75:25 (± 13), with an average total concentration of ∼ 450 (± 150) µg/L. In red wines, R-form levels increased gradually during aging, but no variations were observed in S-form concentrations. To our knowledge, no previous research had determined the enantiomeric distribution of this compound in wine. The olfactory threshold of the S-form in dilute alcohol solution was 21 mg/L, one-third that of the R-form: 63 mg/L. The S- and R-forms had different aromatic nuances. The olfactory threshold of their mixture (85:15, m/m) was 14 mg/L, indicating a simple additive effect in this binary mixture. Furthermore, the concentrations found in red wines were considerably below the olfactory threshold under the same experimental conditions. Sensory analysis revealed that ethyl 3-hydroxybutanoate (S/R, 85:15, m/m) had an enhancing effect on the perception of fruity aromas in the matrices studied. Sensory profiles highlighted the contribution of ethyl 3-hydroxybutanoate to red-berry and fresh-fruit descriptors, despite its subthreshold concentrations.