Sensory, olfactometric and chemical characterization of the aroma potential of Garnacha and Tempranillo winemaking grapes.

Reconstituted polyphenolic and aromatic fractions (PAFs) from 33 different Garnacha and Tempranillo grapes were incubated in strict anoxia (75 °C × 24 h). Obtained hydrolyzates were characterized by sensory analysis, gas chromatography-olfactometry (GC-O) and gas chromatography-mass spectrometry (GC-MS). Five different aroma categories emerged. Garnacha may develop specific tropical/citrus fruit, kerosene and floral and Tempranillo toasty-woody and red-fruit characteristics. Those notes seem to mask alcoholic and fruit-in-syrup descriptors and the common vegetal background. Twenty-seven odorants were detected by GC-O. GC-MS data showed a clustering closely matching the one found by sensory analysis, suggesting the existence of five specific metabolomic profiles behind the five specific sensory profiles. Overall results suggest that 3-mercaptohexanol is responsible for tropical/citrus fruit, TDN for kerosene, volatile phenols for woody/toasty, ß-damascenone and massoia lactone, likely with Z-1,5-octadien-3-one for fruit-in-syrup and alcoholic notes. Nine lipid-derived unsaturated aldehydes and ketones may be responsible for the vegetal background.

Development of a new strategy for studying the aroma potential of winemaking grapes through the accelerated hydrolysis of phenolic and aromatic fractions (PAFs).

Current methods for assessing grape aroma potential are based on the fast hydrolysis of precursor fractions but provide hydrolyzates of poor aromatic quality. A new strategy based on the accelerated hydrolysis of reconstituted phenolic and aromatic fractions (PAFs) extracted from grapes is herein developed. PAFs are obtained by solid phase extraction on 10 g-C18 sorbents of partially dealcoholized "mistelle", obtained from grapes treated with ethanol. Under optimal conditions, PAFs contain all aroma precursors but the most polar ones, such as those of DMS, more than 85% of the total phenolics and just traces of metal cations and of amino acids. PAFs reconstituted in model wine, aged in strict anoxia 7 weeks at 45 °C or 24 h at 75 °C, develop strong aromas. At least 30 different odorants including lipid derivatives, volatile phenols, vanillins, norisoprenoids, terpenes, bencenoids and 3-mercaptohexanol were identified by GC-Olfactometry and GC-MS. Methodological aspects of the extraction, hydrolysis and analysis are optimized and discussed.

How does the addition of antioxidants and other sulfur compounds affect the metabolism of polyfunctional mercaptan precursors in model fermentations?

Polyfunctional mercaptans are powerful wine aroma compounds formed from cysteinylated and glutathionylated precursors present in grapes. The concentration of polyfunctional mercaptans in the wines is not directly correlated to the concentration of their precursors in the juices, which suggests that there are many aspects related to the uptake and use of precursors by yeasts which remain poorly understood. In this work we determine the influence of different sulfur sources on the release of polyfunctional mercaptans and on the consumption of their precursors. A series of fermentations of synthetic juice was prepared containing known concentrations of polyfunctional mercaptans precursors. The effect of different sulfur compounds (elemental sulfur, glutathione, methionine, cysteine and sulfur dioxide), as well as of the amino acids residues that constitute glutathione (cysteine, glycine and glutamic acid) have been studied. Significant differences were found in the concentration of polyfunctional mercaptans and in the metabolization of their precursors when varying the sulfur source. The addition of glutathione and high levels of cysteine caused a significantly lower metabolization of the glutathione precursor of 3-mercaptohexanol. In parallel, in the wines spiked with sulfur dioxide and cysteine, significantly higher levels of polyfunctional mercaptans were observed. These results suggest that the consumption of polyfunctional mercaptans precursors is dependent on the juice composition, and by modifying the sulfur source it is possible to change the concentration of polyfunctional mercaptans.

Gas chromatographic-sulfur chemiluminescent detector procedures for the simultaneous determination of free forms of volatile sulfur compounds including sulfur dioxide and for the determination of their metal-complexed forms.

Three different procedures for the quantitative assessment of free and metal complexed volatile sulfur compounds (VSCs) and for the determination of truly free SO2 have been developed, taking advantage of a GC-sulfur chemiluminescent detector system (GC-SCD) with cryotrapping. The inertness of the inlet systems, together with the column used (SPB-1 sulfur) makes it possible to obtain a non-saturated perfectly Gaussian peak for SO2, well resolved from H2S. In the main procedure, the injection of 1 mL of the headspace of a sample prepared in complete anoxia and equilibrated at 30 °C makes it possible to get highly sensitive signals for all VSCs and free SO2. Detection limits are 3, 35 and 60 ng/L for H2S, MeSH and EtSH, 13 µg/L for truly free SO2 (at pH = 3.4, or 0.46 µg/L for molecular SO2), and better than 1 µg/L for other relevant sulfur volatiles. Method precision is also satisfactory and linearity covers the whole range of occurrence of these compounds. A second procedure, not making use of the cryotrapping unit, gives also satisfactory results, although with higher detection limits (0.03, 0.25 and 0.37 µg/L for free H2S, MeSH and EtSH, respectively). For the analysis of free plus metal-complexed forms, it has been demonstrated that the headspace injection of the vapors on a 1:10 brine dilution of the sample heated at 70 °C for 25 min, gives good estimates of the free + metal-complexed forms of H2S and wine mercaptans.

Micro-oxygenation does not eliminate hydrogen sulfide and mercaptans from wine; it simply shifts redox and complex-related equilibria to reversible oxidized species and complexed forms.

This work seeks to assess the effects of micro-oxygenation (MOX) on the present and potential levels of Volatile Sulfur Compounds (VSCs) of wine. With such purpose, three red wines with a tendency to develop sulfury off-odors were subjected to three different MOX conditions (4.4-20mg/L delivered at 0.05 or 0.2mg/L/day). Samples were further subjected to Accelerated Reductive aging (AR) and analyzed for free and Brine Releasable (BR) VSCs and redox potential. Although MOX induced strong decreases in the levels of all free VSCs, hardly affected the ability of the wine to release back hydrogen sulfide and other mercaptans during AR-aging. During aging BR-levels of MOX samples became in most cases similar or higher than non-oxygenated controls. BR-levels and the fractions free/BR follow characteristic sigmoid plots when represented versus redox potential suggesting that all changes are the result of reversible equilibria between free, metal-complexed and oxidized forms of VSCs.

Elusive Chemistry of Hydrogen Sulfide and Mercaptans in Wine.

This paper summarizes, discusses, and complements recent findings about the fate of H2S and methanethiol (MeSH) during wine storage. Analytical assays to determine free volatile sulfur compounds (VSCs) and brine-releasable (BR-) VSCs in combination with accelerated reductive (AR) aging and micro-oxygenation (MOX) assays allow characterizing the different categories of species able to produce H2S and MeSH and the processes of interconversion. Each wine seems to contain a specific total amount of H2S and MeSH distributed into free, metal-complexed, and oxidized forms (di and polysulfides) interconnected through reversible redox equilibria whose external expression is wine redox potential. Oxidation transforms all mercaptans likely into nonvolatile disulfides and hydrodisulfides. In anoxia, these molecules are spontaneously and quantitatively reduced back. The concomitant accumulation of major wine thiols would provoke complex dissociation and the release of free H2S and MeSH. Additionally, total amounts can increase due to the metal-catalyzed desulfhydration of cysteine and methionine.

Study of the influence of varietal amino acid profiles on the polyfunctional mercaptans released from their precursors.

Polyfunctional mercaptans such as 4-mercapto-4-methyl-2-pentanone, 3-mercaptohexanol and 3-mercaptohexyl acetate contribute strongly to the varietal aroma of wines. These compounds are released during alcoholic fermentation from their precursors and their concentration is strongly linked to the grape variety. The aim of this work was to determine the effect of nine different amino acid profiles of grape varieties on the release of these polyfunctional mercaptans and on the consumption of their precursors as well as to determine which are the preferred precursors. A set-up of fermentations of synthetic must was prepared, which simulated the amino acid profiles of nine different grape varieties, containing known amounts of polyfunctional mercaptan precursors as well as the elements necessary for the yeast metabolism. The yeast assimilable nitrogen was adjusted in percentage to reach 150mgN/L in all the fermentations. Polyfunctional mercaptans and their precursors were analyzed in the final wine by gas chromatography coupled to mass spectrometry with negative chemical ionization and ultrahigh liquid performance chromatography coupled to mass spectrometry, respectively. The results showed significant differences in the release of these polyfunctional mercaptans as well as in the consumption of their precursors according to the amino acid profile. Moreover, it was observed that the preferred precursor of 4-mercapto-4-methyl-2-pentanone was its cysteinylated precursor. These results suggest that the amount of the amino acids as well as the ratio between them could modify the amount of polyfunctional mercaptans released. This could be a tool for the wine industry to vary the aromatic profile of wines by increasing or decreasing these volatile thiols.

The effects of copper fining on the wine content in sulfur off-odors and on their evolution during accelerated anoxic storage.

Three different red wines with reductive character have been treated with two different doses of copper sulfate (0.06 and 0.5mg/L) and with a commercial copper-containing product at the recommended dose (0.6mg/L). Wines were in contact with copper one week, centrifuged and stored at 50°C in strict anoxia for 2weeks (up to 7 in one case). Brine-releasable (BR-) and free fractions of Volatile Sulfur Compounds were determined throughout the process. Relevant increases of BR-H2S suggest that those wines contained other H2S precursors non-detectable by the brine dilution method. Copper treatments had two major effects: 1) immediate decrease the levels of free H2S and methanethiol (MeSH); 2) slow the rate at which free H2S (not MeSH) increases during anoxic storage. After 7weeks of anoxia levels of free H2S and MeSH were high and similar regardless of the copper treatment. Higher copper doses could induce the accumulation of BR-H2S.

Rapid sensory-directed methodology for the selection of high-quality aroma wines.

BACKGROUND: The present work contributes by developing a rapid sensory-directed methodology for the screening and selection of high-quality wines with different sensory profiles. Verdejo and Tempranillo musts were fermented with 50 different yeasts each under controlled laboratory conditions. Resulting samples were firstly categorised according to five levels of quality by a panel of wine professionals. Higher quality samples were described by flash profiling by a semi-trained panel and most distinctive samples were screened by gas chromatography-olfactometry (GC-O). RESULTS: Seven Verdejo and five Tempranillo samples were classified in the highest quality category, presenting different aroma profiles such as citrus, fruit in syrup, boxtree/vegetal, tropical or wet grain aromas for Verdejo and red fruit or fruit in syrup for Tempranillo. ß-Damascenone, 3-mercaptohexyl acetate and ethyl butyrate appeared as distinctive quality compounds linked to dried, tropical and red fruit aromas, respectively. CONCLUSIONS: The categorisation task followed by flash profiling and GC-O analysis was shown to be a rapid and effective sensory-directed methodology for the screening of distinctive and quality wine aroma profiles in a case study of yeast selection. The wine industry could benefit from the use of this methodology as a complementary tool for optimising different technical processes. © 2016 Society of Chemical Industry.

Influence of viticulture practices on grape aroma precursors and their relation with wine aroma.

BACKGROUND: The effects of two different operations in the vineyard (basal leaf plucking and head trimming) on the synthesis of aromatic precursors in the grape and their impact on wine aroma have been studied and compared with a control sample. The study was carried out over two consecutive years with four different varieties. Glycosidic precursors were analysed in grapes and volatile compounds were studied in the wines. ANOVA studies were performed to study the effect of the vintage, variety and treatment for each of the compounds released from their precursors. RESULTS: Regarding treatment, the highest values in the concentration of free aroma compounds were achieved in the leaf plucking grapes, except for Chardonnay. Significant and positive correlations between aromatic precursors of terpenes present in grapes and their released form in wines were found for all varieties. For norisoprenoids, significant and positive correlations were exclusively found for Chardonnay and for phenols and vanillins in the year 2010 the correlations were high in three of the four varieties studied. CONCLUSION: In the assays of the 2 years, more precursors were synthesised in Merlot, Gewurztraminer and Tempranillo grapes if the vineyards were plucked.

Glycosidically bound aroma compounds and impact odorants of four strawberry varieties.

This paper reports the determination of glycosidically bound aroma compounds and the olfactometric analysis in four strawberry varieties (Fuentepina, Camarosa, Candonga and Sabrina). Different hydrolytic strategies were also studied. The results showed significant differences between acid and enzymatic hydrolysis. In general terms, the greater the duration of acid hydrolysis, the higher was the content of norisoprenoids, volatile phenols, benzenes, lactones, Furaneol, and mesifurane. A total of 51 aglycones were identified, 38 of them unreported in strawberry. Olfactometric analyses revealed that the odorants with higher modified frequencies were Furaneol, γ-decalactone, ethyl butanoate, ethyl hexanoate, ethyl 3-methylbutanoate, diacetyl, hexanoic acid, and (Z)-1,5-octadien-3-one. This last compound, described as geranium/green/pepper/lettuce (linear retention index = 1378), was identified for the first time. Differences with regard to fruity, sweet, floral, and green aroma characters were observed among varieties. In Candonga and Fuentepina, the green character overpowered the sweet. In the other two strawberry varieties sweet attributes were stronger than the rest.

Sensory and chemical characterisation of the aroma of Prieto Picudo rosé wines: the differential role of autochthonous yeast strains on aroma profiles.

This study evaluates the specific impact of isolated yeast strains on the aromatic profile of fermented musts from Prieto Picudo, an autochthonous Castilla y León (Spain) red grape variety with an increasing demand in the local marketplace. For this purpose, the aroma profiles of wines elaborated from Prieto Picudo grapes have been studied by sensory analysis, gas chromatography-olfactometry (GC-O) and gas chromatography-mass spectrometry (GC-MS), with the aim of determining the potential of each strain to generate distinctive varietal and fermentation-derived aromatic compounds. The results have shown that the yeast strain exerts a critical influence on the levels of some fermentative (linear and branched ethyl esters, fatty acids, ethyl phenylacetate) and varietal compounds (4-mercapto-4-methyl-2-pentanone, 3-mercaptohexylacetate, ß-damascenone), thus inducing a deep influence on the final aroma of the wine. Combination of both sensory and chemical data arises as a major tool to monitor the different patterns of aroma release and formation from selected yeast strains during the winemaking process.

Effect of aromatic precursor addition to wine fermentations carried out with different Saccharomyces species and their hybrids.

This work explores the ability of different yeast strains from different species of the genus Saccharomyces (S. cerevisiae, S. uvarum and S. kudriavzevii) and hybrids between these species to release or form varietal aroma compounds from fractions of grape odourless precursors. The de novo synthesis by the yeasts of some of the varietal aroma compounds was also evaluated. The study has shown that de novo synthesis affects some lipid derivatives, shikimic derivatives and terpenes in all species and hybrids, with some remarkable differences amongst them. The release or formation of aroma compounds from precursors was found to be strongly linked to the yeast or hybrid used, and the triple hybrid S. cerevisiae × S. bayanus × S. kudriavzevii in particular and secondarily the hybrid S. cerevisiae × S. bayanus were highly efficient in the production of most varietal aroma compounds, including γ-lactones, benzenoids, volatile phenols, vanillin derivatives and terpenols. The presence of precursors in the fermenting media caused a surprising levelling effect on the fermentative aroma composition. Altogether, these results suggest that it is possible to modulate wine aroma by employing different yeast species in order to create new wines with different aromatic notes.

Genetic characterization and phenotypic variability in Torulaspora delbrueckii species: Potential applications in the wine industry.

In this study, several strains of Torulaspora delbrueckii yeast species were evaluated in the laboratory for their enological properties. In a preliminary step, the ability of different molecular methods to discriminate among T.delbrueckii strains was compared. A combination of 7 PCR methods was able to separate 21 strains into 18 groups, while an REA-PFGE method allowed, in one experiment, the separation into 19 groups. The T.delbrueckii strains used presented a wide phenotypic variability in fermentation behaviour, e.g. Lag Phase (LP) duration, T50 parameter (time necessary to ferment half the sugar), and ethanol production. These 3 parameters have to be considered for industrial selection, particularly the LP duration. The majority of T.delbrueckii strains produced 8 to 11% and 7 to 10% ethanol vol. at 17 degrees C and 24 degrees C, respectively, with a maximum ethanol concentration of 12.35 at 17 degrees C and 10.90% vol. at 24 degrees C. The phenotypic variability of this species was also reflected in volatile acidity, glycerol, and aroma production. These experiments confirmed the low volatile acidity and glycerol production of this species and revealed a difference in osmotic stress response, compared to Saccharomyces cerevisiae. T.delbrueckii presented high fermentation purity and produced low levels of undesirable volatile compounds, such as hydrogen sulphide and volatile phenols.

Fate of grape flavor precursors during storage on yeast lees.

The effect of the addition of a grape flavor precursor extract to a grape juice, before or after fermentation with three different Saccharomyces cerevisiae yeast strains, on the evolution of the wine aroma composition during a 9-month aging period on yeast lees has been studied. Wine aroma compounds were determined by gas chromatography-mass spectrometry after alcoholic fermentation and after 3 and 9 months of storage. The aging of wine on lees caused important changes in the aroma profiles of wines, making the concentrations of three terpenes, norisoprenoids (except beta-damascenone and beta-ionone), 4-allyl-2,6-dimethoxyphenol, ethyl vanillate, syringaldehyde, and ethyl cinnamate increase, whereas the concentrations of most of the rest of compounds tended to decrease. Lees are responsible for the observed increasing trends, except for linalool and alpha-terpineol, and also for a large part of the observed decrements. As expected, the addition of precursors brings about an increment in the levels of most terpenes, norisoprenoids, vanillins, and ethyl cinnamate, and it is after an aging time when differences linked to the level of precursors in the must become more evident. The timing of the addition of precursors has a minor influence, except for beta-damascenone, vanillin, and syringaldehyde, for which supplementation after fermentation is more effective. It has also been observed that the precursor fraction makes the levels of vinylphenols decrease. Finally, it has also been found that lees from different yeast strains may have a slightly different abilities to release volatile compounds derived from precursors.

Comparison of the suitability of different hydrolytic strategies to predict aroma potential of different grape varieties.

Precursor extracts obtained from different grape varieties were submitted to harsh acid hydrolysis (pH 2.5, 100 degrees C, 1 h) and enzymatic hydrolysis (AR2000, pH 5, 40 degrees C, 16 h) and were also added to a synthetic must (200 g L(-1) glucose), which was fermented (yeast strain Stellevin NT 116), to compare the "natural hydrolysis" carried by yeast with alternative "fast" hydrolytic strategies. In all cases, released volatile compounds were extracted by SPE and determined by GC-MS. Leaving aside Muscat, differences between varieties were not relevant, although Grenache and Chardonnay presented some key peculiarities. In general, alcoholic fermentation showed the lowest potential to release volatile compounds from aromatic precursors, whereas enzymatic hydrolysis was the most efficient but also the most different. Practically, this implies that the predictive ability of this hydrolytic strategy is rather poor. In contrast, harsh acid hydrolysis can be considered to much more adequately measure the aroma potential of grapes for winemaking, which suggests that transformations taking place during fermentation include relevant chemical rearrangements in acid media that are better predicted by acid hydrolysis.

Release and formation of varietal aroma compounds during alcoholic fermentation from nonfloral grape odorless flavor precursors fractions.

An odorless flavor precursor fraction extracted from different nonfloral grape varietals has been added to a grape must and has been fermented by three different yeast strains. The wines obtained were analyzed by sensory descriptive analysis and by gas chromatography mass spectrometry to determine more than 90 aroma chemicals. The addition of the precursor fraction brought about a significant increase of the wine floral notes, irrespective of the yeast used. The levels of 51 wine aroma chemicals were found to depend on the precursor fraction addition and, in most cases, also on the yeast strain. Only beta-damascenone, beta-ionone, and vinylphenols were produced at concentrations well above threshold. However, the concerted addition of groups of compounds has shown that lactones, cinnamates, vanillins, and terpenes are together active contributors to the floral note. Different observations suggest that the formation of varietal aroma is an integral part of yeast metabolism and not a simple hydrolytical process.

Determination of the biogenic amines in musts and wines before and after malolactic fermentation using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate as the derivatizing agent.

An accurate, rapid and sensitive high-performance liquid chromatographic (HPLC) method is reported for the determination of four biogenic amines in red and white wine samples. The method involves derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) followed by liquid chromatography gradient elution analysis without any other sample pretreatment. The procedure developed permitted the determination of the four amines in 40 min. The relative standard deviation (RSD) values ranged from 3.1% to 6.1% in peak areas and the response was linear (R2>0.994) in a range from 0.1 to 4 mg/l. Leaving aside putrescine in Tempranillo wines, the malolactic fermentation did not exert any effect on the concentration of these compounds. On the contrary, the influence of the grape variety was important, and Tempranillo wines showed higher levels of tyramine and histamine and lesser of putrescine and cadaverine (p<0.05) than wines made with Cabernet Sauvignon.

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.

A procedure has been developed for the GC-MS analysis of the aromas released in fast acid hydrolysis of precursor fractions from grape musts and skins. Different sorbents for the extraction of the precursors were compared. The best results were obtained with LiChrolut EN polymeric resins which displayed two and six-fold more extraction capacity than Amberlite XAD-2 resins and C18 sorbents, respectively. C18 sorbents are more suitable for selective extraction of less polar precursors. The initial version of the method was imprecise and so the imprecision of the different steps was assessed. The maceration of the solid parts and the liquid-liquid extraction of the aromas released in the acid hydrolysis proved to be the critical steps. Greater crushing of the solid parts and solid-phase extraction (SPE) instead of liquid-liquid extraction (LLE) improved reproducibility. In the method finally proposed about 100 aromatic components belonging to four large groups (lipid derivatives, shikimic acid derivatives, norisoprenoids and terpenes) were determined with good reproducibility. Important aroma compounds, such as cis-rose oxide or wine lactone were detected in non-Muscat grapes.

Relationship between varietal amino acid profile of grapes and wine aromatic composition. Experiments with model solutions and chemometric study.

Synthetic solutions containing amino acids, sugar, water, and yeast nutrients have been fermented by Saccharomyces cerevisiae, and the volatile composition of the fermented media has been analyzed by GC. Eleven amino acid compositions imitating the characteristic amino acid profile of 11 different grape varieties were tested. Significant differences in the levels of some important volatile compounds (ethanol, ethyl acetate, acetic acid, higher alcohols and some of their acetates, methionol, isobutyric acid, ethyl butyrate, and hexanoic and octanoic acids) were found. The levels of some of the volatile compounds are well correlated with the aromatic composition of wines made with grapes of the same varieties. A multiple linear regression study produced good models for most of the odorants for which the level was related to the must amino acid composition. Partial least-squares regression models confirm that amino acid composition explains a high proportion of the variance in the volatile composition and show that the relationship between both sets of variables is highly multivariate. According to the different models, the levels of some byproducts of fatty acid synthesis are related to threonine and serine, the level of beta-phenyletanol is closely related to the level of phenylalanine, and methionol is strongly correlated to the must methionine contents. The addition of selected amino acids to different musts confirms the previous observations.