biomolecules

Review The Actual and Potential Aroma of Winemaking Grapes

Vicente Ferreira * and Ricardo Lopez Laboratory for Aroma Analysis and Enology (LAAE), Department of Analytical Chemistry, Universidad de Zaragoza, Instituto Agroalimentario de Aragón (IA2) (UNIZAR-CITA), c/Pedro Cerbuna 12, 50009 Zaragoza, Spain; [email protected] * Correspondence: [email protected]; Tel.: +34-976-762-067



Received: 8 November 2019; Accepted: 30 November 2019; Published: 3 December 2019


Abstract: This review intends to rationalize the knowledge related to the aroma of grapes and to the aroma of wine with specific origin in molecules formed in grapes. The actual flavor of grapes is formed by the few free aroma molecules already found in the pulp and in the skin, plus by those aroma molecules quickly formed by enzymatic/catalytic reactions. The review covers key aroma components of aromatic grapes, raisins and raisinized grapes, and the aroma components responsible from green and vegetal notes. This knowledge is used to explain the flavor properties of neutral grapes. The aroma potential of grape is the consequence of five different systems/pools of specific aroma precursors that during fermentation and/or aging, release wine varietal aroma. In total, 27 relevant wine aroma compounds can be considered that proceed from grape specific precursors. Some of them are immediately formed during fermentation, while some others require long aging time to accumulate. Precursors are glycosides, glutathionyl and cysteinyl conjugates, and other non-volatile molecules.

Keywords: wine aging; glycosides; glutathione; mercaptans; terpenols; norisoprenoids; volatile phenols; vanillin

1. Introduction Winemaking grapes are quite unique fruits because they are grown not to be immediately consumed, but to make wine with them. From this point of view, the study of grape aroma cannot be limited to the pool of molecules directly responsible for the odors and flavors of grape and grape juice but has also to include those other chemical structures that, more or less directly, are specific precursors of relevant wine aroma molecules. This task began more than 40 years ago when French and Australian researchers reported the existence of glycosides and other precursors of linalool [1,2]. The task, however, has proved to be extremely difficult due to many factors, such as the chemical and biochemical complexity of the precursor systems, the long times required to see aging effects in wine, or the analytical challenges associated to obtaining reliable representations of wine sensory properties from analytical data [3,4]. The truth is that nowadays, in spite of many significant advances, there are not accurate criteria or accepted methods able to provide a reliable assessment of the grape aroma potential, except perhaps for aromatic varietals such as Muscat or Gewürztraminer. This is a bit of a paradox; the grape genome was untangled more than 10 years ago [5], but yet, we do not have a clear understanding of all the grape metabolites which will ultimately contribute to the aromatic sensory properties of wine. The reasons for this rather sluggish progress in linking grape molecular systems and wine odorants can be better understood with the help of the schema in Figure1. The schema shows that grape contains at least seven different systems or pools of aroma precursors. Two out of the seven have

Biomolecules 2019, 9, 818; doi:10.3390/biom9120818 www.mdpi.com/journal/biomolecules BiomoleculesBiomolecules2019 2019, 9, 818, 9, 818 2 of2 35of 36

have relevance in grape but are not particularly important in wine aroma (the Strecker amino acid relevancesystem inand grape the butfatty are acid/peroxygenase not particularly important system), inwh wineile the aroma other (the five Strecker play essential amino acid roles system in the anddevelopment the fatty acid of/peroxygenase wine varietal system),aroma during while thewine other aging, five and/or play essential in the development roles in the development of wine flavor of winenotes. varietal If at the aroma light duringof our present wine aging, understanding, and/or in the the development different analytical of wine flavorstrategies notes. and If concepts at the lightapplied of our along present the understanding, years for the study the diofff grapeerent analyticalaroma precursors strategies are and revisited, concepts it appliedwill become along evident the yearsthat for they the provide study of information grape aroma covering precursors a rather are revisited, limited fraction it will become of wine evident varietal that aroma. they In provide fact, the informationgeneral strategy covering followed a rather to limitedanalyze fraction grape glycosidic of wine varietal precursors aroma. deals In with fact, precursors the general belonging strategy to followedjust one to or analyze two out grape of the glycosidic five pools. precursors This is dealsnot to with blame precursors previous belonging research, tomost just of one which or two was outbrilliantly of the five carried pools. out This by is notpioneers, to blame but previous to acknowledge research, the most difficulties of which of was the brilliantly study, which carried with out the bylimited pioneers, analytical but to acknowledge tools available the in ditheffi 1980s,culties 1990s, of the and study, even which the 2000s, with the hardly limited could analytical have been tools done availableany better. in the 1980s, 1990s, and even the 2000s, hardly could have been done any better.

Glutathionyl and cysteinyl DMS precursors Glutathionyl DMS precursors buccal and cysteinyl precursors time enzymes precursors

Glycosides Wine Fatty acids + of aroma enzymatic/cat Grape Free varietal molecules alytic systems Aroma aroma

time Glycosides of buccal time Strecker precursors of enzymes amino acids aroma Glycosides Precursors Other molecules of aroma of aroma precursors molecules molecules

time ©V.Ferreira Glycosides of precursors of aroma molecules

FigureFigure 1. Scheme1. Scheme showing showing the the main main systems systems/pools/pools in grapein grap ofe specificof specific precursors precursors of aromaof aroma molecules molecules andand their their involvement involvement in thein the development developmen oft wineof wine varietal varietal aroma aroma and and flavor. flavor.

TheThe two two first first systematic systematic approaches approaches developed developed to studyto study grape grape aroma aroma precursors, precursors, which which are are yet yet thethe basis basis of of the the methods methods in in use use at present,at present, were were developed developed by by Patrick Patrick Williams Williams and and coworkers coworkers in in AustraliaAustralia [6] and[6] and by Ziya by Ziya Gunata Gunata and coworkers and coworkers in Montpellier in Montpellier [7]. In these [7]. approaches,In these approaches, grape glycosil grape aromaglycosil precursors aroma precursors are extracted are from extracted grape from must grape or macerated must or macerated grape solids grape with solids C18 orwith with C18 XAD-2 or with polymericXAD-2 polymeric sorbents, respectively. sorbents, respectively. Much later, Much the use later, of more the use advanced of more polymeric advanced sorbents polymeric providing sorbents a widerproviding extraction a wider of precursorsextraction wasof precursors proposed was [8], althoughproposed as [8], noted although by Hampel as noted et al.,by noHampel sorbent et wasal., no effectivesorbent for was all glycosideseffective for [9 ].all The glycosides glycosidic [9]. fractions The glycosidic are further fractions hydrolyzed are further well by hydrolyzed acid hydrolysis well by andacid enzymatic hydrolysis treatment and enzymatic [6], and welltreatment exclusively [6], an byd well enzymatic exclusively treatment by enzymatic [7]. treatment [7]. TheThe advantage advantage of of enzymatic enzymatic treatment treatment is that,is that, in in comparison comparison to to acid acid hydrolysis, hydrolysis, it providesit provides a a relativelyrelatively unbiased unbiased composition composition of of the the aglycones aglycones present present in in the the extract, extract, as as far far as as the the correct correct type type of of enzymeenzyme is usedis used [9]. [9]. Under Under this this approach approach the the aroma arom ofa of grape grape is dividedis divided into into the the free free and and the the bound bound fractionsfractions [10 [10,11].,11]. Its Its major major disadvantage disadvantage is that,is that, in manyin many cases, cases, the the aglycone aglycone is notis not an an odorant odorant relevant relevant forfor wine wine aroma, aroma, but but an an aroma-worthless aroma-worthless volatile volatile compound compound such such as as benzyl benzyl alcohol alcohol or or an an odorless odorless precursorprecursor that that only only after after a seriesa series of of reactions, reactions, which which can can take take a longa long time, time, will will form form the the odorant. odorant. AttendingAttending to theto the scheme scheme shown shown in Figurein Figure1, enzymatic 1, enzymati hydrolysisc hydrolysis provides provides a useful a useful estimate estimate of wineof wine aromaaroma molecules molecules derived derived from from the the pool pool of “glycosidesof “glycosides of aromaof aroma molecules”, molecules”, but but not not of thoseof those derived derived fromfrom the the pool pool of “glycosidesof “glycosides of of precursors precursors of of aroma arom molecules”a molecules” or or from from the the other other pools pools of of precursors. precursors. Unfortunately, only some terpenols have direct glycosides, while important wine aroma molecules Biomolecules 2019, 9, 818 3 of 35

Unfortunately, only some terpenols have direct glycosides, while important wine aroma molecules derived from norisoprenoids or grape phenols have not many direct glycosides. Consequently, enzymatic hydrolysis can assess the aroma potential of Muscat and other terpenol-related varietals, but not of “neutral varieties” [12]. For neutral varieties things are slightly more complicated, since the precursors of some relevant aroma molecules, such as norisoprenoids, require acid catalysis to undergo the chemical rearrangement processes through which the odorant is formed. Inevitably, this implies that labile aroma molecules, such as linalool and geraniol, will be degraded [9]. This problem is more evident in the many assays in which acid hydrolysis is carried out at high temperatures (100 ◦C). Under these conditions, as will be later detailed, there is a strong degradation of many relevant wine aroma molecules. Best results from the sensory point of view were obtained in the few studies in which acid hydrolysis was carried out at mild temperatures (45–50 ◦C). Only in these conditions the aroma hydrolysates obtained were able to induce significant sensory changes in wine [13,14]. However, some of the aroma descriptors developed during the hydrolysis, such as honey or tea [13], suggest that oxidation and thermal degradation processes are taking place under those conditions. These observations may question whether those hydrolysates are genuine representatives of wine varietal aroma and hence of grape potential aroma. A recently presented strategy tries to sort out these limitations by using a most powerful extraction strategy, carrying out the hydrolysis in strict anoxia and in the presence of grape polyphenols [15]. Grape polyphenols and most specific aroma precursors, except those of dimethyl sulfide (DMS), are coextracted from dearomatized “mistellas” and reconstituted in synthetic wine. Under these conditions, hydrolysates obtained after 24 h display sensory profiles congruent with unoxidized wine odor nuances and specific for the grape variety (Alegre et al., in preparation). The approach is promising, yet requires proper validation. In the present review we will make a distinction between the actual and the potential aromas of grapes, even if in many instances the boundaries between both categories are relatively blurred. Actual grape aroma integrates those aroma molecules and chemical systems responsible for the aromatic sensory properties (odor and flavor) of grapes and grape juices. On the other hand, potential grape aroma refers to the different grape molecules and grape chemical systems that are specific precursors of relevant wine odorants.

2. The Actual Aroma of Grapes and Musts The concept of actual aroma includes not only the aroma molecules found as free forms in the grape or must, but also those others formed in the short time span in which grapes of grape juices are kept in the mouth during mastication and salivation. This can be better understood with the help of the scheme shown in Figure2. In the figure, the precursor systems able to quickly release free aroma molecules are linked by discontinuous arrows to the “grape free aroma molecules pool”. In common with many fruits, the actual aroma of grapes involves compounds in three related categories:

1. Free aroma, which refers to the aroma molecules found as such in the pulp and skin of the fruit, the grape in our case; 2. Aroma molecules formed by nearly instantaneous enzymatic/catalytical processes triggered during the disruption of fruit tissues [16,17]; 3. Aroma molecules formed in the buccal cavity by the action of salivary or bacterial enzymes [18–20].

Compounds in the second category include a number of aldehydes, ketones and alcohols formed by peroxidation of fatty acids. Numerically the most abundant are compounds with six carbon atoms, so that compounds in this category are often named as C6-compounds [21,22]. It should be noted, however, that some powerful aroma compounds with a different number of carbon atoms can be also formed through this way, such as E-2-nonenal [23] or (E,Z)-2,6-nonadienal [24,25]. These powerful Biomolecules 2019, 9, 818 4 of 35 aroma compounds have much smaller odor thresholds, so that some of the green odors usually

Biomoleculesattributed 2019 to, C69, 818 aldehydes and alcohols could be in fact be caused by C9 aromas. 4 of 36

Glutathionyl and cysteinyl DMS precursors precursors Glutathione Salivary Oxidation enzymes Salivary Glycosides enzymes of aroma Fatty acids + Cell Grape Free Aroma molecules enzymatic/cat- disruption alytic systems Molecules n Raisining tio da ng Glycosides of xi ni O isi precursors of Ra aroma molecules Strecker amino acids Other precursors ©V.Ferreira

FigureFigure 2. 2. SchemeScheme showing showing the the different different aroma aroma precursor precursor sy systemsstems/pools/pools in in grape grape and and their their relationship relationship withwith the the fraction fraction of of free free aroma aroma mole moleculescules which which will will ultimately ultimately be be responsible responsible for for the the odor odor and and flavor flavor ofof grapes grapes and and musts. musts.

InCompounds common with in themany third fruits, category the actual derive aroma from of two grapes different involves types compounds of precursors. in three It has related been categories:demonstrated that glutathionyl and cysteinyl precursors, which are odorless cysteine-S-conjugates, can release1. the aromaticFree aroma, thiol which by the refers action to of the buccal aroma microbiota molecules [18 found]. The as release such in takes the 20–30pulp and s and skin can induce a perceptionof the lasting fruit, the for grape up to in 3 min,our case; which supports the idea that these precursors can have an outstanding2. Aroma role in themolecules persistence formed of grape by nearly and wine instantaneous aroma. In theenzymatic/catalytical case of glycosidic precursorsprocesses of aroma molecules,triggered it has during been the demonstrated disruption of that fruit oral tissues bacteria [16,17]; are able to hydrolyze glycosidic precursors,3. releasing Aroma an molecules array of volatiles formed [in19 ].the In buccal the particular cavity by case the of action glycoconjugates of salivary of or the bacterial volatile phenols derivedenzymes from smoke [18–20]. exposure, it was demonstrated that enzymes in saliva are able to release enoughCompounds volatiles in to the create second a sensory category perception include a [numb26]. Iner theof aldehydes, case of glycoconjugates ketones and alcohols extracted formed from byGewürztraminer peroxidation of grapes,fatty acids. sensory Numerically effects in the the most mouth abundant were only are evidentcompounds when with tested six atcarbon 5-times atoms, wine soconcentration that compounds and in in the this absence category of wineare often volatiles, name whichd as C6-compounds may call into question [21,22]. theIt should sensory be relevance noted, however,of the aroma that volatilessome powerful released aroma from compounds those glycosides with a in different the mouth number [20]. However,of carbon atoms all these can in-mouth be also formedeffects arethrough highly this variable way, such between as E-2-nonenal individuals, [23] so that or (E,Z)-2,6-nonadienal for some sensitive individuals [24,25]. These they powerful may have aromaan effect. compounds Additionally, have a recentmuch reportsmaller [27 odor] has threshol revealedds, that so glycosidesthat some extracted of the green from odors the grape usually marc attributedadded to to the C6 must aldehydes produce and wines alcohols with could longer be aftertaste. in fact be caused This observation by C9 aromas. does not unequivocally demonstrateCompounds the rolein the of glycosidicthird category precursors derive in from aftertaste two butdifferent supports types their of importance precursors. on It wine has flavor.been demonstratedIn the case that of glutathionyl grapes, the free and aroma cysteinyl fraction precursors, is very which small inare most odorless varieties, cysteine-S-conjugates, in agreement with canthe release fact that the most aromatic of them thiol display by the weak action and of ratherbuccal neutralmicrobiota odors [18]. and The flavors. release This takes should 20–30 not s and be a cansurprise, induce since a perception grapes are lasting fruits extremelyfor up to 3 rich min, in waterwhich andsupports do not the contain idea specialthat these cellular precursors or vacuolar can havestructures an outstanding in which nonpolar role in moleculesthe persistence such asof aromagrape compoundsand wine aroma. can be safelyIn the stored. case of Hydrophobic glycosidic precursorsmolecules, of including aroma molecules, many aroma it components,has been demons are stabilizedtrated that inthe oral pulp bacteria and skin are byable forming to hydrolyze covalent glycosidicbonds with precursors, polar molecules, releasing such an array as sugars of volatiles or amino [19]. acids, In the constituting particular ca fractionsse of glycoconjugates of specific aroma of theprecursors volatile whichphenols will derived be extensively from smoke discussed exposure, later it on. was demonstrated that enzymes in saliva are able toIn release the present enough section volatiles we will to focuscreate on a thesensory aroma perception molecules [26]. which In can the be case found of glycoconjugates as free molecules extractedin grapes from or musts Gewürztraminer and which aregrapes, likely sensory contributors effects ofin sensorythe mouth notes. were The only section evident will when be divided tested atinto 5-times four subsections.wine concentration The first and one in addresses the absence the of aroma wine moleculesvolatiles, which of those may types call ofinto grapes question showing the sensory relevance of the aroma volatiles released from those glycosides in the mouth [20]. However, all these in-mouth effects are highly variable between individuals, so that for some sensitive individuals they may have an effect. Additionally, a recent report [27] has revealed that glycosides extracted from the grape marc added to the must produce wines with longer aftertaste. This Biomolecules 2019, 9, 818 5 of 35 clear and distinctive aromas, such as Muscat, Gewurztraminer and some hybrids between Vitis vinifera and labruscana. The second subsection summarizes the knowledge about the aroma molecules of raisins. The third subsection considers aroma molecules responsible for green, herbaceous, and vegetal aroma, many of which form a kind of common background in grapes of all types. The fourth and last subsection will briefly discuss about the aroma molecules responsible for the aroma characteristics of neutral grapes.

2.1. Key Aroma Compounds of Aromatic Grapes Among Vitis vinifera grape varieties, only those of the Muscat group have distinctive aroma and flavor [28]. These grapes contain important amounts of terpenols at levels above the odor threshold, as detailed in Table1. The most important aroma compounds are linalool and geraniol, although those grapes also contain important levels of citral, citronellol, nerol, and α-terpineol. Another component, which attending to recent reports can be present at sensorily relevant levels, is geranic acid [29–31]. Muscat grapes can contain more than 5 mg/kg of these aroma compounds, in clear contrast to non-Muscat varieties which contain in general less than 0.5 mg/kg of these aroma compounds. Another relevant terpenic aroma compound is (Z)-rose oxide, which is responsible for the litchi-like or rose-like characteristic aroma of Gewürztraminer wines [32,33]. Rose oxide is a powerful aroma compound with an odor threshold one order of magnitude smaller than that of linalool [34]. It has been quantified in grapes from the Traminer family at 18 µg/L[35]. It has been recently found also in Muscat grapes [36] and a recent report even suggests that the intensity of Muscat aroma in grapes is strongly correlated to the presence of this molecule [37]. Its aromatic relevance in some aromatic grapes could have been underestimated simply because this molecule has been quantified in a reduced number of cases. Semiquantitative data provided by a recent report suggest that this aroma compound could be in fact relevant in the aroma profile not only of Gewurztraminer and Muscat, but also in Traminette and even in Riesling [38]. Among non Vitis vinifera cultivars there are some varieties known by their specific aroma. One of them is Vitis labruscana Bailey cv. Concord which contains at least four different aroma molecules at sensory-relevant levels. These are o-aminoacetophenone, methylfuraneol, methyl anthranilate, and furaneol [39,40]. Two of them, methyl anthranilate and o-aminoacetophenone, are involved in the characteristic “foxy” aroma of the variety (see Table1). Remarkably, methyl anthranilate was identified as early as 1926 [41], while o-aminoacetophenone was identified in the 1980s [42]. Methyl anthranilate has been identified as one of the aroma components able to attract flies [43]. For its part, o-aminoacetophenone can eventually also develop in wines of Vitis vinifera varieties (mostly of German origin) where it causes a defect known as “untypical aging note” [44]. Furaneol (2,5-dimethyl-4-hydroxy-3(2H)-furanone) has also been identified as key odorant of muscadine (Vitis rotundifolia Michx), together with o-aminoacetophenone [45,46]. The potency and particular sensory characteristics of these aroma compounds make it so that those grapes are much appreciated as table grapes and also for making aromatic grape juice, but they are regarded as nonappropriate for making wine. In a recent paper, Wu et al. [29] study the aroma composition of 20 table grapes, 12 of which are hybrids between V. vinifera and V. labrusca. Interestingly, five of the hybrids showed strawberry aroma and four others foxy aroma, which suggests that the former contain large amounts of furaneol and of methylfuraneol, while the latter may contain methyl anthranilate and o-aminoacetophenone. Unfortunately, and this is a limitation of most recent studies carried out on grapes, all these polar and not very volatile aroma compounds cannot be easily determined by headspace solid phase microextraction (HS-SPME), which has become a kind of standard technique for the analysis of grape aroma. This explains the controversy about the implication of ethyl esters on the strawberry aroma of some of those grapes [29,47] and should warn about the risk of extracting conclusions about the sensory implications of analytical data when known essential aroma compounds have not been quantified: even if the profile of the volatiles quantified by HS-SPME is enough to obtain a highly satisfactory varietal differentiation, this does not mean that the varietal aroma profile is perfectly defined. Biomolecules 2019, 9, 818 6 of 36 Biomolecules 2019, 9, 818 6 of 36 Biomolecules 2019, 9, 818 6 of 36 Biomolecules 2019, 9, 818 6 of 36 Biomoleculesaroma and 2019 four, 9, 818 others foxy aroma, which suggests that the former contain large amounts of furaneol6 of 36 aroma and four others foxy aroma, which suggests that the former contain large amounts of furaneol aromaandaroma of and methylfuraneol,and four four others others foxy foxy while aroma, aroma, the latterwhich which may suggests suggests contai thatn that methyl the the former former anthranilate contain contain andlarge large o-aminoacetophenone. amounts amounts of of furaneol furaneol aromaand of andmethylfuraneol, four others foxy while aroma, the latter which may suggests contai nthat methyl the former anthranilate contain and large o-aminoacetophenone. amounts of furaneol andUnfortunately,and of of methylfuraneol, methylfuraneol, and this while whileis a thelimitation the latter latter may ofmay most contai contai recentn nmethyl methyl studies anthranilate anthranilate carried out and and on o-aminoacetophenone. o-aminoacetophenone.grapes, all these polar andUnfortunately, of methylfuraneol, and this while is a limitation the latter ofmay most contai recentn methyl studies anthranilate carried out and on o-aminoacetophenone.grapes, all these polar Unfortunately,andUnfortunately, not very andvolatile and this this aromais is a alimitation limitation compounds of of most mostcannot recent recent be studies easilystudies determinedcarried carried out out onby on grapes,headspace grapes, all all these solidthese polarphase polar Unfortunately,and not very volatile and this aroma is a limitation compounds of most cannot recent be easilystudies determined carried out by on headspacegrapes, all solidthese phasepolar andmicroextractionand not not very very volatile volatile (HS-SPME), aroma aroma whichcompounds compounds has become cannot cannot a kindbe be easily ofeasily standard determined determined technique by by headspace forheadspace the analysis solid solid of phase grapephase andmicroextraction not very volatile (HS-SPME), aroma which compounds has become cannot a kind be easilyof standard determined technique by forheadspace the analysis solid of phasegrape microextractionaroma.microextraction This explains (HS-SPME), (HS-SPME), the controversy which which has has aboutbecome become the a akindimpl kind icationof of standard standard of ethyl technique technique esters onfor for the the strawberryanalysis analysis of of grapearoma grape microextractionaroma. This explains (HS-SPME), the controversy which has about become the a impl kindication of standard of ethyl technique esters on for the the strawberry analysis of aroma grape aroma.ofaroma. some This This of thoseexplains explains grapes the the controversy[29,47] controversy and shouldabout about the warnthe impl impl abouticationication the of riskof ethyl ethyl of estersextracting esters on on the conclusionsthe strawberry strawberry about aroma aroma the aroma.of some This of those explains grapes the [29,47]controversy and should about warnthe impl aboutication the ofrisk ethyl of extracting esters on theconclusions strawberry about aroma the ofsensoryof some some of implicationsof those those grapes grapes of [29,47] [29,47]analytical and and shoulddata should when warn warn known about about theessential the risk risk of ofaroma extracting extracting compounds conclusions conclusions have about aboutnot beenthe the ofsensory some ofimplications those grapes of [29,47]analytical and data should when warn known about essential the risk ofaroma extracting compounds conclusions have aboutnot been the sensoryquantified:sensory implications implications even if the of of profileanalytical analytical of the data data volatiles when when knownquantified known essential essential by HS-SPME aroma aroma compoundsis compounds enough to have obtainhave not nota highlybeen been sensoryquantified: implications even if the of profile analytical of the data volatiles when quantified known essential by HS-SPME aroma is compounds enough to obtainhave not a highly been quantified:satisfactoryquantified: even evenvarietal if ifthe the differentiation, profile profile of of the the volatilesthis volatiles does quantified noquantifiedt mean by thatby HS-SPME HS-SPME the varietal is is enough enougharoma toprofile to obtain obtain is a perfectly ahighly highly quantified:satisfactory evenvarietal if the differentiation, profile of the thisvolatiles does quantifiednot mean thatby HS-SPME the varietal is enougharoma profileto obtain is perfectlya highly satisfactorydefined.satisfactory varietal varietal differentiation, differentiation, this this does does no not tmean mean that that the the varietal varietal aroma aroma profile profile is is perfectly perfectly Biomoleculessatisfactorydefined. 2019 varietal, 9, 818 differentiation, this does not mean that the varietal aroma profile is perfectly 6 of 35 defined.defined. defined. Table 1. Structures, odor properties, and occurrence of the key odorants of aromatic grapes. Table 1. Structures, odor properties, and occurrence of the key odorants of aromatic grapes. Table 1. Structures, odor properties, and occurrence of the key odorants of aromatic grapes. Table 1. 1. Structures,Structures, odor odor properties, properties, and andoccurrence occurrence of the ofkey the odorants key odorants of aromatic of aromatic grapes.Range grapes. of Table 1. Structures, odor properties, and occurrence of theOdor key odorants of aromatic grapes.Range of Compound Structure Grape Odor Threshold OccurrenceRange of Compound Structure Grape DescriptionOdor Threshold OccurrenceRange of Compound Structure Grape DescriptionOdor Threshold RangeOccurrencein Grapes ofRange of Compound Structure Grape DescriptionOdorOdor Threshold Occurrencein Grapes CompoundCompound Structure Grape Grape Description ThresholdThreshold Occurrencein GrapesOccurrence in DescriptionDescription in Grapes in GrapesGrapes

Hyacinth, 0.06–1.5 mg/L Linalool Muscat Hyacinth, 6 μg/L [48] 0.06–1.5 mg/L Linalool Muscat MuscatHyacinth, wine 6 μg/L [48] 0.06–1.5[28] mg/L Linalool Muscat MuscatHyacinth, wine 6 μg/L [48] 0.06–1.5[28] mg/L Linalool Muscat Hyacinth,MuscatHyacinth, wine 6 μg/L [48] 0.06–1.5[28]0.06–1.5 mg/L mg/L LinaloolLinalool MuscatMuscat Muscat wine 6 μg/L 6[48]µg /L[48] [28] MuscatMuscat wine wine [28] [28]

0.09–1.1 mg/L Geraniol Muscat Citrus, rose 40 μg/L [49] 0.09–1.1 mg/L Geraniol Muscat Citrus, rose 40 μg/L [49] 0.09–1.1[28] mg/L Geraniol Muscat Citrus, rose 40 μg/L [49] 0.09–1.1[28]0.09–1.1 mg/L mg/L GeraniolGeraniol MuscatMuscat Citrus, Citrus, rose rose 40 μg/L 40 [49]µg /L[490.09–1.1] [28] mg/L Geraniol Muscat Citrus, rose 40 μg/L [49] [28] [28] [28]

0.5 (l form) or 0.5 (l form) or 0.5 (l form) or μ (Z)-Rose oxide Traminer Rose, litchi 0.5 50(l form)μ0.5g/L (l (dor form) or7–29μ g/L [35] (Z)-Rose oxide Traminer Rose, litchi 0.550 (l μ form)g/L (d or 7–29 g/Lμ [35] (Z)-Rose(Z)-Rose oxide oxide TraminerTraminer Rose, Rose, litchi litchi form)50 μg/L50 [34] µ(dg /L (d 7–29μ 7–29g/L [35]µg/L[ 35] (Z)-Rose oxide Traminer Rose, litchi 50form) μg/L [34] (d 7–29 μg/L [35] (Z)-Rose oxide Traminer Rose, litchi 50form) μg/Lform) [34](d [347–29] g/L [35] form) [34] form) [34]

μ o- Sweet, 10–20μ g/L o- Concord Sweet, 0.2 μg/L [50] 10–20 g/Lμ Aminoacetophenoneo-Aminoacetophenoneo- ConcordConcord caramel Sweet,Sweet, caramel 0.2 μg/L 0.2 [50]µg/ L[50]10–20[46] 10–20μ g/Lµ g/L[46] Aminoacetophenoneo- Concord caramelSweet, 0.2 μg/L [50] 10–20[46] μg/L Aminoacetophenoneo- Concord Sweet,caramel 0.2 μg/L [50] 10–20[46] g/L Aminoacetophenone Concord caramel 0.2 μg/L [50] [46] Aminoacetophenone caramel [46]

0.8 mg/kg0.8 mg [39]/kg [39] Orangine,Orangine, 0.8 mg/kg [39] MethylMethyl anthranilate anthranilate ConcordConcord Orangine, 3 μg/L3 µ[51]g/L[ 51] 0.80.5–6 mg/kg mg/kg0.5–6 [39] mg /kg Methyl anthranilate Concord Orangine,sweetsweet 3 μg/L [51] 0.80.5–6 mg/kg mg/kg [39] Methyl anthranilate Concord Orangine,sweet 3 μg/L [51] 0.80.5–6 mg/kg[52] mg/kg [39][52 ] Methyl anthranilate Concord Orangine,sweet 3 μg/L [51] 0.5–6[52] mg/kg Methyl anthranilate Concord sweet 3 μg/L [51] 0.5–6 mg/kg[52] sweet [52] [52]

Some of these compounds can be also present, albeit at much smaller levels, in grapes from Some of these compounds can be also present, albeit at much smaller levels, in grapes from neutralSomeSomeSome varieties. of of these thesethese For compounds compoundscompounds instance, furaneol can can can be be bealsowas also also proposedpresent, present, present, al timealbeitbeit albeit agoat at much asmuch ata potential muchsmaller smaller smaller markerlevels, levels, infor levels,in grapes thegrapes detection in from grapesfrom from neutralSome varieties. of these For compounds instance, furaneol can be was also proposed present, timealbeit ago at asmuch a potential smaller marker levels, forin thegrapes detection from neutralneutralofneutral forbidden varieties. varieties. hybrids For ForFor instance, instance, (Vitis vinifera furaneol furaneol furaneol × non-vinifera) was was was proposed proposed proposed for time timemaking ago time ago as wine agoas a apotential potential as [53]. a potential Furaneol marker marker markerforca forn the bethe detectionpresent fordetection the at detection neutralof forbidden varieties. hybrids For instance, (Vitis vinifera furaneol × non-vinifera) was proposed for time making ago aswine a potential [53]. Furaneol marker ca forn bethe present detection at ofoflevelsof forbiddenforbidden forbidden above hybrids hybrids1 hybrids mg/kg ( Vitisin (Vitis non-viniferas, vinifera vinifera vinifera × ×non-vinifera) non-vinifera) whilenon-vinifera) it rarely for for makingwill formaking reach making wine wine 0.05 [53].wine mg/kg[53]. Furaneol Furaneol [53 in]. vinifera Furaneol ca can nwinesbe be present canpresent [54]. be at presentat at oflevels forbidden above 1hybrids mg/kg (inVitis non-viniferas, vinifera × non-vinifera)× while it rarely for will making reach wine 0.05 mg/kg[53]. Furaneol in vinifera ca nwines be present [54]. at levelslevelslevels aboveRecentabove above 1 1and 1mg/kg mg mg/kg quite/kg in inin non-viniferas,extensive non-viniferas, reports while while whilefrom it itrarely Chineserarely it rarely will will researchers reach will reach reach 0.05 0.05 mg/kghave 0.05mg/kg mgconfirmed in in vinifera/kg vinifera in viniferawinesthat wines some [54]. wines[54]. table [54 ]. levelsRecent above and1 mg/kg quite in extensive non-viniferas, reports while from it rarelyChinese will researchers reach 0.05 havemg/kg confirmed in vinifera that wines some [54]. table grapesRecentRecent contain and and quitea quiterange extensive extensive of ethyl reports reportsesters fromat from concentrations Chinese Chinese researchers researchers above thhave eirhave thresholdsconfirmed confirmed [2that 9–31,47].that some some tableThese table grapesRecentRecent contain and and a quite quiterange extensive extensiveof ethyl reportses reportsters atfrom concentrations from Chinese Chinese researchers above researchers th haveeir thresholds confirmed have confirmed [2 that9–31,47]. some that These table some table grapesaromagrapes contain compoundscontain a arange range are offound of ethyl ethyl mainly es estersters asat at freeconcentrations concentrations compounds above inabove the thpulp theireir thresholdsand, thresholds in terms [2 [29–31,47]. of9–31,47]. odor activityThese These grapesgrapesaroma containcompoundscontain aa rangerange are found ofof ethylethyl mainly estersesters as atat free concentrationsconcentrations compounds inabove above the pulpth theireir and,thresholds thresholds in terms [2 [of9–31,47].29 –odor31,47 activity ].These These aroma aromavaluesaroma compounds (OAVs),compounds can are areamount found found tomainly mainlya rele vantas as free freefraction compounds compounds of the odorantsin in the the pulp pulp present and, and, in in theterms terms grape. of of odor odorThis activity fractionactivity compoundsaromavalues compounds(OAVs), are can found areamount found mainly to mainly a rele as freevant as free compoundsfraction compounds of the in odorants in the the pulp pulp present and, and, in inin the termsterms grape. of of odorThis odor fractionactivity activity values valuesvalues (OAVs), (OAVs), can can amount amount to to a arele relevantvant fraction fraction of of the the odorants odorants present present in in the the grape. grape. This This fraction fraction (OAVs),values (OAVs), can amount can amount to a relevant to a rele fractionvant fraction of the of odorantsthe odorants present present in in the the grape. grape. This This fractionfraction seems to be particularly high in “foxy” aroma grapes derived from V. labruscana [29] and also in some unfamiliar table-grapes [30]. For instance, in the cultivar “Honey Black”, these compounds account for more than 70% of the total OAV measured by the researchers. It is not clear, however, whether this aromatic power translates into specific aroma nuances. Ethyl esters are relatively ubiquitous aroma compounds and are normal constituents of the aroma of many fruits, so that they will likely contribute to generic fruity aroma nuances to grape flavor.

2.2. Key Aroma Compounds of Raisins and of “Raisinized” Grapes Another type of grapes with intense and specific aroma and flavors are raisins, which are grapes naturally dried under the sun or by different artificial means. Some raisins are used to make dessert wines, such as Pedro Ximenez, and are, therefore, genuine winemaking grapes. Many other raisins are produced to be directly consumed as sweet grapes and confectionery ingredients. Their aroma composition is, however, of general interest for the wine industry, since winemaking grapes can undergo naturally spontaneous drying processes on the vine (raisining, as indicated in Figure2) as Biomolecules 2019, 9, 818 7 of 35 the consequence of different maturation problems. As those problems become more frequent due to climate change, unwanted raisining will be an emerging problem in many vine growing areas [55]. In the event these raisinized grapes are fermented together with healthy grapes, the wine will eventually develop raisin and prune notes. Raisins can contain different groups of key aroma compounds [23,56–58], which explains the high diversity of aroma nuances observed between different types of raisins and also supports the general complexity of raisin aroma. Leaving aside key terpenic odorants, such as linalool, geraniol, and rose oxide, which come directly from the fresh grape in the frequent case in which the raisins are made of aromatic grapes (Muscat and derivatives, Traminer and derivatives, Pedro Ximenez) [23], raisins can contain relevant odorants or groups of odorants produced or accumulated well during the own raisining process, during the last stages of grape maturation, and even during the storage of raisins. The first aroma compound particularly relevant in raisins is β-damascenone, which seems to be a quite ubiquitous and key aroma component of many sun-dried grapes [23,57] and of the wines made with them [59]. β-Damascenone is a norisoprenoid derived from the degradation of carotenoids. It has a quite low odor threshold, close to the ng/L, and an odor reminding of prunes or overmatured plums. As will be later discussed, this molecule plays also a relevant role in the flavor of neutral grapes and in the sensory properties of wines. Its structure and odor properties, together with those of other important aroma compounds from the same family, can be seen in Table2. Di fferent studies confirm that β-damascenone tends to accumulate in grapes in the last periods of maturation [60–63], particularly in the case of late season berry dehydration (or raisining) [64,65], during the storage of the raisins [58], or even during the aging of wines made with raisins [66]. Its levels, however, have no clear relationship with sun exposure on the vine [67,68]. β-Damascenone plays an outstanding role in the fruity aroma characteristics of wine. At low concentrations it acts as aroma enhancer [69] but at levels above 2–3 µg/L it can induce the perception of overmatured fruit, particularly if methional is Biomolecules 2019, 9, 818 8 of 36 alsoBiomolecules present 2019 [70, 9]., 818 8 of 36 Biomolecules 2019, 9, 818 8 of 36 Biomolecules 2019, 9, 818 8 of 36 TableTable 2. Structures,2. Structures, odor odor properties, properties, and an occurrenced occurrence of of norisoprenoids norisoprenoids found found above above their their threshold threshold TableTable 2. 2. Structures, Structures, odor odor properties, properties, an andd occurrence occurrence of of norisoprenoids norisoprenoids found found above above their their threshold threshold valueTablevalue in in wine.2. wine.Structures, odor properties, and occurrence of norisoprenoids found above their threshold valuevalue in in wine. wine. value in wine. Threshold Range of Occurrence in Compound Structure Odor Descriptor Threshold Range of OccurrenceRange of in Compound Structure Odor Descriptor Threshold Range of Occurrence in CompoundCompound StructureStructure OdorOdor Descriptor Descriptor ThresholdThresholdin Wine in WineRangeOccurrence of WineOccurrence in in Compound Structure Odor Descriptor inin Wine Wine WineWine in Wine WineWine β- Plum, cooked 50 ng/L β- Plum, cooked 50 ng/L n.d. to 10.5 μg/L [71] Damascenoneβ- Plum,apple cooked 50 [32]ng/L n.d. to 10.5 μg/L [71] Damascenoneβ- Plum,Plum,apple cooked cooked 50[32] ng/L n.d.n.d. to 10.5 to 10.5μg/Lµ g[71]/L βDamascenone-Damascenone apple 50[32] ng/L[ 32] n.d. to 10.5 μg/L [71] Damascenone appleapple [32] [71]

90 ng/L β-Ionone Violet, woody 90 ng/L n.d. to 1.2 μg/L [71] β-Ionone Violet, woody 90 ng/L n.d. to 1.2 μg/L [71] ββ-Ionone-Ionone Violet,Violet, woody woody 9090 ng[72] ng/L/L[ 72] n.d.n.d. to to1.2 1.2 μg/Lµg/ L[[71]71 ] β-Ionone Violet, woody [72][72] n.d. to 1.2 μg/L [71] [72]

n.d. to 255 µg/L TDN Kerosene-likeKerosene-like 2 2μµg/Lg/L[ [73]73] n.d. to 255 μg/L [74] TDNTDN Kerosene-likeKerosene-like 22 μ μg/Lg/L [73] [73] n.d. n.d. to to 255 255 [μ 74μg/Lg/L] [74] [74] TDN Kerosene-like 2 μg/L [73] n.d. to 255 μg/L [74]

40 ng/L n.d. to 233 ng/L TPB Green,Green, cut-grass cut-grass 40 4040 ng/L ngng/L/L[ 75] n.d. to 233 ng/L [76] TPBTPB Green,Green, cut-grass cut-grass 40[75] ng/L n.d.n.d. to to 233 233 [ng/L 76ng/L] [76] [76] TPB Green, cut-grass [75][75] n.d. to 233 ng/L [76] [75]

n.d.: Not detected. n.d.: Not detected. n.d.:n.d.: Not Not detected. detected. n.d.: Not detected. The second group of powerful aroma compounds likely formed during grape dehydration are The second group of powerful aroma compounds likely formed during grape dehydration are StreckerThe secondaldehydes group derived of powerful from the aroma Strecker compounds degradation likely of formed amino duringacids. The grape most dehydration relevant from are StreckerThe aldehydes second group derived of powerful from the aromaStrecker compounds degradation likely of amino formed acids. during The grape most dehydration relevant from are Streckerthe aromatic aldehydes point ofderived view arefrom phenylacetaldehyde the Strecker degradation (honey of odor) amino and acids. methiona The mostl (raw relevant potato odor),from theStrecker aromatic aldehydes point of derived view are from phenylacetaldehyde the Strecker degradation (honey odor) of amino and methionaacids. Thel (rawmost potatorelevant odor), from thewhich aromatic are important point of aromaview are constituents phenylacetaldehyde of Pedro Ximenez (honey odor)wines and made methiona with sun-driedl (raw potato grapes odor), [59]. whichthe aromatic are important point of aroma view constituentsare phenylacetaldehyde of Pedro Ximenez (honey wines odor) made and methiona with sun-driedl (raw potatograpes odor),[59]. whichPhenylacetaldehyde are important aromahas been constituents also found of atPedro levels Ximenez well above wines its made threshold with sun-driedin raisins grapes[23,57]. [59]. The Phenylacetaldehydewhich are important has aroma been constituents also found ofat Pedrolevels Ximenezwell above wines its thresholdmade with in sun-dried raisins [23,57]. grapes The [59]. Phenylacetaldehydeformation of these compounds has been also can befound particularly at levels intense well above in the its frequent threshold case in in raisins which dehydration[23,57]. The formationPhenylacetaldehyde of these compounds has been can also be found particularly at levels intense well inabove the frequent its threshold case inin which raisins dehydration [23,57]. The formationoccurs after of orthese during compounds the attack can of bethe particularly fungus Botrytis intense cinerea in the [77–79], frequent which case explains in which the dehydration high levels occursformation after ofor theseduring compounds the attack canof the be fungusparticularly Botrytis intense cinerea in [77–79],the frequent which case explains in which the dehydrationhigh levels occursof both after compounds or during in the wines attack from of the Sauternes. fungus BotrytisThese compounds cinerea [77–79], arise which by the explains reaction the of high the aminolevels ofoccurs both compoundsafter or during in winesthe attack from of Sauternes. the fungus Thes Botrytise compounds cinerea [77–79], arise whichby the explainsreaction theof the high amino levels of both compounds in wines from Sauternes.α These compounds arise by the reaction of the amino ofacid both precursor compounds with ain quinone wines from or other Sauternes.α -dicarbonyl. These Incompounds grapes, the arise major by source the reaction of dicarbonyls of the amino is the acidacid precursor precursor with with a a quinone quinone or or other other α-dicarbonyl.-dicarbonyl. In In grapes, grapes, the the major major source source of of dicarbonyls dicarbonyls is is the the acidquinones precursor from withoxidizing a quinone polyphenols. or other Theα-dicarbonyl. oxidation Incan grapes, begin bythe photoactivation major source of (normaldicarbonyls raisining) is the quinonesquinones from from oxidizing oxidizing polyphenols. polyphenols. The The oxidation oxidation can can begin begin by by photoactivation photoactivation (normal (normal raisining) raisining) quinonesor by enzymatic from oxidizing action, whichpolyphenols. will be The particularly oxidation intense can begin in theby photoactivationpresence of the (normalpowerful raisining) phenol- oror by by enzymatic enzymatic action, action, which which will will be be particularly particularly intense intense in in the the presence presence of of the the powerful powerful phenol- phenol- oroxidase by enzymatic from Botrytis action, (laccase). which willRecent be particularlyresults suggest intense that thein the formation presence may of thetake powerful place after phenol- some oxidaseoxidase from from Botrytis Botrytis (laccase). (laccase). Recent Recent results results suggest suggest that that the the formation formation may may take take place place after after some some oxidasetime of fromthe solarBotrytis irradiation, (laccase). Recentsince inresults a study suggest of thethat effectsthe formation of the maystorage take on place raisin after aroma, some timetime ofof thethe solarsolar irradiation,irradiation, sincesince inin aa studystudy ofof thethe effectseffects ofof thethe storagestorage onon raisinraisin aroma,aroma, timephenylacetaldehyde of the solar irradiation, strongly accumu since latedin a onlystudy after of the12 monthseffects ofof storagethe storage of sun-dried on raisin raisins aroma, but phenylacetaldehydephenylacetaldehyde strongly strongly accumu accumulatedlated only only after after 12 12 months months of of storage storage of of sun-dried sun-dried raisins raisins but but phenylacetaldehydenot in air-dried raisins strongly [58]. These accumu compoundslated only are after relatively 12 months difficult of storage to analyze of sun-dried because of raisins their high but notnot in in air-dried air-dried raisins raisins [58]. [58]. These These compounds compounds are are relatively relatively difficult difficult to to analyze analyze because because of of their their high high activity towards many chromatographic phases and because of the adducts they form with SO2. This activitynot in air-driedtowards manyraisins chromatographic [58]. These compounds phases areand relatively because ofdifficult the adducts to analyze they formbecause with of SO their2. This high activityexplains towards why many many reports chromatographic fail in their detection,phases and particularly because of inthe the adducts case of they methional, form with so SOthat2. Thistheir explainsactivity whytowards many many reports chromatographic fail in their detection, phases and particularly because of in the the adducts case of methional,they form with so that SO 2their. This explainsimportance why may many be underestimated.reports fail in their detection, particularly in the case of methional, so that their importanceexplains why may many be underestimated. reports fail in their detection, particularly in the case of methional, so that their importanceThe third may group be underestimated. of aroma components of raisins is formed by two odorous lactones derived from importanceThe third may group be ofunderestimated. aroma components of raisins is formed by two odorous lactones derived from grapeThe lipids, third groupnamely of aromaγ-nonalactone components and ofmassoia raisins islactone. formed γ by-Nonalactone two odorous is lactones a well-known derived fromwine grape Thelipids, third namely group ofγ-nonalactoneγ aroma components and massoia of raisins lactone. is formed γ-Nonalactoneγ by two odorous is alactones well-known derived wine from grapecomponent lipids, [72] namely of coconut γ-nonalactone aroma andwhose massoia levels lactone.in wine γ-Nonalactonewere first tentatively is a well-known related towine the componentgrape lipids, [72] namely of coconut -nonalactone aroma whose and massoialevels inlactone. wine were-Nonalactone first tentatively is a well-known related to winethe componentdevelopment [72] of pruneof coconut character aroma by Ponswhose et levelsal. [80]. in The wine contribution were first to tentatively dry-fruit aromarelated has to beenthe developmentcomponent [72]of prune of coconut character aroma by Pons whose et al. levels [80]. Thein wine contribution were first to dry-fruittentatively aroma related has tobeen the developmentrecently shown of toprune happen character by perceptual by Pons interaction et al. [80]. withThe contributionfuraneol and tohomofu dry-fruitraneol aroma [81]. has Its levelsbeen recentlydevelopment shown of to prunehappen character by perceptual by Pons interaction et al. [80]. with The furaneol contribution and homofu to dry-fruitraneol aroma [81]. Its has levels been recentlyare increased shown in to wines happen made by fromperceptual grapes interaction affected by with Botrytis furaneol [78,79], and in homofu late harvestraneol wines [81]. Its[82], levels and arerecently increased shown in wines to happen made by from perceptual grapes affectedinteraction by Botrytiswith furaneol [78,79], and in latehomofu harvestraneol wines [81]. [82], Its levelsand arein wines increased made in from wines raisinized made from grapes grapes [64]. affected Remarkably, by Botrytis γ-nonalactone [78,79], in is late also harvest a constituent wines [82],of raisins and inare wines increased made fromin wines raisinized made fromgrapes grapes [64]. Remarkably,affected by Botrytis γ-nonalactoneγ [78,79], isin alsolate aharvest constituent wines of [82], raisins and in[57]; wines its levelmade andfrom fate raisinized much depends grapes [64]. on Remarkably,the type of grape, γ-nonalactone its pretreatment, is also a constituent time of storage of raisins and [57];in wines its level made and from fate raisinized much depends grapes on[64]. the Remarkably, type of grape, -nonalactone its pretreatment, is also a time constituent of storage of raisins and [57];packaging its level material and fate [58,83]. much Massoia depends lactone on the (5,6-d typeihydro-6-pentyl-2H-pyran-2-one) of grape, its pretreatment, time has of beenstorage recently and packaging[57]; its level material and [58,83].fate much Massoia depends lactone on (5,6-dthe typeihydro-6-pentyl-2H-pyran-2-one) of grape, its pretreatment, time has ofbeen storage recently and packagingidentified asmaterial key aroma [58,83]. component Massoia lactonein musts (5,6-d showingihydro-6-pentyl-2H-pyran-2-one) clear over-ripe characters of cooked has been plums recently and identifiedpackaging as material key aroma [58,83]. component Massoia in lactone musts showing(5,6-dihydro-6-pentyl-2H-pyran-2-one) clear over-ripe characters of cooked has been plums recently and identified as key aroma componentγ in musts showing clear over-ripe characters of cooked plums and identifieddried figs [84].as key Both aroma components, componentγ -nonalactone in musts showing and massoia clear over-ripe lactone, hacharactersve been foundof cooked at higher plums levels and drieddried figs figs [84]. [84]. Both Both components, components, -nonalactoneγ-nonalactone and and massoia massoia lactone, lactone, ha haveve been been found found at at higher higher levels levels dried figs [84]. Both components, γ-nonalactone and massoia lactone, have been found at higher levels Biomolecules 2019, 9, 818 8 of 35

The second group of powerful aroma compounds likely formed during grape dehydration are Strecker aldehydes derived from the Strecker degradation of amino acids. The most relevant from the aromatic point of view are phenylacetaldehyde (honey odor) and methional (raw potato odor), which are important aroma constituents of Pedro Ximenez wines made with sun-dried grapes [59]. Phenylacetaldehyde has been also found at levels well above its threshold in raisins [23,57]. The formation of these compounds can be particularly intense in the frequent case in which dehydration occurs after or during the attack of the fungus Botrytis cinerea [77–79], which explains the high levels of both compounds in wines from Sauternes. These compounds arise by the reaction of the amino acid precursor with a quinone or other α-dicarbonyl. In grapes, the major source of dicarbonyls is the quinones from oxidizing polyphenols. The oxidation can begin by photoactivation (normal raisining) or by enzymatic action, which will be particularly intense in the presence of the powerful phenol-oxidase from Botrytis (laccase). Recent results suggest that the formation may take place after some time of the solar irradiation, since in a study of the effects of the storage on raisin aroma, phenylacetaldehyde strongly accumulated only after 12 months of storage of sun-dried raisins but not in air-dried raisins [58]. These compounds are relatively difficult to analyze because of their high activity towards many chromatographic phases and because of the adducts they form with SO2. This explains why many reports fail in their detection, particularly in the case of methional, so that their importance may be underestimated. The third group of aroma components of raisins is formed by two odorous lactones derived from grape lipids, namely γ-nonalactone and massoia lactone. γ-Nonalactone is a well-known wine component [72] of coconut aroma whose levels in wine were first tentatively related to the development of prune character by Pons et al. [80]. The contribution to dry-fruit aroma has been recently shown to happen by perceptual interaction with furaneol and homofuraneol [81]. Its levels are increased in wines made from grapes affected by Botrytis [78,79], in late harvest wines [82], and in wines made from raisinized grapes [64]. Remarkably, γ-nonalactone is also a constituent of raisins [57]; its level and fate much depends on the type of grape, its pretreatment, time of storage and packaging material [58,83]. Massoia lactone (5,6-dihydro-6-pentyl-2H-pyran-2-one) has been recently identified as key aroma component in musts showing clear over-ripe characters of cooked plums and dried figs [84]. Both components, γ-nonalactone and massoia lactone, have been found at higher levels in wines made from partially dehydrated (raisinized) Shiraz grapes [65]. Massoia lactone has been also identified in the hydrolysates of phenolic and aromatic fractions (PAFs) extracted from grapes [15]. The fourth group of relevant aroma compounds formed during grape dehydration are some pyrazines with roasted aromas derived from Maillard reactions between sugars and amino acids. Wang et al. [23,57] identified at sensory-relevant levels 3-ethyl-2,5-dimethyl pyrazine and 2,6-diethylpyrazine. Both compounds were found to increase with storage of raisins [58]. Finally, and in common with any kind of grapes, raisins contain a relatively wide array of aldehydes and alcohols derived from the oxidation of grape fatty acids (FAOs). According to Wang et al. [23,57], pentanal, hexanal, heptanal, nonanal, decanal, (E)-2-hexenal, (E)-2-heptenal, (E)-2-octenal, (E)-2-nonenal, and 1-octen-3-ol can be found at levels above sensory thresholds. The effects of dehydration on aroma composition are strongly dependent on many factors poorly controlled, such as the previous physiological state of the grape or the environmental conditions. Such variability has been observed for terpenols [85]. There are reports in which no changes in these compounds are observed during dehydration [86], others in which dramatic decreases were seen [87], and even others in which slight increases were measured [37,88]. A similar degree of diversity of patterns was also identified in the case of β-damascenone. Increased levels of this component, and also of γ-nonalactone [64] and of massoia lactone [65], have been observed and related to the prevalence of prune and fig character of the wines made with partially raisinized grapes [80]. In contrast, other studies have shown that shriveled grapes did not produce wines with higher β-damascenone content [89]. In the case of Strecker aldehydes, levels formed will be likely strongly related to the levels of the amino acid precursors (methionine and phenylalanine) present in the grape. Biomolecules 2019, 9, 818 9 of 35

Regarding aldehydes and alcohols from FAOs, these compounds in general decrease during grape dehydration [56,86,88,90,91]. Such decreases may be attributed to a reduction in the lipoxygenase activity [86,90,91] of the raisinized grapes which cannot compensate for the general and continuous decrease of aldehydes by reaction with, among others, grape polyphenols.

2.3. Aroma Compounds Responsible for Vegetal and Green Aroma and Flavors There are two families of aroma compounds which play a role in the vegetal, herbaceous, and green–unripe characteristics of grapes, musts and, eventually, wine: alkylmethoxypyrazines along with aldehydes and alcohols derived from the oxidation of fatty acids, or fatty acid oxidation-derived odorants (FAOs). Alkylmethoxypyrazines are extremely powerful aroma molecules which accumulate in some grapes. They were first found in wines from Cabernet Sauvignon [92] and were further identified in Sauvignon Blanc juices and wines [93]. These compounds are 3-isobutyl-2-methoxypyrazine (IBMP), 3-secbutyl-2-methoxypyrazine (SBMP), and 3-isopropyl-2-methoxypyrazine (IPMP). Their properties are listed in Table3. These compounds accumulate preferably in fruits grown under cool conditions and their levels decrease during ripening. They have been blamed for the specific green bell pepper character associated with Cabernet varieties, with a threshold for this character estimated to be just 15 ng/L[94]. Carmenere wines, which also belong to the Cabernet family, contain large amounts of these compounds too. Levels of IBMP were found to be strongly affected by climatic conditions and by vine genotype [95]. Temperatures during spring were found to be an important driver of green characters [96]. Levels of IBMP have been also positively related to altitude [97] and negatively related to light exposure, which limits accumulation but does not promote degradation [98]. Consequently, leaf removal significantly reduces accumulation of IBMP but only if it is carried out before veraison [99]. The relationship with nitrogen fertilization seems to be indirect, through the higher vigor [100]. Anecdotally, huge levels of IPMP can be induced by some foreign ladybeetles, causing great concern [101]. The levels of these compounds in wines from Spain and other southern countries are very low. It should be remarked,Biomolecules however, 2019, 9 that, 818 strong negative correlations between the levels of these compounds—notably10 of 36 IBMP—andBiomolecules thedi 2019fferent, 9, 818 fruity and liquorice attributes of wines have been found in a recent10 of work 36 [102]. Such negativewines have correlation been found would in a recent suggest work that[102]. these Such negative compounds correlation could would be relevant suggest that suppressors these at winescompounds have beencould found be relevant in a recent suppres worksors [102]. at subthreshold Such negative level. correlation would suggest that these subthresholdcompounds level. could be relevant suppressors at subthreshold level. Table 3. Structures, odor properties, and occurrence of alkylmethoxypyrazines. TableTable 3. Structures, 3. Structures, odor odor properties, properties, andand oc occurrencecurrence of ofalkylmethoxypyrazines. alkylmethoxypyrazines. Range of Odor Range of Range of Compound Structure Odor Odor Threshold Occurrence in CompoundCompound Structure OdorDescriptor Descriptor Odor Threshold Odor Threshold OccurrenceOccurrence in in Descriptor Grape Juice Grape JuiceGrape Juice 2 ng/L (in water) 2 ng/L (in water) 3-Isobutyl-2- Bell pepper, 2 ng/L[103]; (in water) 3-Isobutyl-2- Bell pepper, [103]; n.d. to 79 ng/L [93] methoxypyrazine3-Isobutyl-2- Bellearthy pepper, 15 ng/L2[103]; ng (in/L wine) (in water) [n.d.103]; to 79 ng/L [93] 3-Isobutyl-2-methoxypyrazinemethoxypyrazine Bellearthy pepper, earthy15 ng/L (in wine) n.d. to 79n.d. ng/L to [93] 79 ng/L[93] methoxypyrazine earthy 15 ng/L15[94] (in ng /wine)L (in wine) [94] [94]

0.74–1.11 (hybrid 3-Isopropyl-2- Green pea, 0.74–1.110.74–1.11 (hybrid (hybrid grape 3-Isopropyl-2- Green pea, grape0.74–1.11 juice) (hybrid [104]; n.d. to 6.8 ng/L [93] 3-Isopropyl-2-methoxypyrazinemethoxypyrazine3-Isopropyl-2- GreenGreenearthy pea, pea, earthy grape juice)juice) [104]; [104 ];n.d. to 6.8n.d. ng/L to [93] 6.8 ng/L[93] methoxypyrazine earthy grape juice) [104]; n.d. to 6.8 ng/L [93] methoxypyrazine earthy 2 ng/L in2 ngwine/L in[105] wine [105] 2 ng/L in wine [105]

3-Secbutyl-2- 1–2 ng/L (in water) 3-Secbutyl-2-methoxypyrazine3-Secbutyl-2- BellBell pepper pepper 1–2 ng/L 1–2 ng(in/ Lwater) (in water) n.d. [106 to] 1.3n.d. ng/L to [93] 1.3 ng/L[93] methoxypyrazine Bell pepper [106] n.d. to 1.3 ng/L [93] methoxypyrazine [106]

n.d.: n.d.: NotNot detected.detected. n.d.: Not detected. The second family of compounds is formed by a relatively large number of aroma compounds, The second family of compounds is formed by a relatively large number of aroma compounds, most of them aldehydes, derived from the oxidation of fatty acids or FAOs. Since quantitatively the most ofabundant them aldehydes, were C6 derivedalcohols from and thealdehydes, oxidation the of fattyfamily acids was or first FAOs. referred Since quantitativelyas the C6-family, the most abundant were C6 alcohols and aldehydes, the family was first referred as the C6-family, however, some of the most powerful aroma compounds have nine carbon atoms, such as E-2-nonenal however,or (E,Z)-2,6-nonadienal. some of the most For powerful instance, aroma the most compound relevants have aroma nine compound carbon atom ofs, Cabernet such as E-2-nonenal Sauvignon or (E,Z)-2,6-nonadienal. For instance, the most relevant aroma compound of Cabernet Sauvignon must, as assessed by aroma extract dilution analysis was (E,Z)-2,6-nonadienal [107]. Chemical must,structures as assessed and basic by properties aroma extract of these dilution compound analysiss are givenwas (E,Z)-2,6-nonadienalin Table 4. This group [107]. of compounds Chemical structures and basic properties of these compounds are given in Table 4. This group of compounds derives from the enzymatic oxidation of fatty acids during must processing [22] and are well-known derivesfor the green from odorthe enzymatic of green leaves oxidation particularly of fatty acidsevident during in some must teas processing [21]. The [22]most and powerful are well-known in aroma for the green odor of green leaves particularly evident in some teas [21]. The most powerful in aroma are the aldehydes, as usual, which have odor thresholds hundreds of times smaller than those of the arecorresponding the aldehydes, alcohols. as usual, These which aldehydeshave odor threshare suoldsrely hundreds responsible of times for smaller the herbaceous than those ofnote the corresponding alcohols. These aldehydes are surely responsible for the herbaceous note characteristics of some musts, particularly of thosethose producedproduced fromfrom unripeunripe grapes.grapes. However,However, characteristicsaldehydes are mostlyof some eliminated musts, particularlyduring fermentati of thoseon, inproduced which they from are unripe enzymatically grapes. reducedHowever, to aldehydes are mostly eliminated during fermentation, in which they are enzymatically reduced to the corresponding alcohols. Consequently, the role ofof thethe familyfamily onon thethe greengreen andand herbaceousherbaceous the(negative) corresponding aroma characteristics alcohols. Consequently, of wines has theyet torole be clearlyof the demonstrated.family on the FAOgreen odorants and herbaceous decrease (negative) aroma characteristics of wines has yet to be clearly demonstrated. FAO odorants decrease with maturity. Their levels are strongly related to grape variety [108] and also to the position in the withbunch maturity. [109], being Their richer levels in arethe stronglyshoulder. related to grape variety [108] and also to the position in the bunch [109], being richer in the shoulder.

1 Table 4. Structures, odor properties, and occurrence of FAO-related 1 family of compounds. Table 4. Structures, odor properties, and occurrence of FAO-related 1 family of compounds. Ranges of Occurrence in Odor Threshold in Ranges of Occurrence in Compound Structure Odor Threshold in Grape [23,25,57,61,110– Compound Structure Descriptor Water Grape [23,25,57,61,110– Descriptor Water 112] 112] Hexanal Herbaceous 5 μg/L [113] 8–1300 μg/kg Hexanal Herbaceous 5 μg/L [113] 8–1300 μg/kg

0.25 μg/L (Z)-3-Hexenal Grass 0.25 μg/L 4–20 μg/kg (Z)-3-Hexenal Grass [48] 4–20 μg/kg [48]

Biomolecules 2019, 9, 818 10 of 36 Biomolecules 2019, 9, 818 10 of 36 wines have been found in a recent work [102]. Such negative correlation would suggest that these wines have been found in a recent work [102]. Such negative correlation would suggest that these compounds could be relevant suppressors at subthreshold level. compounds could be relevant suppressors at subthreshold level.

Table 3. Structures, odor properties, and occurrence of alkylmethoxypyrazines. Table 3. Structures, odor properties, and occurrence of alkylmethoxypyrazines. Range of Odor Range of Compound Structure Odor Odor Threshold Occurrence in Compound Structure Descriptor Odor Threshold Occurrence in Descriptor Grape Juice Grape Juice 2 ng/L (in water) 2 ng/L (in water) 3-Isobutyl-2- Bell pepper, [103]; 3-Isobutyl-2- Bell pepper, [103]; n.d. to 79 ng/L [93] methoxypyrazine earthy 15 ng/L (in wine) n.d. to 79 ng/L [93] methoxypyrazine earthy 15 ng/L (in wine) [94] [94]

0.74–1.11 (hybrid 3-Isopropyl-2- Green pea, 0.74–1.11 (hybrid 3-Isopropyl-2- Green pea, grape juice) [104]; n.d. to 6.8 ng/L [93] methoxypyrazine earthy grape juice) [104]; n.d. to 6.8 ng/L [93] methoxypyrazine earthy 2 ng/L in wine [105] 2 ng/L in wine [105]

3-Secbutyl-2- 1–2 ng/L (in water) 3-Secbutyl-2- Bell pepper 1–2 ng/L (in water) n.d. to 1.3 ng/L [93] methoxypyrazine Bell pepper [106] n.d. to 1.3 ng/L [93] methoxypyrazine [106]

Biomolecules 2019, 9, 818 10 of 35

n.d.: Not detected. n.d.: Not detected. The second family of compounds is formed by a relatively large number of aroma compounds, TheThe second second familyfamily of compounds is is formed formed by by a relatively a relatively large large number number of aroma of aroma compounds, compounds, most of them aldehydes, derived from the oxidation of fatty acids or FAOs. Since quantitatively the mostmost of of them them aldehydes,aldehydes, derived derived from from the the oxidation oxidation of fatty of fattyacids acidsor FAOs. or FAOs.Since quantitatively Since quantitatively the most abundant were C6 alcohols and aldehydes, the family was first referred as the C6-family, themost most abundant abundant were were C6 C6alcohols alcohols and and aldehydes, aldehydes, the family the family was wasfirst firstreferred referred as the as C6-family, the C6-family, however, some of the most powerful aroma compounds have nine carbon atoms, such as E-2-nonenal however,however, some some of of the the mostmost powerful aroma aroma compound compoundss have have nine nine carbon carbon atom atoms,s, such such as E-2-nonenal as E-2-nonenal or (E,Z)-2,6-nonadienal. For instance, the most relevant aroma compound of Cabernet Sauvignon oror (E,Z)-2,6-nonadienal. (E,Z)-2,6-nonadienal. For For instance, instance, the the most most relevant relevant aroma aroma compound compound of of Cabernet Cabernet Sauvignon Sauvignon must, must, as assessed by aroma extract dilution analysis was (E,Z)-2,6-nonadienal [107]. Chemical asmust, assessed as assessed by aroma by extract aroma dilution extract analysisdilution was analysis (E,Z)-2,6-nonadienal was (E,Z)-2,6-nonadienal [107]. Chemical [107]. structuresChemical and structures and basic properties of these compounds are given in Table 4. This group of compounds basicstructures properties and basic of these properties compounds of these are compound given in Tables are given4. This in group Table of4. This compounds group of derivescompounds from the derives from the enzymatic oxidation of fatty acids during must processing [22] and are well-known derives from the enzymatic oxidation of fatty acids during must processing [22] and are well-known enzymaticfor the green oxidation odor of of green fatty leaves acids particularly during must evi processingdent in some [22 teas] and [21]. are The well-known most powerful for the in aroma green odor for the green odor of green leaves particularly evident in some teas [21]. The most powerful in aroma ofare green the leavesaldehydes, particularly as usual, evidentwhich have in some odor teasthresh [21olds]. The hundreds most powerful of times smaller in aroma than are those the of aldehydes, the are the aldehydes, as usual, which have odor thresholds hundreds of times smaller than those of the ascorresponding usual, which havealcohols. odor These thresholds aldehydes hundreds are su ofrely times responsible smaller than for thosethe herbaceous of the corresponding note corresponding alcohols. These aldehydes are surely responsible for the herbaceous note alcohols.characteristics These aldehydesof some musts, are surely particularly responsible of forthose the produced herbaceous from note unripe characteristics grapes. However, of some musts, characteristics of some musts, particularly of those produced from unripe grapes. However, particularlyaldehydes ofare those mostly produced eliminated from during unripe fermentati grapes.on, However, in which aldehydes they are enzymatically are mostly eliminated reduced to during aldehydes are mostly eliminated during fermentation, in which they are enzymatically reduced to fermentation,the corresponding in which alcohols. they areConsequently, enzymatically the reduced role of tothe the family corresponding on the green alcohols. and herbaceous Consequently, the corresponding alcohols. Consequently, the role of the family on the green and herbaceous (negative) aroma characteristics of wines has yet to be clearly demonstrated. FAO odorants decrease the(negative) role of the aroma family characteristics on the green of andwines herbaceous has yet to be (negative) clearly demonstrated. aroma characteristics FAO odorants of wines decrease has yet to with maturity. Their levels are strongly related to grape variety [108] and also to the position in the bewith clearly maturity. demonstrated. Their levels FAO are strongly odorants related decrease to grape with variety maturity. [108] Their and also levels to arethe position strongly in related the to bunch [109], being richer in the shoulder. grapebunch variety [109], being [108] andricher also in the to theshoulder. position in the bunch [109], being richer in the shoulder.

Table 4. Structures, odor properties, and occurrence of FAO-related 1 family1 of compounds. TableTable 4. 4.Structures, Structures, odor properties, properties, and and occurrence occurrence of ofFAO-related FAO-related 1 familyfamily of compounds. of compounds. Ranges of Occurrence in Odor Threshold in Ranges ofRanges Occurrence of Occurrence in Compound Structure Odor ThresholdThreshold in inGrape [23,25,57,61,110– CompoundCompound Structure OdorDescriptor Descriptor Water Grape [23,25,57,61,110–in Grape Descriptor WaterWater 112] [23112],25 ,57,61,110–112] BiomoleculesHexanal 2019 , 9, 818 Herbaceous 5 μg/L [113] 8–1300 μg/kg 11 of 36 Biomolecules 2019, 9, 818 µ µ11 of 36 BiomoleculesHexanalHexanal 2019 , 9, 818 HerbaceousHerbaceous 5 μg/L 5 [113]g/L[ 113] 8–1300 μ 8–1300g/kg 11g /ofkg 36 Biomolecules 2019, 9, 818 11 of 36 Biomolecules 2019, 9, 818 11 of 36 Biomolecules 2019, 9, 818 0.25 μg/L 11 of 36 Biomolecules(E)-2-Hexenal(Z)-3-Hexenal(Z)-3-Hexenal 2019 , 9, 818 GrassGrass 170.25 μg/L 0.25 μ g/L[113]µ g/ L[48] 13–38004–20 μ g/kgμ 4–20g/kg 11 µg /ofkg 36 (Z)-3-Hexenal(E)-2-Hexenal GrassGrass 17 [48]μg/L [113] 4–20 13–3800 μg/kg μg/kg (E)-2-Hexenal Grass 17 μ[48]g/L [113] 13–3800 μg/kg (E)-2-Hexenal Grass 17 μg/L [113] 13–3800 μg/kg (E)-2-Hexenal Grass 17 μg/L [113] 13–3800 μg/kg (E)-2-Hexenal Grass 17 μg/L [113] 13–3800 μg/kg (E)-2-Hexenal(E)-2-Hexenal(E,E)-2,4- GrassGrass 17 μg/L 17 [113]µg/L[ 113] 13–3800 13–3800 μg/kg µg/kg (E,E)-2,4- Grass 60 μg/L [114] 50–120 μg/kg Hexadienal(E,E)-2,4- Grass 60 μg/L [114] 50–120 μg/kg (E,E)-2,4-Hexadienal Grass 60 μg/L [114] 50–120 μg/kg (E,E)-2,4-Hexadienal Grass 60 μg/L [114] 50–120 μg/kg Hexadienal(E,E)-2,4- Grass 60 μg/L [114] 50–120 μg/kg (E,E)-2,4-HexadienalHexadienal(E,E)-2,4- GrassGrass 60 μg/L 60 [114]µg/L[ 114] 50–120 μ 50–120g/kg µg/kg Hexadienal Grass 60 μg/L [114] 50–120 μg/kg (Z)-3-HexenolHexadienal Grass 70 μg/L[48] 4–79 μg/kg (Z)-3-Hexenol Grass 70 μg/L[48] 4–79 μg/kg (Z)-3-Hexenol Grass 70 μg/L[48] 4–79 μg/kg (Z)-3-Hexenol Grass 70 μg/L[48] 4–79 μg/kg (Z)-3-Hexenol(Z)-3-Hexenol GrassGrass 70 μg/L[48] 70 µg/ L[48]4–79 μg/kg 4–79 µg/kg (Z)-3-Hexenol Grass 70 μg/L[48] 4–79 μg/kg (Z)-3-Hexenol Grass 70400 μg/L[48] μg/L 4–79 μg/kg (E)-2-Hexenol Green 400 μg/L (E)-2-Hexenol Green 400[114] μg/L (E)-2-Hexenol Green 400 μ[114]g/L (E)-2-Hexenol(E)-2-Hexenol GreenGreen 400 400[114] μg/Lµ g /L[114] (E)-2-Hexenol Green 400[114] μg/L (E)-2-Hexenol Green 2500400[114] μμg/Lg/L (E)-2-Hexenol1-Hexanol Green 2500[114] μ g/L 45–214 μg/kg 1-Hexanol Green 2500[113][114] μ g/L 45–214 μg/kg 1-Hexanol1-Hexanol GreenGreen 2500 2500 [113]μg/Lµg /L[113]45–214 45–214 μg/kg µ g/kg 1-Hexanol Green 2500[113] μg/L 45–214 μg/kg 1-Hexanol Green 25000.17[113] μμg/Lg/L 45–214 μg/kg E-2-Nonenal1-Hexanol Green,Green fatty 25000.17[113] μ g/Lμ g/L 45–214 μg/kg E-2-Nonenal1-HexanolE-2-Nonenal Green,Green,Green fatty fatty 0.17[113] 0.17 μ g/Lµg/ L[113] 45–214 μ g/kg E-2-Nonenal Green, fatty 0.17[113] [113]μg/L E-2-Nonenal Green, fatty 0.17[113] μg/L E-2-Nonenal Green, fatty 0.17[113] μg/L E-2-Nonenal Green, fatty 0.17[113] μg/L E-2-Nonenal Green, fatty [113] (E,Z)-2,6- 0.01[113] μg/L (E,Z)-2,6-Nonadienal(E,Z)-2,6- CucumberCucumber 0.01 0.01 μµg/Lg/L[ 115]113–482 113–482μg/kg µg/kg Nonadienal(E,Z)-2,6- Cucumber 0.01[115] μ g/L 113–482 μg/kg (E,Z)-2,6-Nonadienal Cucumber 0.01 [115]μg/L 113–482 μg/kg (E,Z)-2,6-Nonadienal Cucumber 0.01[115] μg/L 113–482 μg/kg Nonadienal(E,Z)-2,6- Cucumber 0.01[115] μg/L 113–482 μg/kg Nonadienal(E,Z)-2,6- Cucumber 0.01[115] μg/L 113–482 μg/kg Nonadienal 1 FAO:1 FAO: Fatty FattyCucumber acid acid oxidation oxidation. [115] 113–482 μg/kg Nonadienal 1 FAO: Fatty acid oxidation[115] 1 FAO: Fatty acid oxidation 1 FAO: Fatty acid oxidation The vegetal aromas of Cabernet Sauvignon1 FAO: Fatty and acid other oxidation wines are, however, much more complex TheThe vegetal vegetal aromas aromas of of Cabernet Cabernet1 Sauvignon FAO:Sauvignon Fatty acid and oxidation other wines wines are, are, however, however, much much more more complex complex and cannotThe vegetal be completely aromas ofexplained Cabernet just 1Sauvignon FAO: by Fattyanalyzing acidand oxidationother IPMP wines and IBMPare, however, [116], or muchC6-alcohols. more complex While andand cannotThe cannot vegetal be be completely completelyaromas of explained Cabernet explained Sauvignon just just by by analyzinganalyzing and other IPMP wines and and are, IBMP IBMP however, [116], [116 much ],or or C6-alcohols. 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Sonomamajor between Inchallenge and fact,the IBMPsensory a for comprehensive winelevels, vegetative science more aromarecentunderstanding notesreports of Cabernethave of the not green Sauvignonbeen and unripeable grapes to characters find grown an yof in correlationwines five sitesremains of[116]. Sonoma a major In challengefact,and IBMPa comprehensive for levels, wine morescience today.recentaromaunderstanding Preliminarynotesreports of Cabernethaveof the reports greennot Sauvignon been fromand unripe ableour grapes groupto characters find grownsuggest an ofy inwines correlationthat five (a) remainssites C6-alcohols of[116]. aSonoma major In togetherchallenge fact,and IBMPa withcomprehensive for levels, wine IBMP science more can recentunderstandingtoday. reports Preliminary ofhave the greenreportsnot andbeen from unripe able our charactersto group find suggestan ofy wines correlation that remains (a) C6-alcohols[116]. a major In challenge fact,together a forcomprehensive with wine IBMP science can impartunderstandingrecenttoday. herbaceous reportsPreliminary ofhave the notes greenreportsnot to andbeenred from unripewine able our [117]; characterstogroup find(b) thesuggest an of concertedy wines correlation that remains (a)action C6-alcohols [116]. a of major hexanol, In challenge togetherfact, the amajor for comprehensivewith wine C6 IBMP alcohol, science can understandingtoday.impart Preliminary herbaceous of the reports notesgreen toand from red unripe wineour group characters[117]; (b)suggest the of winesconcerted that remains (a) actionC6-alcohols a major of hexanol, challengetogether the formajorwith wine IBMP C6 science alcohol, can withtoday.understandingimpart dimethyl Preliminary herbaceous sulfide of the reportsnotes green and methanethiol,to andfrom red unripe wine our [117];groupcharacters opposed (b) suggest the ofto concerted winesthe that action remains(a) action ofC6-alcohols acetaldehyde a majorof hexanol, challengetogether and the linear major forwith wine fatty IBMPC6 sciencealcohol, acids, can today.impartwith dimethylPreliminaryherbaceous sulfide notesreports and to redfrommethanethiol, wine our [117]; group opposed (b) suggest the concerted to thatthe action (a) action C6-alcohols of acetaldehydeof hexanol, together the and major withlinear C6 IBMP fatty alcohol, acids,can couldimparttoday.with bedimethyl Preliminaryherbaceous related sulfide to the notesreports vegetaland to methanethiol,redfrom character wine our [117]; group of opposed wine(b) suggest the [70]. concerted to the that action (a) action C6-alcohols of acetaldehyde of hexanol, together the and major linearwith C6 IBMPfatty alcohol, acids, can impartwithcould dimethyl herbaceous be related sulfide to notes the and vegetalto methanethiol, red wine character [117]; opposed of (b) wine the toconcerted[70]. the action action of acetaldehyde of hexanol, andthe major linear C6fatty alcohol, acids, withimpartcould Theredimethyl beherbaceous related is alsosulfide to notesstrong the and vegetal to methanethiol,evidence red winecharacter demonstrating[117]; opposed of (b) wine the [70]. toconcerted theth e action implication action of acetaldehyde of hexanol,of 1,8-cineole, andthe majorlinear a terpineol C6fatty alcohol, acids, of withcould dimethyl beThere related is sulfide alsoto the strongand vegetal methanethiol, evidence character demonstrating ofopposed wine [70]. to the th actione implication of acetaldehyde of 1,8-cineole, and linear a fattyterpineol acids, of eucalyptuscouldwith dimethyl Therebe related odor, is sulfidealso to in the thestrong and vegetal green methanethiol, evidence characterand minty demonstrating ofopposed characterswine [70]. to the of th actionwine.e implication ofIn acetaldehydemany of instances, 1,8-cineole, and thelinear origina terpineolfatty of acids, this of couldeucalyptusThere be related is odor,also to thestrongin thevegetal greenevidence character and demonstratingminty of wine characters [70]. th eof implicationwine. In many of 1,8-cineole, instances, thea terpineolorigin of ofthis moleculecouldeucalyptusThere be relatedis isexogenous, odor, also to inthestrong the vegetal coming green evidence character fromand mintyleavesdemonstrating of wine charactersof Eucalyptus [70]. th ofe treesimplicationwine. [118] In many or of from 1,8-cineole,instances, invasive the plants,a originterpineol such of thisasof eucalyptusmoleculeThere isodor, exogenous,also in strong the green coming evidence and from minty demonstrating leaves characters of Eucalyptus thofe wine.implication trees In [118] many ofor instances, from1,8-cineole, invasive the a originplants,terpineol of such this of as ArtemisiaeucalyptusmoleculeThere verlotiorum is odor,is exogenous, also in strong the[119]. greencoming evidenceHighest and from mintylevels demonstrating leaves charactersare of related Eucalyptus th oftoe wine.theimplication trees presence In [118] many or ofof frominstances, the1,8-cineole, Eucalyptusinvasive the a plants,origin terpineolleaves ofsuch or this of as eucalyptusmoleculeArtemisia is verlotiorumodor,exogenous, in the [119].coming green Highest and from minty leaves levels characters ofare Eucalyptus related of towine. trees the Inpresence[118] many or from instances,of the invasive Eucalyptus the plants, origin leaves suchof thisor as of smallmoleculeeucalyptusArtemisia quantities is verlotiorum odor,exogenous, of in the the [119].plant coming green inHighest the andfrom fermentation minty leaveslevels characters areof Eucalyptus related tanks, of butto wine. treesthe the presence moleculeIn[118] many or from ofcaninstances, the accumulateinvasive Eucalyptus the plants, origin in leaves the such of berry orthis as of moleculeArtemisiasmall quantities isverlotiorum exogenous, of the[119]. coming plant Highest in from the levels fermentationleaves are of Eucalyptusrelated tanks, to but thetrees thepresence [118] molecule or offrom the can invasiveEucalyptus accumulate plants, leaves in suchthe or berry asof skinArtemisiamoleculesmall at sensorilyquantities isverlotiorum exogenous, relevant of the [119]. comingplant levels Highest in [120]. fromthe fermentationlevels leavesAdditionally, are of relatedEucalyptus ta nks,recent to but thetrees evidence the presence [118] molecule has or of fromshown thecan invasive Eucalyptusaccumulate that the plants, molecule leaves in the such or berry can asof Artemisiasmallskin quantities at sensorilyverlotiorum of relevantthe [119]. plant levelsHighest in the [120]. fermentation levels Additionally, are related tanks, recent to but the the evidencepresence molecule has of can theshown accumulateEucalyptus that the leaves inmolecule the berryor ofcan besmallArtemisiaskin found atquantities sensorily verlotiorumin unripe of relevant the berries[119]. plant levels Highest inof the Cabernet[120]. fermentation levels Additionally, Sauvignonare related tanks, recent andto but the theMerlotevidence presence molecule [1 has19], of can shownthecontributing accumulate Eucalyptus that the to moleculeleavesin the the green berryor canof smallskinbe atfound quantities sensorily in unripe ofrelevant the plantberries levels in the of[120]. Cabernet fermentation Additionally, Sauvignon tanks, recent but and evidencethe Merlot molecule has [119], shown can contributing accumulate that the molecule into the the berry greencan perceptionskinsmallbe foundat quantities sensorily viain unripeperceptual ofrelevant the berriesplant levels interaction in of the[120]. Cabernet fermentation Additionally,with IBMP.Sauvignon ta nks,A recent third butand evidence formationthe Merlot molecule has[1 route19], shown can contributingof accumulate 1,8-cineole that the molecule toin in thethe wine berrygreen can as skinbeperception found at sensorily in viaunripe relevantperceptual berries levels interactionof [120].Cabernet Additionally, with Sauvignon IBMP. recent A andthird evidenceMerlot formation [1 has19], routeshown contributing of that 1,8-cineole the moleculeto the in winegreen can as productbeskinperception found at sensorily of inthe viaunripe reaction perceptualrelevant berries of levelslimonene interaction of [120].Cabernet and Additionally, with othe Sauvignon rIBMP. terpenols recentA thirdand has evidenceMerlot formationbeen also [1 has19], reported route shown contributing of that[66,121].1,8-cineole the moleculeto thein winegreen can as beperceptionproduct found ofin via theunripe perceptual reaction berries of interaction limonene of Cabernet andwith otheSauvignon IBMP.r terpenols A third and has formationMerlot been [1also 19],route reported contributing of 1,8-cineole [66,121]. to inthe wine green as perceptionbeproduct found ofin via the unripe perceptual reaction berries of interactionlimonene of Cabernet and with othe Sauvignon IBMP.r terpenols A third and has formationMerlot been also[1 19],route reported contributing of 1,8-cineole [66,121]. to inthe wine green as perceptionproduct of thevia reactionperceptual of limoneneinteraction and with othe IBMP.r terpenols A third has formation been also routereported of 1,8-cineole [66,121]. in wine as 2.4.productperception Compounds of thevia reactionperceptualResponsible of limonene forinteraction the Flavor and with of othe Neutral IBMP.r terpenols Grapes A third has formation been also routereported of 1,8-cineole [66,121]. in wine as product2.4. Compounds of the reaction Responsible of limonene for the Flavor and othe of Neutralr terpenols Grapes has been also reported [66,121]. product2.4. Compounds of the reaction Responsible of limonene for the Flavor and otheof Neutralr terpenols Grapes has been also reported [66,121]. 2.4. CompoundsThe subtle Responsibleflavor of neutral for the grapesFlavor ofis Neutralthe conseq Grapesuence of the presence of very small amounts of 2.4. 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Monastrell,solid of somephase In fact,Tempranillo,extraction studies performed onlyAglianico, find onat or thequantifiable Uva aroma di compositionTroia, levels using C6 of compoundsdirect neutral liquid–liquid varietals, together such extractionwith as Grenache, minor or levels solid Monastrell, ofphase some Tempranillo,fact,extraction studies onlyperformed Aglianico, find aton orquantifiable the Uva aroma di compositionTroia, levels using C6 compoundsof direct neutral liquid–liquid varietals, together such withextraction as Grenache,minor or levels solid Monastrell, of phase some Tempranillo,extraction only Aglianico, find at quantifiableor Uva di Troia,levels usingC6 compounds direct liquid–liquid together with extraction minor orlevels solid of phasesome extractionTempranillo, only Aglianico, find at quantifiableor Uva di Troia,levels usingC6 compounds direct liquid–liquid together with extraction minor orlevels solid of phasesome extraction only find at quantifiable levels C6 compounds together with minor levels of some extraction only find at quantifiable levels C6 compounds together with minor levels of some Biomolecules 2019, 9, 818 11 of 35 green and unripe characters of wines remains a major challenge for wine science today. Preliminary reports from our group suggest that (a) C6-alcohols together with IBMP can impart herbaceous notes to red wine [117]; (b) the concerted action of hexanol, the major C6 alcohol, with dimethyl sulfide and methanethiol, opposed to the action of acetaldehyde and linear fatty acids, could be related to the vegetal character of wine [70]. There is also strong evidence demonstrating the implication of 1,8-cineole, a terpineol of eucalyptus odor, in the green and minty characters of wine. In many instances, the origin of this molecule is exogenous, coming from leaves of Eucalyptus trees [118] or from invasive plants, such as Artemisia verlotiorum [119]. Highest levels are related to the presence of the Eucalyptus leaves or of small quantities of the plant in the fermentation tanks, but the molecule can accumulate in the berry skin at sensorily relevant levels [120]. Additionally, recent evidence has shown that the molecule can be found in unripe berries of Cabernet Sauvignon and Merlot [119], contributing to the green perception via perceptual interaction with IBMP. A third formation route of 1,8-cineole in wine as product of the reaction of limonene and other terpenols has been also reported [66,121].

2.4. Compounds Responsible for the Flavor of Neutral Grapes The subtle flavor of neutral grapes is the consequence of the presence of very small amounts of a relatively large list of aroma compounds. The list includes nearly all the aroma compounds described in the three previous subsections, the difference being that neutral grapes do not contain any odorant at the concentrations at which it can be regarded to act as impact aroma compound. In fact, studies performed on the aroma composition of neutral varietals, such as Grenache, Monastrell, Tempranillo, Aglianico, or Uva di Troia, using direct liquid–liquid extraction or solid phase extraction only find at quantifiable levels C6 compounds together with minor levels of some hydrocarbons, alcohols, ketones, esters, and terpenes [122–125]. Methods using SPME can more easily find other nonpolar volatiles, because of its intrinsic higher concentration power [126], but at the expense of losing the most polar and less volatile ones, such as furaneol or vanillin derivatives. Many neutral grapes contain low amounts of free furaneol, limonene, linalool, geraniol and other terpenols, β-damascenone, β-ionone and other norisoprenoids, and also of ethyl esters, such as ethyl butyrate, ethyl hexanoate, some volatile phenols, and vanillin derivatives. All these compounds, together with FAO derivatives, contribute concertedly to the subtle fruity flavor of neutral grapes. For instance, in one of the few works published about the gas chromatography-olfactometric (GCO) profiles of neutral grapes, the most relevant odorants were β-damascenone, β-ionone, ethyl hexanoate, ethyl octanoate, and different FAO derivatives (hexanal, decanal, and (E,Z)-2,6-nonadienal) [25]. With no impact aroma compound present, but with a relatively wide array of fruity–sweet–citric–flowery smelling aroma compounds present at low levels, there is a perceptual cooperation between all of them as described by Loscos et al. [127], whose outcome is a subtle sweet–fruity flavor. There is also some evidence that neutral grapes of specific varieties contain eventually sensorily-relevant levels of rotundone. Rotundone is a sesquiterpene that is also present in grapes and can give a peppery aroma to grapes and wines [128]. In certain varieties, like Shiraz or Duras, and under favorable agronomical conditions [129,130], rotundone can accumulate in the berry exocarp in levels in the order of 600 ng/kg [128]. The synthesis pathway of rotundone in grape is not clear, but α-guaiene has been proposed as a potential precursor [131]. During the red wine winemaking maceration process, rotundone is extracted and can reach levels well above its perception threshold of 16 ng/L[128,132]. This characteristic peppery aroma is usually perceived positively among wine consumers [133]. Following the idea of aromatic series proposed by different authors [29,31,125], it can be stated that the aroma of neutral grapes is the consequence of the concerted action of 25–30 aroma compounds, with aroma nuances classifiable into seven odor categories:

1. Fruity: ethyl isobutyrate, ethyl butyrate, ethyl 3-methylbutyrate, ethyl hexanoate, ethyl octanoate, and eventually others; Biomolecules 2019, 9, 818 12 of 35

2. Jammy, very sweet fruit: furaneol, homofuraneol, β-damascenone, γ-nonalactone, and massoia lactone; 3. Sweet–floral: vanillin, ethyl vanillate, β-ionone, β-phenylethyl acetate, and phenylacetaldehyde; 4. Floral–citric aroma compounds: linalool, geraniol, limonene, nonanal, and eventually others; 5. Herbaceous: hexanal, (Z)-3-hexenal, (E)-2-hexenal, (Z)-3-hexenol, (E)-2-nonenal, (E,Z)-2,6- nonadienal; 6. Peppery: rotundone; 7. Unspecific: 3-methylbutanal, ethyl acetate, diacetyl.

3. Grape Potential Aroma: Specific Aroma Precursors

3.1. Specific vs. Unspecific Precursors Grape specific aroma precursors are non-volatile and hence odorless molecules which may rend a specific odoriferous molecule by the hydrolysis of a chemical bond, by spontaneous chemical rearrangement, or by a combination of both mechanisms. Many grape and grape-derived wine aroma molecules have specific aroma precursors. Remarkably, some of them have a relatively complex pool of different “specific precursors”. This is common in nature; for instance, apples contain more than eight different non-volatile molecules which by hydrolysis and further chemical rearrangement lead to β-damascenone [134]. A higher level of complexity regarding the number and type of precursor molecules is found in grapes. Such a pool of molecules is the pool of β-damascenone precursors. Similarly, there is a pool of precursors for linalool, for geraniol, for (Z)-rose oxide, for β-ionone, for furaneol, for TDN, for 3-mercaptohexanol, and for many other relevant grape-derived wine aroma compounds. The word specific has an important meaning here. “Specific” means that the aroma compound will be formed by simple incubation of the pool of precursors extracted from grape at normal wine pH, or alternatively, by incubation in the presence of an enzyme. This definition deliberately excludes those precursor molecules which can be transformed into aroma compounds only by a complex metabolic action of yeast, bacteria or other micro-organisms. For instance, the amino acid isoleucine can be metabolized by Saccharomyces producing as byproducts isovaleric acid, isoamyl alcohol and isoamyl acetate. But isoleucine cannot be regarded as a specific precursor for these important aroma compounds, because their final levels are extraordinarily constrained by the metabolic requirements of yeast. In fact, yeast is able to produce all those compounds even if there is no isoleucine in the fermentation media. We rather should consider it as an unspecific precursor of the aroma molecule. This differentiation has a paramount importance for defining grape aroma potential. In general, wines made from grapes containing higher levels of specific precursors will develop higher levels of the aroma molecules derived from those precursors and/or will keep levels of those molecules for longer aging periods.

3.2. Grape Aroma vs. Grape-Derived Wine Aroma As was schematized in Figure1, grapes contain seven relatively well di fferentiated chemical/ biochemical aroma precursor systems. As discussed previously, two of the systems—the fatty acid/enzymatic system and the Strecker amino acid system—have a major role in the development of the actual aroma of grapes, but to the best of our knowledge, they seem to have a rather limited role as wine aroma precursors. Both systems will influence wine aroma insofar as they form grape aroma molecules or precursors of aroma molecules, which will eventually pass to wine, but the systems as such do not survive fermentation. This explains why if the grape has not suffered raisination or over-ripening, the wine, generally, will not develop prune and overmatured character. On the contrary, the five other systems or molecular pools will be transferred to wine with different degrees of change induced by fermentation and will release or produce the specific aroma molecules at different moments of the winemaking process. Biomolecules 2019, 9, 818 13 of 35

The wine odorants for which there is more or less strong evidence about the implication of grape specific precursors in their formation are summarized in Tables5–7. The list includes 27 compounds: four norisoprenoids, five terpenes, six volatile phenols, four vanillin derivatives, ethyl cinnamate, two ethyl esters, two lactones, furaneol, DMS, and three polyfunctional mercaptans. Some compounds in the list, such as polyfunctional mercaptans, DMS, linalool, rose oxide, or TDN, can reach odor-impact levels. Some others, such as volatile phenols or vanillin derivatives, rather exert a cooperative effect on wine aroma. Mint lactones, recently identified at low levels in red wines from Bordeaux [135], limonene and 1,4- and 1,8-cineol, as well as some megastigmatrienones, may also play a role in minty, balsamic, and tobacco notes [66], but evidence about their implication is yet weak. The tables summarize information relative to the presence of the odorants in hydrolysates obtained by enzymatic, harsh, or mild (long term) acid hydrolysis. This information is relevant to understand the genesis of the aroma compound and also to assess the relevance of the findings of the different reports. In some of the few studies using long term acid hydrolyses, there is additional information about the pattern of accumulation of the odorant with time. This information is crucial to understand the evolution of these aroma molecules during wine aging. As can be seen in Table5, none of the four norisoprenoid odorants were present in enzymatic hydrolysates. Only in grapes kept frozen before the analysis, or in raisins, were these odorants found after enzymatic hydrolysis. In the case of terpenes (Table5), volatile phenols, and vanillin derivatives (Table6), enzymatic hydrolysis in general produced much higher levels than harsh acid hydrolysis. By contrast, most compounds are found at reasonable levels in hydrolysates obtained by long-term acid hydrolysis. Large differences between compounds are also found regarding the pattern of accumulation during aging. Linalool and geraniol reach maximal levels immediately after fermentation or after a short aging time, and afterwards their levels decay dramatically. β-Damascenone and β-ionone reach maximal levels also after a relatively short aging period, but their levels decay slowly or stay stable (Table5). By contrast, TDN, TPB, and most volatile phenols and vanillin derivatives steadily increase during aging (Tables5 and6). 4-Vinylphenol and 4-vinylguaiacol follow more complex evolutions with at least two maxima, likely because of the number of precursors they have and their chemical reactivity. The evolution with time of some relevant odorants, such as (Z)-rose oxide, geranic acid, or piperitone is mostly unknown. Data summarized in the tables also reveal the existence of huge variabilities in the levels of most compounds, regarding variety, vintage, location, or maturity. While some differences may be attributed just to the different analytical methodologies followed by the researchers, some others truly reflect a large diversity. Differences between Muscat grapes and “neutral” grapes regarding levels of terpenols are known, as well as those of furaneol between hybrids and Vitis vinifera varieties. However, data in Table6 suggest that di fferences in the levels of some volatile phenols and vanillin derivatives are well above the order of magnitude. Finally, Table7 contains some odorants for which the existence of precursors can be expected but has not been demonstrated. The following four sections deal with the different types of precursors responsible for all those odorants. The first section deals with glycosidic precursors, the second with other precursors, and the two last sections with glutathionyl and cysteinyl precursors and DMS precursors. Biomolecules 2019, 9, 818 14 of 35

Table 5. Wine norisoprenoid and terpene odorants coming from specific precursors.

Mild/Long Term Acid Aroma Molecule Enzymatic Hydrolysis Harsh Acid Hydrolysis Hydrolysis Norisoprenoids Detected by GCO [142]; 26 ppb [138]; detected by GCO maxima (3.3 ppb) after short [139]; 4–28 ppb depending aging, then steady decrease varieties, unclear pulp/skin [14]; steady increase all the Not found; yes in raisins [23,57] distribution [140]; 4–20 ppb aging in fermented samples and frozen grapes [136]; not in β-Damascenone depending location [140]; [143]; maxima 7.1–7.3 ppb wines [137]; 0.17–0.5 ppb in levels correlated to total after medium aging in frozen grapes [12] norisoprenoids by enzymatic unfermented controls [143]; [141]; 2–4.5 ppb depending formed soon and stable, varieties [12] maxima 17 ppb [15]; idem, with maxima 7 ppb [66] Maxima (1.9 ppb) after short Not found; yes in frozen grapes Generally yes; not found in aging, stable with time [14]; β-Ionone [136]; not in wines [137]; <0.11 [12] formed soon, stable for a ppb in frozen grapes [12] while, maxima 7.7 ppb [15] 8 ppb [138]; detected by GCO Not found; yes in frozen grapes [139]; 1–35 ppb depending on [136]; not in wines [137]; 1–6 varieties, unclear pulp/skin Linear increase with time, max TDN ppb (5–30% of levels found in distribution [140]; n.d. to 26 140 ppb [143]; idem, max at 61 harsh acid hydrolysis) in frozen ppb depending on place [140]; ppb [15]; idem [66] grapes [12] 8–89 ppb depending on varieties [12] Not found; 0.2–3 ppb (2–22% of 3 ppb [138]; 2–23 ppb Continuously formed, maxima TPB levels found in harsh acid depending varieties [12] 9 ppb [66] hydrolysis) in frozen grapes [12] Terpenes Found only in mild acid Generally present; not found in hydrolysis [141]; maxima after Portuguese reds [140]; not found 3% levels found in enzymatic fermentation, sharp decrease in Melon B [141]; not found in Linalool [138]; 10–50% of levels found in aging [14]; in Grenache, Shiraz [144]; found at low levels in enzymatic [12] maxima after short aging [143]; (less than 7% geraniol 1% total formed very soon, sharp terpenes) [144] decrease [15,66] Maxima in fermentation, sharp Always found; up to 10% of No [138]; 3–30% of levels decrease in aging [14,143]; Geraniol total terpenes in Shiraz, 14% in found in enzymatic [12] formed very soon, sharp Muscat [144] decrease [15,66] 0.04 ppb in Muscat, 0.01 ppb in 11–29 ppb in Muscat, depending Grenache; not found in (Z)-Rose oxide on maturity [145]; unrelated to Verdejo, Tempranillo, free form in raisins [23] Chardonnay, Cabernet Sauvignon, or Merlot [12] Up to 2–3 ppm [146,147]; also found in raisins [23]; <4 ppb Not found [138]; 0.5–50% of 1.5 ppb in Chardonnay juices Geranic acid [145]; up to 7.5% total terpenes levels found in enzymatic [12] [148] in Shiraz, 18% in Muscat [144] Derived from limonene, unknown accumulation pattern [149]; limonene Piperitone accumulates in the first periods of aging, then slight decrease [66] n.d.: Not detected. Biomolecules 2019, 9, 818 15 of 35

Table 6. Wine benzenoid odorants coming from specific precursors.

Aroma Molecule Enzymatic Hydrolysis Harsh Acid Hydrolysis Mild/Long Term Acid Hydrolysis Volatile Phenols Not found [146]: only in Brachetto, not in Aleatico, Malvasia, or Moscato [147]; <2 Detected by GCO [142]; Steady ppb [125]; up to 60 ppb in Rojal wine [137]; Detected by GCO [139]; increase with time, maxima 4.3 ppb Guaiacol 0–41 ppb [150]; 10–76 ppb depending on <0.61 ppb, unrelated to [14]; idem, maxima 6.3 ppb [143]; vintage [151]; 15–44 ppb depending on enzymatic levels [12] idem, maxima 14 ppb [15] vintage [152]; 17 ppb in Shiraz [144]; 0.4–2.3 ppb depending on varieties [12] 1–8.3 ppb [146,147]; not found [125]; up to 33 ppb in Rojal wine [137]; present in less than half varieties, up to 16 ppb [150]; 84–216 ppb depending on vintage [151]; Detected by GCO [139]; Eugenol 12–20 ppb in Bobal depending on vintage <0.36 ppb, unrelated to Steady increase, maxima 1.25 ppb [15] [152]; n.d. to 9.4 ppb depending on variety enzymatic levels [12] [140]; 2.7–18 ppb depending on location [140]; 10 ppb in Shiraz [144]; 0.4–7 ppb depending on variety [12] Up to 14 ppb in Rojal wine [137]; 7.6–26 ppb depending on vintage [151]; 5–25 ppb <0.58 ppb, unrelated to Isoeugenol Detected by GCO [142] depending on vintage [152]; 0.4–4.8 ppb enzymatic levels [12] depending on varieties [12] n.d. to 5.5 ppb Detected by GCO [142]; steady 3–60 ppb [147]; n.d. to 13 ppb depending 2,6-Dimethoxyphenol depending on varieties increase with time, maxima 33 ppb on varieties [12] [12] [14]; idem, maxima 142 ppb [15] 65–357 ppb [147]; <24 ppb [150]; 56–378 ppb depending on vintage [151]; 56–64 ppb 40% of enzymatic [138]; A maxima (21 ppb) after short aging, depending on vintage in Bobal [152]; 2–114 detected by GCO [139]; then decrease and steady increase [14]; 4-Vinylguaiacol ppb depending on varieties [140]; 2–178 10–38 ppb depending on continuous increase, maxima 5.5 ppm ppb depending on location [140]; 21 ppb in varieties, unrelated to [143]; formed soon and stable, Shiraz [144]; 39–162 ppb on depending enzymatic [12] maxima at 1.3 ppm [15] varieties [12] A maxima after short aging (45 ppb), 28–266 ppb [150]; 5–222 ppb depending on then decrease and steady increase, 9–21 ppb depending on varieties [140]; 19–310 ppb depending on maxima 80 ppb [14]; continuous 4-Vinylphenol varieties, unrelated to location [140]; 6 ppb in Shiraz [144]; increase, maxima 4.4 ppm [143]; enzymatic [12] 121–1739 ppb depending on varieties [12] formed very soon, later steady decrease, maxima at 102 ppb [15] Vanillin Derivatives 27–42 ppb [147]; 361 ppb in skin of Uva di Troia [125]; 31–61 ppb [137]; <37 ppb [150]; Detected by GCO [142]; linear 50% enzymatic [138]; 48–68 ppb depending on vintage [151]; increase with time, maxima 45 ppb Vanillin detected by GCO [139]; 60–160 ppb depending on vintage in Bobal [14]; idem, maxima 91 ppb [143]; <1.5 ppb [12] [152]; 31 ppb in Shiraz [144]; 40 ppb in idem, maxima 123 ppb [15] Muscat [144]; <4.1 ppb [12] 4–7 ppb [147]; <7 ppb [125]; up to 205 ppb in Rojal wine [137]; <42 ppb [150]; 12–147 ppb depending on vintage [151]; 9–143 Methyl vanillate <3.4 ppb [12] 6 ppb in Chardonnay juices [148] depending on vintage in Bobal [152]; 25 ppb in Shiraz [144]; 154 ppb in Muscat [144]; <18 ppb [12] Up to 45 ppb in Rojal wine [137]; n.d. to 10 Ethyl vanillate ppb depending on vintage in Bobal [152]; <3.1 ppb <12 ppb [12] Up to 205 and 260 ppb in Rojal and Tortosí Detected by GCO [139]; wines [137]; 1–12 ppb depending on vintage Unclear pattern [15]; 5 ppb in Acetovanillone <2.5 ppb, unrelated to [151]; 42 ppb in Muscat, none in Shiraz Chardonnay juices [148] enzymatic [12] [144]; 8–34 ppb depending on variety [12] Cinnamic Acid Derivatives 7 ppb only in pulp from Uva di Troia [125]; Detected by GCO [142][15]; steady <0.8 ppb [12]; its precursor, cinnamic acid 12 ppb [138]; <0.11 ppb increase with time in some varietals, Ethyl cinnamate has been found up to 7 ppb in fractions [12] maxima 3.3 ppb [14]; maxima 3.3 ppb from wine, levels depending on vintage after short aging [143] [137,151,152] n.d.: Not detected. Biomolecules 2019, 9, 818 16 of 35

Table 7. Wine miscellaneous odorants coming from specific precursors.

Mild/Long Term Acid Aroma Molecule Enzymatic Hydrolysis Harsh Acid Hydrolysis Hydrolysis Its precursor, ethyl Ethyl cyclohexanoate cyclohexanoic acid, found in unfermented mistellas [153] Its precursor, ethyl 4-methylpentanoic acid, Ethyl 4-methylpentanoate found in unfermented mistellas [153] γ-Decalactone No [125] Identified [8] Detected by GCO [15,142] Massoia lactone Detected by GCO [15] Aglianico up to 2 ppm in pulp and 0.6 in skin, Uva di Furaneol Troia 1,2 ppm in pulp, 90 Detected by GCO [139] Detected by GCO [15] ppb in skin [125]; 15–51 ppm in muscadine [46] Only found in grape or DMS grape mistellas not in precursor fractions [15] Polyfunctional Mercaptans 4-Methyl-4-mercaptopentan-2-one Mostly released by yeast. Released by yeast. Detected 3-Mercaptohexanol by GCO in mild-acid hydrolyzates [15,142] 3-Mercaptohexyl acetate Formed by yeast from 3MH

3.3. Glycoconjugates as Aroma Precursors Some good reviews on these questions have been recently published [154–156]. Glycoconjugation is a clever way to solubilize and fix nonpolar and volatile aroma molecules and it is very common in nature [157]. Many secondary metabolites of plants are glycoconjugated, and in fact, glycoconjugation can be considered a relatively common last step of plant secondary metabolism and seems to be a primary sedative mechanism used by plants to maintain metabolic homeostasis [158] and to detoxify from potentially toxic (lipophilic and/or reactive nucleophiles) molecules [159]. Glycoconjugation takes place by reaction between a reactive functional group and an “activated” sugar. Activated sugars are UDP-glucose, UDP-rhamnose, UDP-galactose, UDP-xyloxe and glucuronic acid, where UDP stands for uracil-diphosphate glucose. The reactive functional groups are -COOH, -NH2, -SH, and -OH, among others. In the case of grapes, little is known about the real activities and selectivities of glycosyltransferases, but at least 240 different types of these enzymes are coded in the grape genome [160]. Although glycosides may be more easily handled and transported by plant transport systems, recent evidences suggest that grape aroma glycosides are integrally formed in the grape. Of course, major grape glycosides are those of flavonoids, phenolic acids, and anthocyanins, while aroma compounds represent quantitatively a quite modest fraction. In the case of aroma compounds, to date, all grape aroma-related derivatives have been found to be bound to a β-D-glucose, and such glucose can be further bound to malonic acid, arabinose, apiofuranose, or rhamnose to form the structures indicated in Figure3. BiomoleculesBiomolecules2019 2019, 9, 818, 9, 818 1718 of 35of 36

Figure 3. Sugar moieties of glycoside precursors. Adapted from [154].

Recently, two trisaccharidesFigure 3. Sugar have moieties been alsoof glycoside tentatively precursors. identified Adapted in grape from [ 161[154].,162 ]. According to the type of aglycone, glycoconjugates in grapes can be broadly classified into the followingRecently, categories: two trisaccharides have been also tentatively identified in grape [161,162]. According to the type of aglycone, glycoconjugates in grapes can be broadly classified into the 1. Aliphatic alcohol derivatives; following categories: 2. Terpenes;1. Aliphatic alcohol derivatives; 3. Norisoprenoids;2. Terpenes; 4. Benzenoids,3. Norisoprenoids; which can be further subdivided into: 4. Benzenoids, which can be further subdivided into: a. Benzyl and phenyl derivatives; a. Benzyl and phenyl derivatives; b. Volatile phenols; b. Volatile phenols; c. Vanillins; c. Vanillins; d. Ethyl cinnamate.d. Ethyl cinnamate. 5. Miscellaneous5. Miscellaneous compounds. compounds. Aliphatic alcohol derivatives can be quantitatively important, but they are quite unimportant fromAliphatic the aromatic alcohol point derivatives of view. can Compounds be quantitatively in this group, important, among but others, they are include quite isoamyl unimportant alcohol, fromhexanol, the aromatic (Z)-3-hexenol, point of (E)-2-hexenol, view. Compounds 1-octen-3-ol, in this group,heptanol, among and others,octanol include[138]. isoamyl alcohol, hexanol,Terpenes (Z)-3-hexenol, include (E)-2-hexenol, a quite complex 1-octen-3-ol, array of terpenes heptanol, in anddifferent octanol oxidation [138]. states. The list includes severalTerpenes terpenic include diols aquite which complex will be arraypresented of terpenes in the next in di ffsection,erent oxidation together states.with linalool, The list α includes-terpineol, severalnerol, terpenic geraniol, diols and which several will of be their presented oxides, in including the next section, c-rose oxide. together The with most linalool, importantα-terpineol, from the nerol,sensory geraniol, point andof view several are the of theirsame oxides,four as in including the free fraction, c-rose oxide. namely The linalool, most important geraniol, c-rose from oxide, the sensoryand geranic point ofacid. view Note are that the some same of four these as compou in the freends fraction,will suffer namely chemical linalool, rearrangements geraniol, c-roseat acidic oxide,pHs. and Different geranic reports acid. Note have that estimated some of thesethat between compounds 77% willto 83% suffer of chemical the total rearrangements terpenic content at in acidicRiesling pHs. grapes Different are reports present have as gl estimatedycosides [163–165]. that between Some 77% of tothem, 83% such of the as total different terpenic hydroxylated content in Rieslingforms of grapes the main are presentterpenols as or glycosides of geranic [163 acid,–165 se].em Some to ofbe them, majorly such or as even diff erentexclusively hydroxylated found as formsglycosides of the main[147]. terpenolsFrom the quantitative or of geranic point acid, of seemview, tomajor be majorly aglycones or of even terpenes exclusively in neutral found varietals as glycosidesare those [147 of ].geraniol From the (Figure quantitative 4), (Z)-8-hydroxy-linalool point of view, major (or aglycones (2Z)-2,6-dimethylocta-2,7-diene-1,6-diol), of terpenes in neutral varietals areand those p-menthene-7,8-diol of geraniol (Figure 4with), (Z)-8-hydroxy-linalool account to more than (or 60% (2Z)-2,6-dimethylocta-2,7-diene-1,6-diol), of the peak area, eventually followed by andthose p-menthene-7,8-diol of linalool and geranic with acid account and tothose more of the than (E)- 60% and of (Z)-pyran the peak linalool area, eventually oxides [137,140,150,166]. followed by thoseA glycoside of linalool precursor and geranic of acid1,8-cineole, and those namely of the 2-exo- (E)- andhydroxy-1,8-cineole, (Z)-pyran linalool has oxides been [137 also,140 identified,150,166]. in A glycosideFalanghinna precursor grapes of[167]. 1,8-cineole, namely 2-exo-hydroxy-1,8-cineole, has been also identified in Falanghinna grapes [167]. Biomolecules 2019, 9, 818 18 of 35 Biomolecules 2019, 9, 818 19 of 36

Figure 4. Release of geraniol via acid-catalyzed hydrolysis of the geranyl-β-D-glucopyranoside. Figure 4. Release of geraniol via acid-catalyzed hydrolysis of the geranyl-β-D-glucopyranoside. There are a number of recent reports about the evolution of these precursors during grape There are a number of recent reports about the evolution of these precursors during grape maturation. Results show that the patterns of accumulation depend largely on the aroma maturation. Results show that the patterns of accumulation depend largely on the aroma compound compound [145], on the variety of grape [147], and on the vintage [146], which makes difficult [145], on the variety of grape [147], and on the vintage [146], which makes difficult to extract sound to extract sound conclusions. In general, it can be said that glycosidic forms tend to increase with conclusions. In general, it can be said that glycosidic forms tend to increase with maturation following maturation following more regular accumulation patterns than free forms, which can show erratic more regular accumulation patterns than free forms, which can show erratic patterns of evolution patterns of evolution during maturation. during maturation. As summarized in Table5, levels of linalool and geraniol are maximal in the wines immediately or As summarized in Table 5, levels of linalool and geraniol are maximal in the wines immediately shortly after fermentation, and levels decrease due to the poor stability of these molecules at wine pH. or shortly after fermentation, and levels decrease due to the poor stability of these molecules at wine The pool of precursors which survived the fermentation seems to be essential for keeping the levels of pH. The pool of precursors which survived the fermentation seems to be essential for keeping the these relevant aromas longer times [14,143]. levels of these relevant aromas longer times [14,143]. Aglycones in the norisoprenoid family can be also extraordinarily complex and, not surprisingly, Aglycones in the norisoprenoid family can be also extraordinarily complex and, not there are not aglycones representing the most relevant aroma compounds in this family, such surprisingly, there are not aglycones representing the most relevant aroma compounds in this family, as β-damascenone, β-ionone, TDN, or TPB. The major aglycones are 3-hydroxy-β-damascone, such as β-damascenone, β-ionone, TDN, or TPB. The major aglycones are 3-hydroxy-β-damascone, dihydro-β-ionone, and different ionols, particularly 3-oxo-a-ionol and vomifoliol [140,150]. This dihydro-β-ionone, and different ionols, particularly 3-oxo-a-ionol and vomifoliol [140,150]. This represent quite a nuisance, since the direct analysis of the aglycones (after careful enzymatic hydrolysis) represent quite a nuisance, since the direct analysis of the aglycones (after careful enzymatic or the direct HPLC-MS of the unaltered glycosidic precursors do not give clear information about the hydrolysis) or the direct HPLC-MS of the unaltered glycosidic precursors do not give clear aroma potentiality of this important precursor fraction. information about the aroma potentiality of this important precursor fraction. There is large difference between the four major nor-isoprenic odorants regarding the pattern There is large difference between the four major nor-isoprenic odorants regarding the pattern of of accumulation during aging. β-Damascenone and β-ionone reach maximal levels soon and then accumulation during aging. β-Damascenone and β-ionone reach maximal levels soon and then remain stable or steadily decrease with aging. By contrast, TDN and TPB are formed much more remain stable or steadily decrease with aging. By contrast, TDN and TPB are formed much more slowly during aging, with levels steadily increasing, as indicated in Table5. A recent report has shown slowly during aging, with levels steadily increasing, as indicated in Table 5. A recent report has that fermented samples form TDN faster than unfermented controls, which suggests that some of the shown that fermented samples form TDN faster than unfermented controls, which suggests that first chemical reactions in the sequence required to form TDN from 3,6-dihydroxy-β-ionone, its main some of the first chemical reactions in the sequence required to form TDN from 3,6-dihydroxy-β- precursor [168], are accelerated by yeast [143]. Such a report also demonstrates that levels of TDN ionone, its main precursor [168], are accelerated by yeast [143]. Such a report also demonstrates that formed during aging can be modulated by yeast. levels of TDN formed during aging can be modulated by yeast. Within the group of benzenoids (Table6) there are several subgroups of volatile compounds Within the group of benzenoids (Table 6) there are several subgroups of volatile compounds usually identified in the hydrolysates of grape precursor fractions [8,12,14,142,147]. usually identified in the hydrolysates of grape precursor fractions [8,12,14,142,147]. Benzyl and phenyl derivatives include benzaldehyde, benzoic acid, benzyl alcohol, and Benzyl and phenyl derivatives include benzaldehyde, benzoic acid, benzyl alcohol, and 2- 2-phenylethanol. In many neutral grape varieties these compounds, particularly the latter two, phenylethanol. In many neutral grape varieties these compounds, particularly the latter two, are the are the major constituents of the glycosidic aroma fraction [137,140,150]. This has some practical major constituents of the glycosidic aroma fraction [137,140,150]. This has some practical relevance relevance since the contribution of these glycosides to wine flavor can be considered negligible. since the contribution of these glycosides to wine flavor can be considered negligible. One the one One the one hand, the odor thresholds of both odorants are relatively high, and on the other hand, hand, the odor thresholds of both odorants are relatively high, and on the other hand, 2- 2-phenylethanol is a main secondary product of yeast metabolism, so that levels derived from grape phenylethanol is a main secondary product of yeast metabolism, so that levels derived from grape glycosides represent a quantitatively marginal fraction. The consequence is that indirect measures for glycosides represent a quantitatively marginal fraction. The consequence is that indirect measures the aromatic potential of neutral grapes [169] may be not related to the true aromatic potential but just for the aromatic potential of neutral grapes [169] may be not related to the true aromatic potential but to the general secondary metabolic activity of the grape. just to the general secondary metabolic activity of the grape. Volatile phenols, such as guaiacol, eugenol, isoeugenol, 2,6-dimethyoxyphenol, 4-vinylguaiacol, and 4-vinylphenol,Volatile phenols, are such relevant as guaiacol, components eugenol, of the isoeugenol, hydrolysates 2,6-dimethyoxyph obtained from fractionsenol, 4-vinylguaiacol, of precursors extractedand 4-vinylphenol, from grapes are or wines,relevant as detailedcomponents in Table of 6the. All hydrolysates or some of them obtain tended tofrom score fractions high in the of diprecursorsfferent GCO extracted studies from carried grapes out on or hydrolysates wines, as detail [139ed,142 in,170 Table]. Reported 6. All or levels some of of all them these tend compounds to score havehigh in ranges the different of variation GCO depending studies carried on vintage out on and hydrolysates varieties close [139,142,170]. to two orders Reported of magnitude, levels of all as these compounds have ranges of variation depending on vintage and varieties close to two orders of magnitude, as summarized in Table 6. These compounds cannot be determined by harsh acid Biomolecules 2019, 9, 818 19 of 35 summarized in Table6. These compounds cannot be determined by harsh acid hydrolysis, even though most of them accumulate steadily during aging. The case of vinylguaiacol and vinylphenol is particularly interesting. Both can be considered detrimental for wine quality if present at high levels [171]. As recently documented, they can be formed via yeast phenolic acid decarboxylases from phenolic acids and also by enzymatic or acid hydrolyses of their glycosides [143]. Vanillin and other related compounds are also formed from different precursors. Although the levels of these important aroma compounds derived from the grape cannot rival with levels released by some types of oak wood, grapes contain a large number of precursors able to release significant levels of these compounds. Vanillin is one of the odorants of acid hydrolysates which always scores very high by GCO [15,155,158,159]. In the enzymatic hydrolysates obtained from some varieties, such as those of skins from Uva di Troia [125] vanillin can be found at high levels (more than 360 µg/kg). Additionally, vanillin can be also formed by oxidation of 4-vinylguaiacol [172]. Ethyl cinnamate has been also found at minor levels in the hydrolysates of precursor fractions extracted from grapes (see Table6). Since cinnamic acid was also identified as aglycone after enzymatic hydrolysis, the precursor should be a glycoside. A glycoside of cinnamic acid has been recently identified in wine made from Korean black raspberries [173]. Within the miscellaneous section (Table7), the most relevant odorant is furaneol. Furaneol glucopyranoside has been recently identified and quantified in the must of Muscat Bailey A (V. labrusca (Bailey) V. vinifera (Muscat Hamburg)) [174]. The gene encoding the UDP-glucose: furaneol × glucosyltransferase was also determined [175]. The same authors were also able to quantify this precursor in different grape varieties and in the parental concord. Concentrations of the precursor were much higher in the labrusca and in the hybrids, but normal grapes also contain low amounts of this precursor. This aroma compound has been systematically identified by GC olfactometry in the hydrolyzed precursor fractions extracted from Grenache [142], Aragonez [139], Pinot Noir [170], or Tempranillo [15], and it has been found as aglycone released by enzymatic hydrolysis of the precursor fraction from Aglianico and Uva di Troia [125]. Finally, it should be noted that several authors have reported the presence of glycosides of some fatty acids at relatively large levels in the enzymatic hydrolysates of precursor fractions extracted from wines. For instance, isovaleric acid was found at 109 µg/L, butyric acid at 412 µg/L, hexanoic at 336 µg/L, and octanoic acid at 295 µg/L[150,152]. These amounts are just slightly smaller than those formed by yeast.

3.4. Other Precursors: Molecules Which by Chemical Rearrangement or Esterification Form the Aroma Molecule The first type of molecules includes a series of polyols discovered more than 30 years ago which by chemical rearrangements induced by the acid hydrolysis at wine pH produce different aroma active terpenols [2]. As shown in Figure5, one of the diols (3,7-dimethyloct-1-ene-3,7-diol) rearranges to give linalool and α-terpineol. The other molecules are different terpenols of lesser olfactory importance such as myrcenol or ocimenol. The diols were also found to be present as glycosides [176]. Some C13-triols with a megastimagne structure were also further identified as potential precursors for some norisoprenoids such as vitispiranes and TDN [177]. At wine pH, these precursors can spontaneously form TDN, responsible for the kerosene–off odor developed by some wines during aging. Also a megastimagne structure, megastigm-5-en-7-yne-3,9-diol, was identified as precursor for β-damascenone [178]. This was later confirmed by synthesis of the pure molecule [179]. The dienyne derivative and the allenic diol, shown in Figure6, were further identified in 2005 [ 180]. Both proceed from an allenic triol derived from the degradation of carotenoids such as neoxanthin [181]. Biomolecules 2019, 9, 818 21 of 36

Biomolecules 2019, 9, 818 21 of 36 Biomolecules 2019, 9, 818 20 of 35

Figure 5. Polyol rearrangement at pH 3.2. Adapted from [2].

Figure 5. Polyol rearrangement at pH 3.2. Adapted from [2].

Figure 5. Polyol rearrangement at pH 3.2. Adapted from [2].

Figure 6. Formation of β-damascenone from allenic triol. Adapted from [181].

Notably, AustralianFigure 6. Formation researchers of haveβ-damascenone recently demonstrated from allenic triol. that Adapted a ketone from and [181]. a diketone derived from diol 5 can be transformed by the action of yeast in β-damascenone [182]. AsNotably, previously Australian mentioned, researchers most of thesehave molecules recently aredemonstrated also found asthat glycosides, a ketone which and supposedlya diketone amount to a larger fraction of precursors. derived from diol 5 can be transformed by the action of yeast in β-damascenone [182]. Finally,As previously in this sectionmentioned, we should most mentionof these themo twolecules lactones are also and thefound two as ethyl glycosides, esters listed which in Figure 6. Formation of β-damascenone from allenic triol. Adapted from [181]. Tablesupposedly7: γ-decalactone amount to anda larger massoia fraction lactone of precursors. and ethyl cyclohexanoate and ethyl 4-methylpentanoate. The two lactones are primarily formed during grape dehydration, but since they accumulate in some Finally,Notably, in Australianthis section researcherswe should mentionhave recently the two demonstrated lactones and that the twoa ketone ethyl andesters a listeddiketone in wines or precursor fractions, it can be suggested that the corresponding γ-hydroxy or δ-hydroxy acids Tablederived 7: γ from-decalactone diol 5 can and be massoia transformed lactone by andthe actionethyl cy ofclohexanoate yeast in β-damascenone and ethyl 4-methylpentanoate. [182]. are present as precursors. As different glycosidic precursors of whisky lactones (γ-methyloctalactone) The twoAs lactonespreviously are primarilymentioned, formed most during of these grape mo dehydration,lecules are butalso since found they as accumulate glycosides, in somewhich have been described in oak wood [183,184] the presence of some glycosidesγ of the acidsδ cannot be winessupposedly or precursor amount fractions, to a larger it ca fractionn be suggested of precursors. that the corresponding -hydroxy or -hydroxy acids ruled out. In the case of the esters, the corresponding acids have been quantified inγ unfermented grape are presentFinally, as precursors.in this section As differentwe should glyc mentionosidic precursors the two lactones of whisky and lactones the two ( -methyloctalactone)ethyl esters listed in must [185]. haveTable been 7: γ -decalactonedescribed in andoak massoiawood [183,184] lactone andthe presethylence cyclohexanoate of some glycosides and ethyl of 4-methylpentanoate.the acids cannot be ruled out. In the case of the esters, the corresponding acids have been quantified in unfermented 3.5.The S-Derivatives two lactones of are Cysteine primarily or Glutathione formed during grape dehydration, but since they accumulate in some grapewines must or precursor [185]. fractions, it can be suggested that the corresponding γ-hydroxy or δ-hydroxy acids are Twopresent recent as precursors. reviews [186 As,187 different] have been glyc publishedosidic precursors on cysteinyl of whisky or glutathionyl lactones (γ derivatives.-methyloctalactone) Grapes 3.5. S-Derivatives of Cysteine or Glutathione containhave been some described cysteinyl orin glutathionyloak wood [183,184] derivatives the whichpresence by hydrolysisof some glycosides of the S–C of bond the acids in the cannot cysteine be partruled canTwo out. give recent In some the reviews case of the of [186,187] mostthe esters, powerful have the correspbeen aroma publisonding moleculeshed acids on of cysteinyl have wine been and or of quantifiedgl natureutathionyl in in general. unfermentedderivatives. The aromaGrapesgrape molecules mustcontain [185]. some are 4-methyl-4-mercaptopentan-2-onecysteinyl or glutathionyl derivatives (4MMP), which by 3-mercaptohexanol hydrolysis of the S–C (3MH), bond and in 3-mercaptohexylthe cysteine part acetatecan give (3MHA). some of The the aromamost powerful properties aroma of these molecules relevant of aromawine and compounds of nature are in summarizedgeneral.3.5. S-Derivatives The inaroma the of following Cymoleculessteine or Table Glutathioneare8 [4-methyl-4-mercaptopentan-2-one187]: (4MMP), 3-mercaptohexanol There are at least three or four more other varietal polyfunctional mercaptans in wine with far less (3MH),Two and recent 3-mercaptohexyl reviews [186,187] acetate have (3MHA). been publis The hedaroma on propertiescysteinyl or of gl theseutathionyl relevant derivatives. aroma aromatic importance. compoundsGrapes contain are summarized some cysteinyl in the or glutathionylfollowing Table derivatives 8 [187]: which by hydrolysis of the S–C bond in All these aroma compounds are released by the action of β-lyase enzymes from yeasts from their the cysteine part can give some of the most powerful aroma molecules of wine and of nature in specific precursors presentTable 8. Structures, in the grape odor must. properties, The 3MHA and occurrence requires, of in varietal addition, thiols. the acetylation of the general. The aroma molecules are 4-methyl-4-mercaptopentan-2-one (4MMP), 3-mercaptohexanol alcohol 3MH by action of an acyltransferase also from yeast, as summarized in Figure7. (3MH), and 3-mercaptohexyl acetate (3MHA). The aroma properties of these relevant aroma compounds are summarized in the following Table 8 [187]:

Table 8. Structures, odor properties, and occurrence of varietal thiols. Biomolecules 2019, 9, 818 22 of 36

Threshold in Range of Odor Compound Structure Model Wine Occurrence in Descriptor (ng/L) [188] Wine (ng/L) [189] 4-Methyl-4- Biomoleculesmercaptopentan-2-2019, 9, 818 Box tree 0.8 n.d. to21 90 of 35 one

Biomolecules 2019, 9, 818 22 of 36 Biomolecules 2019, 9, 818 22 of 36 Biomolecules 2019, 9, 818 22 of 36 3-MercaptohexanolTable 8. Structures, odor properties, and occurrenceGrapefruit of varietal 60 thiols. n.d. to 7300 Threshold in Range of Odor Threshold in Range of Compound Structure Odor ThresholdModel WineThreshold in inOccurrenceRange of Rangein of Compound Structure DescriptorOdor Model Wine Occurrence in CompoundCompound Structure Descriptor Odor Descriptor Model(ng/L) Wine[188]Model WineWineOccurrence (ng/L)Occurrence [189] in in Descriptor (ng/L) [188] Wine (ng/L) [189] 3-Mercaptohexyl Box tree, (ng/L) [188](ng/ L) [188Wine] (ng/L)Wine [189] (ng /L) [189] 4-Methyl-4- 4 n.d. to 440 4-Methyl-4- acetatemercaptopentan-2-4-Methyl-4- passionBox tree fruit 0.8 n.d. to 90 mercaptopentan-2- Box tree 0.8 n.d. to 90 4-Methyl-4-mercaptopentan-2-onemercaptopentan-2-one BoxBox tree tree 0.8 0.8 n.d. to 90 n.d. to 90 one one n.d.: Not detected.

3-Mercaptohexanol Grapefruit 60 n.d. to 7300 3-Mercaptohexanol3-Mercaptohexanol GrapefruitGrapefruit 60 60 n.d. to 7300 n.d. to 7300 3-Mercaptohexanol Grapefruit 60 n.d. to 7300 There are at least three or four more other varietal polyfunctional mercaptans in wine with far

less aromatic3-Mercaptohexyl importance. BoxBox tree,tree, passion 3-Mercaptohexyl3-Mercaptohexyl acetate Box tree, 4 4 n.d. to 440 n.d. to 440 All these3-Mercaptohexyl aromaacetate compounds are released by thepassion Boxaction tree, fruitfruit of β-lyase4 enzymes n.d.from to 440 yeasts from their acetate passion fruit 4 n.d. to 440 acetate passion fruit specific precursors present in the grapen.d.: must.n.d.: Not TheNot detected. detected. 3MHA requires, in addition, the acetylation of the n.d.: Not detected. alcohol 3MH by action of an acyltransferasen.d.: also Not fromdetected. yeast, as summarized in Figure 7. There are at least three or four more other varietal polyfunctional mercaptans in wine with far There are at least three or four more other varietal polyfunctional mercaptans in wine with far less aromaticThere are importance. at least three or four more other varietal polyfunctional mercaptans in wine with far less aromatic importance. less aromaticAll these importance.aroma compounds are released by the action of β-lyase enzymes from yeasts from their All these aroma compounds are released by the action of β-lyase enzymes from yeasts from their specificAll precursorsthese aroma present compounds in the aregrape released must. byThe the 3MHA action requires, of β-lyase in enzymes addition, from the acetylation yeasts from of their the specific precursors present in the grape must. The 3MHA requires, in addition, the acetylation of the specificalcohol 3MHprecursors by action present of an in acyltransferase the grape must. also The from 3MHA yeast, requires, as summarized in addition, in Figurethe acetylation 7. of the alcohol 3MH by action of an acyltransferase also from yeast, as summarized in Figure 7. alcohol 3MH by action of an acyltransferase also from yeast, as summarized in Figure 7.

Figure 7. Biogenesis pathways of 4MMP, 3MH, and 3MHA. Adapted from [187]. Figure 7. Biogenesis pathways of 4MMP, 3MH, and 3MHA. Adapted from [187]. Figure 7. Biogenesis pathways of 4MMP, 3MH, and 3MHA. Adapted from [187]. Figure 7. Biogenesis pathways of 4MMP, 3MH, and 3MHA. Adapted from [187]. ApartFigure from 7. Biogenesis the precursors pathways described of 4MMP,in Figure 3MH, 7, very and recent 3MHA. reports Adapted demonstrate from [187]. also the Apart from the precursors described in Figure 7, very recent reports demonstrate also the existenceApart of fromthe glutathione the precursors precursor described of 4-mercapto in Figure-4-methylpentan-2-ol 7, very recent reports [190] demonstrate and of hexanal also [191]. the existence of the glutathione precursor of 4-mercapto-4-methylpentan-2-ol [190] and of hexanal [191]. Apart fromexistenceThe thefirst precursorsof the glutathione discovered described precursor were in theFigureof 4-mercapto cysteinylate7, very-4-methylpentan-2-old recentones [192], reports and for demonstrate [190] over and 10 years of hexanal also thiols the [191].were existence ApartThe from first precursors the precursors discovered described were the cysteinylate in Figured ones 7, very[192], andrecent for over reports 10 years demonstrate thiols were also the of the glutathioneThethought first precursor toprecursors be formed ofdiscovered exclusively 4-mercapto-4-methylpentan-2-ol were from the cysteine cysteinylate conjugates.d ones [192],Glutathione [190 and] for and precursors over of 10 hexanal years were thiols identified [191 were]. The first existence thoughtof the glutathioneto be formed exclusively precursor from of 4-mercaptocysteine conjugates.-4-methylpentan-2-ol Glutathione precursors [190] were and identified of hexanal [191]. thoughtmuch later to beand formed definitive exclusively evidence from of theircysteine effe conjugates.ctive role as Glutathione precursors precursorsof 3MH and were 4MM4P identified was precursorsThe first discovered precursorsmuch later and werediscovered definitive the cysteinylated evidence were the of theircysteinylate ones effective [192 ],roled and ones as precursors for [192], over and 10of 3MH yearsfor overand thiols 4MM4P 10 years were was thoughtthiols were muchobtained later only and some definitive years ago evidence [193–195]. of their Recently, effective a glutamyl–cysteine role as precursors dipeptide of 3MH S-conjugate and 4MM4P to 3MH was to be formedobtained exclusively only some from years cysteine ago [193–195]. conjugates. Recently, a Glutathioneglutamyl–cysteine precursors dipeptide S-conjugate were identified to 3MH much thought toobtainedhas be also formed been only identified some exclusively years in ago must [193–195]. from[196]. Fromcysteine Recently, the qu conjugates.aantitative glutamyl–cysteine point Glutathione of view, dipeptide Glu–3MH precursorsS-conjugate precursor to wereis3MH the identified has also been identified in must [196]. From the quantitative point of view, Glu–3MH precursor is the latermuch and definitivelaterhas andalso been evidencedefinitive identified ofevidence in their must e [196].ff ectiveof Fromtheir role the effe qu asctiveantitative precursors role point as ofprecursors view, 3MH Glu–3MH and of 4MM4P 3MHprecursor and was is the4MM4P obtained was onlyobtained some years only agosome [193 years–195 ago]. [193–195]. Recently, aRecently, glutamyl–cysteine a glutamyl–cysteine dipeptide dipeptide S-conjugate S-conjugate to 3MH to has 3MH alsohas been also identified been identified in must in [must196]. [196]. From From the quantitative the quantitative point point of view, of view, Glu–3MH Glu–3MH precursor precursor is the is the most concentrated, being present at levels between 8 and 35 times higher than those of the Cys–3MH precursor. In the case of MP, both can be at similar levels [197] (see Table9). Biomolecules 2019, 9, 818 22 of 35

Table 9. Mean concentration of 4MMP and 3MH cysteinylated and gluthanionylated precursors in µg/L RSD% (n = 2) in eight grape varieties [197]. ± Variety CYS–MH CYS–MMP GLU–MH GLU–MMP Sauvignon Blanc 174 7 12.6 1.4 1557 86 7.7 1.3 ± ± ± ± Gewürztraminer 89 6 8.0 1.5 1154 56 6.6 0.8 ± ± ± ± Muscat 157 8 n.d. 1673 71 8.3 0.9 ± ± ± Grenache 172 5 7.9 1.2 1422 63 9.4 1.2 ± ± ± ± Albariño 158 3 7.2 0.7 1462 80 8.4 0.7 ± ± ± ± Tempranillo 205 8 6.1 1.8 1284 76 10.3 1.1 ± ± ± ± Verdejo 215 9 7.3 1.0 3397 102 n.d. ± ± ± Chardonnay 32 4 0.4 0.2 1405 97 n.d. ± ± ± n.d.: Not detected.

The conjugated thiol precursors are produced in the grape and concentrations are highest in the skin [198]. Little is known, however, about their biosynthesis and about the factors determining their accumulation during grape maturation. Levels are varietal-dependent, being highest in Sauvignon Blanc and Verdejo and close to null in Malvasia del Lazio, and increase during maturation [190]. Levels are also related to picking time [199], being maximum at early morning and later decreasing during the day. Interesting changes in amino acid levels during the day have been also identified as a consequence of leaf photosynthesis [200]. As it is also suggested in the previous figure, there is an additional prefermentative pathway leading to the in situ formation of 3MH precursors during grape processing before fermentation. According to this pathway, 3MH precursors form once the berry is damaged by reaction between E-2-hexenal formed via enzymatic oxidation of grape fatty acids and cysteine or glutathione present in the must. The existence of such pathway resulted as evident by the observed paradox that hand-picked grapes from Sauvignon Blanc produced wines much less aromatic than those harvested by machine [201]. The relative importance of the two different “kinds” of precursors, those already present in the grape and those formed in situ during early grape processing, is not clear. Subileau et al. showed that in their conditions (E)-2-hexenal was not a major contributor [194], while different studies confirm that machine-harvested grapes contain higher levels, with excessive oxidation being detrimental [201,202]. The effects of maceration time and pressing have been also studied by several authors, mostly concluding that prolonged maceration times leaded to higher levels of precursors [203,204]. More recently, Larcher et al. demonstrated that oxygen at harvest was essential for increased levels of precursors [205]. The apparent contradictory observations could be related to the existence of several concurrent factors not yet well controlled in the experiments such as the E-2-hexenal formation rate of the grape (dependent on grape lipoxygenases, oxygen, and grape fatty acids) and the cysteine and glutathione availability of the must. Cysteinyl and glutathionyl precursors are poorly metabolized by most yeasts, so that levels of the precursors in the final wines can be high [206], particularly if the must contains high levels of glutathione [15]. It should be noted that there is evidence, some old [142,207] and some new [15], suggesting that the powerful polyfunctional mercaptans could be also formed by acid hydrolysis of the precursors. The role of this pool of compounds to keep longer levels of these powerful aroma compounds should not be ruled out.

3.6. S-Methylmethionine and Other DMS Precursors Dimethyl sulfide is a quite remarkable wine aroma compound. It has been repeatedly identified as a powerful aroma enhancer [117,208] and, more specifically, as a contributor to blackberry and blackcurrant aroma nuances of red wines [209]. This compound can be formed by spontaneous hydrolysis of different precursors (very fast at alkaline medium) [210], of which S-methylmethionine (vitamin U) has been identified as the most important [211]. There are nearly no other reports about the occurrence and factors affecting the levels Biomolecules 2019, 9, 818 23 of 35 of this precursor in grapes, although its level has been found to be related to water deficit of vines [212]. Vines with moderate water deficit have higher potential for this compound and the concomitant higher levels of yeast assimilable nitrogen contained in the musts from those vines seem additionally to avoid the destruction (metabolization) of the precursor during fermentation.

3.7. The Action of Fungus and Other Exogenous Factors on Grape Actual and Potential Aroma Finally, the aroma of the must or grapes reaching the cellar can be strongly affected by the presence of fungus or by some other exogenous factors. Wines made from grapes affected by noble rot have higher levels of 3MH, furaneol, sotolon, methional, and phenylacetaldehyde [59,77,78], while wines made from grapes affected by uncontrolled fungal attacks can develop fungal odors. Some of them, at smaller levels, are of course also present in noble rot wines, such as 1-octen-3-ol [78]. The infection with Botrytis cinerea also changes some must enzymes with effect on aroma (esterase and β-glucosidase). Grapes affected by noble rot have also increased levels of cysteinyl precursors [213] and can have even an expanded number of this type of precursor [214], which helps explaining their particular aroma. Regarding negative odors related to fungal attacks, 3-octanone, 1-octen-3-one, (E)-2-octenol, 1-octen-3-ol, 2-methyl isoborneol, TCA, geosmin, TBA, and pentachloroanisole are usually targeted as responsible for off-odors [215]. The type and levels are related to the strain of fungus; 50% of Botrytis cinerea strains induce geosmin, one strain induces anisol [216] Following the exposure of vineyards to forest or bushfires, the occurrence of the smoke taint has been detected repeatedly; one review has been published recently about this off-flavor in wine [217]. Volatile phenols, like phenol, guaiacol, and their derivatives, that usually appear in wines as a consequence of barrel toasting or contamination with Brettanomyces yeasts, are present in greater quantities in wines produced with grapes exposed to smoke [218]. The evidence that free run juice of smoked grapes had trace levels of volatile phenols, while the same juice after several days of maceration showed levels in the range of hundreds of µg/L, proved that volatile phenols were stored in the skin rather that in the pulp [219]. Several studies have confirmed that the accumulation of volatile phenols takes place in the form of different glycoconjugates [220–222]. The release of volatile phenols from their precursor forms takes place not only during fermentation via enzymatic hydrolysis, but also via acid hydrolysis during post-bottle aging [223].

4. Final Conclusions Both grape aroma and grape-derived wine aroma are formed by a relatively large group of odorants belonging to different chemical and biochemical families. Only in the specific cases of aromatic grapes are there clear impact compounds or families of compounds defining the aroma profile. In neutral varieties, grape aroma profiles are rather the consequence of the presence of more than 20 odorants imparting at least seven different types of aroma nuances. In the case of wine, up to 27 relevant wine odorants have specific origin in grape molecules or specific aroma precursors. Those odorants have, however, a much larger aromatic diversity than that observed between grape odorants, introducing or contributing to many different wine odor nuances such as fruity, jammy, floral, citrus, phenolic, spicy, empyreumatic, or green, and hence contributing decisively to wine quality. Additionally, grape-derived wine aroma molecules accumulate in quite different time periods of winemaking; some of them are mostly released during fermentation, while some others accumulate only after long periods of aging. Within the first, remaining precursors in wine can have a crucial effect on keeping levels of odorants during aging, and therefore, in wine shelf-life. Within the latter, some of the odorants accumulating during aging, such as DMS, TDN, or TPB, may have controversial effects on wine quality, and may therefore have also a major influence on wine longevity. For of all these reasons, the control of grape-derived wine aroma is an essential piece for controlling wine quality and wine shelf-life. Comprehensive analytical strategies for such a control have to face demanding challenges, which at present are not satisfactorily solved. On the one hand, aroma molecules with different chemophysical properties have to be simultaneously determined, which is Biomolecules 2019, 9, 818 24 of 35 nearly impossible using a single isolation strategy. On the other hand, the strategy has to sort out the difficult and non-obvious link between specific precursors and wine odorants. Surely this will require combining metabolomic approaches with new, comprehensive hydrolytical strategies. Both techniques are at hand but will require from researchers a clear awareness of all the dimensions of the analytical problem.

Funding: Funded by the Spanish Ministry of Economy and Competitiveness (MINECO) (project AGL2017-87373-C3-1-R). LAAE acknowledges the continuous support of Diputación General de Aragón (T29) and European Social Fund. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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