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2021 125 Inés Pereira Biscaia de Oliveira Contribution of wine microorganisms to the aroma composition of wine and its sensory impact Director/es Ferreira González, Vicente Fischer, Ulrich © Universidad de Zaragoza Servicio de Publicaciones ISSN 2254-7606 Inés Pereira Biscaia de Oliveira CONTRIBUTION OF WINE MICROORGANISMS TO THE AROMA COMPOSITION OF WINE AND ITS SENSORY IMPACT Director/es Ferreira González, Vicente Fischer, Ulrich Tesis Doctoral Autor 2019 Repositorio de la Universidad de Zaragoza – Zaguan http://zaguan.unizar.es UNIVERSIDAD DE ZARAGOZA Escuela de Doctorado Programa de Doctorado en Ciencia Analítica en Química Contribution of wine microorganisms to the aroma composition of wine and its sensory impact PhD Thesis by Inês Pereira Biscaia de Oliveira January 2019 Supervisors: Dr. Vicente Ferreira González Dr. Ulrich Fischer 2 Contribution of wine microorganisms to the aroma composition of wine and its sensory impact by Inês Pereira Biscaia de Oliveira Dissertation presented for the degree of Doctor of Philosophy in Analytical Chemistry January 2019 Supervisors: Dr. Vicente Ferreira González Dr. Ulrich Fischer D. VICENTE FERREIRA GONZÁLEZ, Catedrático del Departamento de Química Analítica y D. ULRICH FISCHER, director en el Instituto DLRRheinpfalz, Alemania CERTIFICAN: Que la presente memoria, titulada “Contribution of wine microorganisms to the aroma composition of wine and its sensory impact” correspondiente al plan de investigación aprobado por la Comisión de Doctorado del Departamento de Química Analítica y presentada para optar al grado de doctora en Ciencia Analítica en Química, ha sido realizada bajo nuestra dirección por Dª. Inês Pereira Biscaia de Oliveira, autorizando su presentación para proseguir los trámites oportunos y proceder a su calificación por el tribunal correspondiente. Zaragoza, 17 enero de 2019. Fdo. Dr. Vicente Ferreira González Fdo. Dr. Ulrich Fischer Index Index Index PRESENTATION ................................................................................................................ 3 INTRODUCTION ................................................................................................................ 7 1. WINE FLAVOUR AND ITS PERCEPTION .............................................................................. 9 1.1 Wine aroma genesis............................................................................................... 10 1.2 Grape variety-related non-specific precursors ....................................................... 12 1.3 Glycosidic precursors ............................................................................................ 13 1.4 Cysteinylated and glutathionylated precursors ....................................................... 14 1.5 Effects of external additives on wine aroma ........................................................... 15 2. WINE AROMA FORMATION FROM SPECIFIC AND NON-SPECIFIC PRECURSORS DURING ALCOHOLIC FERMENTATION .............................................................................................. 16 2.1 Spontaneous versus inoculation fermentations ....................................................... 17 2.2 Yeast metabolism during alcoholic fermentation .................................................... 18 2.3 Release of aroma compounds from glycosidic precursors ....................................... 22 2.3.1 Release and formation of monoterpenes ................................................................. 22 2.3.2 Release and formation of norisoprenoids................................................................ 24 2.3.3 Release and formation of volatile phenols .............................................................. 26 3. SELECTION OF NON-SACCHAROMYCES YEAST STRAINS .................................................. 27 3.1 Torulaspora delbrueckii ........................................................................................ 28 3.2 Pichia kluyveri ...................................................................................................... 28 3.3 Lachancea thermotolerans..................................................................................... 28 4. BIBLIOGRAPHY ............................................................................................................. 30 OBJECTIVES ................................................................................................................... 39 SECTION I ........................................................................................................................ 41 STRECKER ALDEHYDES ARE NORMAL BY-PRODUCTS OF ALCOHOLIC FERMENTATION LINKED TO YEAST SULPHITE METABOLISM 1. INTRODUCTION ............................................................................................................. 43 2. METHODOLOGY ............................................................................................................ 46 2.1 Reagents and standards ......................................................................................... 46 2.2 Culture conditions: ................................................................................................ 46 Index 2.2.1 Synthetic must composition ...................................................................................46 2.2.2 Yeast culture and fermentation set-up.....................................................................47 2.3 Analytical methods ................................................................................................ 48 2.3.1 Classical oenological characterization ....................................................................48 2.3.2 Quantification of total aldehydes ............................................................................49 2.3.3 Analysis of major volatile compounds ....................................................................50 2.4 Data treatment ...................................................................................................... 50 3. RESULTS AND DISCUSSION ............................................................................................ 53 3.1 Strecker aldehydes are normal fermentative compounds ........................................ 55 3.2 Role of SO2 ........................................................................................................... 60 3.3 Role of Zn on SA formation ................................................................................... 63 3.4 Fatty acids and their ethyl esters ........................................................................... 63 4. CONCLUSIONS .............................................................................................................. 66 5. BIBLIOGRAPHY ............................................................................................................ 68 SECTION II ....................................................................................................................... 73 ROLES OF YEAST ON THE FORMATION AND EVOLUTION OF THE AROMA OF RIESLING AND GARNACHA WINES INTRODUCTION AND METHODOLOGY .................................................................... 75 1. SECTION II – INTRODUCTION ........................................................................................ 77 2. GOALS ......................................................................................................................... 80 3. SECTION II – METHODOLOGY ....................................................................................... 81 3.1 Reagents and standards ........................................................................................ 81 3.2 Glycosidic precursors extraction ........................................................................... 81 3.2.1 Grape processing ...................................................................................................81 3.2.2 Glycosidic extraction using a SPE based method ....................................................82 3.3 Synthetic must fermentation................................................................................... 83 3.3.1 Synthetic must composition ...................................................................................83 3.3.2 Yeast culture .........................................................................................................84 3.3.3 Fermentation set-up and accelerated aging .............................................................84 3.3.4 Unfermented controls ............................................................................................85 3.4 Analytical methods ................................................................................................ 86 3.4.1 Analysis of minor and trace volatile compounds .....................................................86 3.4.2 Chromatographic method.......................................................................................86 Index 3.5 Data treatment ...................................................................................................... 89 4. BIBLIOGRAPHY ............................................................................................................. 90 CHAPTER 1 ....................................................................................................................... 95 EFFECTS OF SEQUENTIAL INOCULATION WITH DIFFERENT NONSACCHAROMYCES ON THE FORMATION AND FURTHER EVOLUTION OF RIESLING WINE AROMA 1. RESULTS AND DISCUSSION ............................................................................................ 97 1.1 Classic oenological parameters of final synthetic wines ......................................... 98 1.2 Overview of the aroma composition of final wines .................................................. 99 1.3 Fermentative compounds in Riesling.................................................................... 102 1.4 Varietal compounds in Riesling ........................................................................... 110 2. CONCLUSIONS ............................................................................................................ 121 3. BIBLIOGRAPHY ........................................................................................................... 122 CHAPTER 2 ..................................................................................................................... 127 EFFECTS OF SEQUENTIAL INOCULATION WITH DIFFERENT NONSACCHAROMYCES ON THE FORMATION AND FURTHER EVOLUTION OF GARNACHA WINE AROMA 1. RESULTS AND DISCUSSION .......................................................................................... 129 1.1 Classical oenological parameters of final synthetic wines .................................... 130 1.2 Overview of the aroma composition of final wines ................................................ 131 1.3 Fermentative compounds in Garnacha wines ....................................................... 135 1.4 Considerations about potential sensory effects of different yeast strains ............... 142 1.5 Varietal compounds in Garnacha wines ............................................................... 144 2. CONCLUSIONS ............................................................................................................ 153 3. BIBLIOGRAPHY ........................................................................................................... 156 CHAPTER 3 ..................................................................................................................... 159 OBSERVATIONS, QUESTIONS AND CONCLUSIONS DERIVED FROM THE COMPARISON BETWEEN VARIETIES 1. OBJECTIVE ................................................................................................................. 161 2. RESULTS AND DISCUSSION .......................................................................................... 161 Index 2.1 Norisoprenoids formation and evolution in Riesling and Garnacha...................... 164 2.2 Monoterpenes in Riesling and Garnacha ............................................................. 172 2.3 Volatile phenols in Riesling and Garnacha .......................................................... 175 2.4 Medium chain fatty acids ethyl esters in Riesling and Garnacha .......................... 177 2.5 Fusel alcohols in Riesling and Garnacha ............................................................ 178 3. CONCLUSIONS ............................................................................................................ 180 4. BIBLIOGRAPHY .......................................................................................................... 182 SECTION III ................................................................................................................... 185 AROMA OF GERMAN RIESLING WINES AND INFLUENCE OF THE TERROIR 185 INTRODUCTION AND METHODOLOGY 1. SECTION III - INTRODUCTION ...................................................................................... 189 2. SECTION III - METHODOLOGY CHAPTER 4 ................................................................... 193 2.1 Project collaboration .......................................................................................... 193 2.2 Riesling commercial wines .................................................................................. 193 2.3 Sensory analysis: ................................................................................................ 193 2.4 Chromatography-olfactometry analysis ............................................................... 195 2.5 Volatile compounds quantification ...................................................................... 196 2.6 Data analysis ...................................................................................................... 196 3. SECTION III - METHODOLOGY FROM CHAPTER 5 .......................................................... 197 3.1 Project collaboration .......................................................................................... 197 3.2 Harvest and wine fermentation ............................................................................ 197 3.3 Chemical analysis ............................................................................................... 198 3.4 Data analysis ...................................................................................................... 200 4. BIBLIOGRAPHY .......................................................................................................... 202 CHAPTER 4 .................................................................................................................... 205 SENSORY AND CHEMOSENSORY CHARACTERIZATION OF COMMERCIAL YOUNG RIESLING WINES FROM DIFFERENT GERMAN APPELLATION 1. RESULTS AND DISCUSSION .......................................................................................... 207 1.1 Sensory Descriptive Analysis ............................................................................... 207 1.2 Semiquantitative Gas Chromatography-Olfactometry and GC quantitative analysis ................................................................................................................................. 211 Index 1.3 Integration of sensory, semiquantitative and quantitative data.............................. 215 2. CONCLUSIONS ............................................................................................................ 219 3. BIBLIOGRAPHY ........................................................................................................... 220 CHAPTER 5 ..................................................................................................................... 223 IMPACT OF VINEYARD VERSUS CELLAR MICROBIOTA FROM DIFFERENT HARVESTS ON THE DISTINCTION OF DIFFERENT RIESLING VINEYARD 1. RESULTS AND DISCUSSION .......................................................................................... 225 1.1 Importance of harvest year effect and fermentation location ................................. 225 1.2 Vineyard versus winery ....................................................................................... 230 2. CONCLUSIONS ............................................................................................................ 236 3. BIBLIOGRAPHY ........................................................................................................... 238 SUPPLEMENTARY DATA ............................................................................................ 241 1. ODOUR THRESHOLDS .................................................................................................. 243 2. SUPPLEMENTARY DATA FROM SECTION I ..................................................................... 245 3. SUPPLEMENTARY DATA FROM SECTION II: CHAPTER 1 ................................................. 248 4. SUPPLEMENTARY DATA FROM SECTION II: CHAPTER 2 ................................................. 253 5. SUPPLEMENTARY DATA FROM SECTION III: CHAPTER 2 ................................................ 259 Presentation Introduction 9 Introduction 1. Wine flavour and its perception Flavour of foods has long been described as the complex interaction of odour, aroma, taste and mouthfeel. Odour comprises the perception of volatiles directly by ortho-nasal via while the aroma entails the perception of volatiles which are ingested and detected by retro-nasal via. Major factors differentiating odour and aroma are volatilization temperature and the distinctive mass transfer conditions between the product and the product spread in buccal mucosa. Taste is well known by its attributes sweet, sour, salty, bitter and umami sensed by tongue receptors and mouthfeel describes the tactile sensation produced by food (Baert et al., 2012). It is not an easy task to define flavour since it depends on some aspects which are specific to each individual, such as the sensitivities of their olfactory and taste receptors and their degree of experience. The flavour is related not only with the presence of taste and odour-active molecules in the product and to their concentration profile, but also with the way in which those molecules interact with the matrix, to the possible existence of different species in equilibrium and to their specific ability to break the wine aroma buffer. Furthermore, perceptual interactions between the individual perceptions elicited by each odorant will also affect the way they are perceived by human senses (Ferreira, 2010). All the aroma-sensory attributes found in wine are caused by one or more molecules which were present in sufficient concentration to surpass the wine aroma buffer. The wine aroma buffer refers to the specific sensory properties of the mixture of 27 compounds from different chemical families found in all wines and alcoholic beverages at the concentrations produced in a normal alcoholic fermentation. These molecules are the main secondary products of alcoholic Introduction 10 fermentation and are responsible for important processes of aroma suppression, particularly of fruity and woody notes (de-la-Fuente-Blanco et al., 2016). The composition of the wine buffer can slightly change since it depends on yeast metabolism and other oenological practices, but, all in all, its sensory profile does not change much and is described as “vinous” (Ferreira et al., 2019). An aroma vector is defined as “a perceptual unit constituted by one or several molecules with similar aroma descriptors, which altogether and in an integrated form, are responsible for a specific set of sensory features of a type of products; wine in our case” (Ferreira et al., 2019). Wine flavour is one of the most complex and difficult to characterize and manage, since there is a huge variability associated with its formation. Factors like grape variety, vine management, sanitary conditions, location and soil type, microorganisms involved in fermentation, technological choices of wine making practices, additives, wood barrels and wine preservation are only a few examples of important sources of aroma variability (Fischer et al., 1999; Robinson et al., 2014). 1.1 Wine aroma genesis Wine volatiles can be classified according to their chemical structure, their odour into aroma families, or according to their contribution to a specific aroma vector. However, in this work we are more interested in a classification according to their genesis into the following categories: 1. Aroma compounds derived from specific precursors in the grape 2. Fermentative aroma compounds from unspecific precursors: a. Related to the grape variety b. Unrelated to the grape variety 3. Aroma compounds formed or extracted during aging Introduction 11 The most important grape varieties for winemaking have a rather neutral aroma character, however, they contain a series of specific aroma precursors, which after a more or less complex chemical process including hydrolysis, enzymatic cleavage or spontaneous chemical rearrangement, render the odorant. Compounds in this category are easily identified as varietal aroma compounds because chemically they have been built by the grape. Specific aroma precursors are glycosidic precursors and cysteinylated and glutathionylated precursors being the former responsible for the formation of terpenes, norisoprenoids and volatile phenols and the later for polyfunctional mercaptans (Ferreira, 2010). A third compound in this category is the amino acid S-methyl methionine, the precursor of dimethyl sulphide (DMS) (Landaud et al., 2008). However, grapes also contain a more or less specific profile of nutrients which will determine yeast metabolism and hence, also the fermentative aroma profile. Those grape components influencing yeast metabolism can be regarded as unspecific aroma precursors related to the grape variety. These compounds are most often classified as fermentative, since structurally they have been built by the yeast and not by the grape, however, they may have a very important role in the identity of the specific aroma of the variety. Important non-specific grape precursors are amino acids which will lead to the formation of compounds like higher alcohols, branched acids, their ethyl esters and the acetate esters of higher alcohols. The specific profile of compounds of these chemical families formed during fermentation is strongly linked to the specific grape amino acid profile, however its formation occurs due to alcoholic and/or malolactic fermentation (Ferreira, 2010; Hernández-Orte et al., 2002; Swiegers et al., 2005). There are of course, other fermentative compounds formed from unspecific aroma precursors not related with the grape variety. Ethanol is the most important in this category, but hydrogen sulphide (H2S), whose levels are Introduction 12 strongly determined by the residues of elemental sulphur sprayed to the vine, can be also classified in this category (Jiranek et al., 1995; Mendes-Ferreira et al., 2009). Finally, aged-related aroma is formed by molecules extracted from the wood such as whiskylactones, or formed by oxidation of different precursors, such as strecker aldehydes or sotolon, or formed by the reaction of wine components, such as furfurylthiol which is formed by reaction between H2S and furfural. Little amounts of H2S or Methanethiol (MeSH) formed by slow catalytical decomposition of S-amino acids also belong to this category (Ferreira, 2010; Loscos et al., 2009). 1.2 Grape variety-related non-specific precursors The main non-specific grape precursors are grape amino acids (Albers et al., 1996; Hernández-Orte et al., 2002). Other compounds which may be regarded as unspecific precursors are grape lipids, notably phytosterols. A very recent report demonstrates that the wine aroma signature strongly depends on the presence and type of these compounds (Fairbairn, 2018). However, and to the best of our knowledge, there are no further clues about the aroma compounds related to the presence of those compounds, although it can be hypothesized that they are fatty acids and their corresponding esters. Amino acids are the most well-known grape elements related with odorants. Early studies revealed that levels of higher alcohols, their acetates, branched acids and their ethyl esters were linked to the grape variety with which the wine was made (Ferreira et al., 2000). Later, it was demonstrated that the fermentation of synthetic musts containing the characteristic amino acid profiles of each variety, effectively produced specific aroma profiles containing all these aroma compounds (Hernández-Orte et al., 2002). Introduction 13 1.3 Glycosidic precursors Glycosylation is a common transport and detoxification plant mechanism and thus has been described in several plant species (Sarry and Günata, 2004; Winterhalter and Skouroumounis, 1997). Glycosidic precursors are formed by units containing a β-D-glucose moiety (sugar moiety) and an aglycone which will produce the volatile molecule (Winterhalter and Skouroumounis, 1997). Common sugar moieties in grapes are α-L-arabinofuranosyl-β-Dglucopyranoside, α-L-rhamnopyranosyl-β-D-glucopyranoside, β-Dxylopyranosyl-β-D-glucopyranoside, β-D-apiofuranosyl-β-D-glucopyranoside, and β-D-glucopyranoside-β-D-glucopyranoside. Regarding the aglycone, the diversity is immense and includes terpenes, C13-norisoprenoids, C6-alcohols, volatile phenols and benzyl derivatives (Liu et al., 2017; Wilson et al., 1984; Winterhalter and Rouseff, 2001; Winterhalter and Skouroumounis, 1997). Glycosidic precursors contribute to the varietal expression of wine aroma and are a huge source of aroma variability, due to the high number of factors affecting the composition of this fraction and also to high number of factors affecting the odorants finally produced from it. These precursors were initially identified and studied in the aromatic variety Muscat liking monoterpenes like linalool and geraniol with its varietal character. Their importance was further confirmed in other varieties like Gewürztraminer and Riesling (Günata et al., 1985; Strauss et al., 1986). Other relevant aroma compounds like β-damascenone, cis-rose oxide, 1,1,6-trimethyl-1,2dyhydronaphthalene (TDN) or 4-vinylphenol were further identified in Riesling, Gewürztraminer and other grape varieties and were also linked to glycosidic precursors (Parker et al., 2017; Sefton et al., 2011; Winterhalter and Rouseff, 2001; Winterhalter and Skouroumounis, 1997). Introduction 14 Some of these precursors are able to produce a free odorant by direct hydrolysis of the aglycone (Strauss et al., 1986; Wilson et al., 1984), however other compounds can have multiple intermediary precursors, generally non-volatile, which by a series of rearrangements will ultimately originate a free volatile. One of the most studied cases are the compounds derived from carotenoids (Winterhalter and Rouseff, 2001). However, normal glycosylation mechanism that occur in grapevines (Winterhalter and Skouroumounis, 1997), are not the only source of glycosidic precursors. The plant is also able to glycosylate volatile molecules existing in the external environment being the best-known case from smoke. The appearance of smoke-taint in wines after large wildfires close to vineyards was attributed to the glycosylation of volatile phenols, such as guaiacol and 4methylguaiacol, present in the atmosphere. The study conducted by Kennison et al., 2008 comparing grape juice and corresponding final wine from vineyards exposed to smoke and unsmoked grapevines, has shown that volatile phenols present in smoke were accumulated as non-volatile grape glycosidic precursors in exposed grapes, but they were only revealed as free odorants in final wine after must fermentation, reaching levels resulting in consumer rejection. Further analysis on bottled aged wine showed that levels continued to increase indicating that these molecules kept on being hydrolysed from the glycosides by slow but spontaneous chemical hydrolysis. 1.4 Cysteinylated and glutathionylated precursors Cysteinylated and glutathionylated precursors were first discovered in Sauvignon Blanc and are responsible for the formation of the potent varietal polyfunctional mercaptans in wine: 4-mercapto-4-methyl-2-pentanone (4M4M2P), 4-mercapto-4-methyl-2-pentanol (4M4M2POH), 3- Introduction 15 mercaptohexanol (3MH) and 3-mercaptohexyl acetate (Darriet et al., 1993; Peña-Gallego et al., 2012). These compounds are present in wine in ranges of ng/L but have an extreme odorant potential of tropical fruits, guava, passion fruit, grapefruit or boxtree. Depending on the concentration, they can also be perceived as sweat or onion (Ferreira and San Juan, 2012; Mateo-Vivaracho et al., 2010; Peña-Gallego et al., 2012). 1.5 Effects of external additives on wine aroma In addition to grape composition, also external additives can influence the formation of aroma compounds during alcoholic fermentation. These elements include metal cations often added as fertilizers or pesticides, SO2 added to preserve wine quality or oak barrels to mention only a few examples. These elements integrate wine production process and can highly affect the microbiome activity by modulating their enzymatic response signal by the presence of certain metal cations (De Nicola et al., 2009) or by having antiseptic action as in the case of SO2. Oak barrels can contribute highly to the extraction of wood molecules such as lactones, however they were also linked with the appearance of off-flavours by spoilage yeast activity (Ferreira, 2010; MalfeitoFerreira, 2011; Ribéreau-Gayon et al., 2006). Zinc, for instance, is present in vineyards and consequently in grape must. It has a wide range of sources from water pipes to pesticides (De Nicola et al., 2009; Hopfer et al., 2015). Yeast cells regulate the uptake of zinc by membrane transporters and the activation or inhibition of several yeast metabolic pathways are regulated by the zinc external concentration through a specific metalresponsive regulatory protein (De Smidt et al., 2008). This cation is crucial for the development of yeast strains, since it integrates the active-site of 6 important Introduction 16 classes of enzymes: oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases (De Nicola et al., 2009). SO2 is commonly used in winemaking due to its antiseptic and antioxidant properties. When added to the must in sufficient concentration, SO2 destroys most of wild spontaneous yeast and bacteria, preventing spontaneous fermentation (Henick-Kling et al., 1998). Later on, during storage, this compound is also added to the wine for preventing the proliferation of spoilage microorganism like acetic acid bacteria or Brettanomyces spp., thus, avoiding the formation of off-flavours like vinegar and horse sweat, respectively (Malfeito-Ferreira, 2011; Ribéreau-Gayon et al., 2006). Additionally, SO2 is highly reactive with oxygen and carbonyl compounds, thus it prevents direct oxidation by binding with dissolved oxygen and by inhibiting oxidation enzymes. On an indirect manner, it prevents typical oxidation flavours in wine by binding to aldehydes such as acetaldehyde (Ferreira et al., 2015; Ribéreau-Gayon et al., 2006). 2. Wine aroma formation from specific and non-specific precursors during alcoholic fermentation Fermentations have a crucial role on the formation of wine aroma. On one hand, microorganisms are directly responsible for the formation of many aroma compounds found transversely in all wines (fermentative compounds), but they are also important modulators in the production of varietal flavours from specific precursors. The formation of these last compounds is not, however, fully understood, despite large research conducted to date. Introduction 17 2.1 Spontaneous versus inoculation fermentations In nature, several yeast genera have been isolated both from vineyards and wineries, which demonstrates the existence of a complex microbiome in both locations (Barata et al., 2012; Fleet, 2003). S. cerevisiae has been described as the “wine yeast” due to its high fermentative vigour and resistance to other competitor microorganism and even antiseptics like SO2 (Fleet, 2003; HenickKling et al., 1998; Jolly et al., 2014). However, other yeast genera, mostly nonSaccharomyces yeasts, were shown to be active during the latent phase of normal alcoholic fermentations after which S. cerevisiae takes over, carrying most part of the alcoholic fermentation (Barata et al., 2012). However, not all the yeast active were considered positive and rather were often associated with the formation of off-flavours like acetic acid and ethyl acetate (Jolly et al., 2014). For this reason, for years, traditional winemaking has preferred to use commercial dried yeast products of isolated S. cerevisiae or S. bayanus strains. The addition of a large number of cells to the grape must ensures that any spontaneous microorganisms are surpassed, obtaining a controlled fermentation and avoiding stuck or problematic fermentations. This approached ensures indeed higher control over fermentative processes, but has been demonstrated to lead to standardization of wines and thus, new studies have been done to assess which non-Saccharomyces genera could lead to positive traits in wine (Henick-Kling et al., 1998; Hernández-Orte et al., 2008; Jolly et al., 2014; Padilla et al., 2016). During alcoholic fermentation, S. cerevisiae is not only responsible for the conversion of glucose and fructose into ethanol and CO2, but also for the formation of important volatiles due to the secondary yeast metabolism. The wine metabolome is dependent on the pool of grape precursors and on the efficiency with which they are converted into volatile molecules during alcoholic fermentation. The formation of volatile compounds through yeast Introduction 24 But, acid catalysis is also responsible for the transformation of relevant aroma volatiles, such as geraniol, which after being released from the glycoside, will spontaneously transform into the much less odorant a-terpineol, as shown in Figure 4. 2.3.2 Release and formation of norisoprenoids The most important aroma compounds derived from norisoprenoids are βdamascenone, β-ionone and TDN. Some other aroma molecules with less aromatic relevance are a-ionone, Riesling acetal and vitispiranes. All these molecules are chemically ketones, hydrocarbons or ethers, meaning that they cannot be part of aglycones, which by nature have to have a hydroxyl group (alcohols or carboxylic acids). This implies that the formation of these aroma Figure 4 Formation of monoterpenes from glycosidic precursors due to enzymatic or acidic hydrolysis (a) and (b) followed by chemical rearrangements to form other monoterpene molecules due to acidic environment (a) adapted from Waterhouse et al., 2016. Introduction 25 molecules will, necessarily, take place after different reactions other than the cleavage of the glycosidic precursors. For instance, one possible mechanism for β-damascenone formation in wine has been shown to be from the carotenoid neoxanthin, requiring oxidative cleavage, followed by enzymatic reduction and finally acid hydrolysis in order to become volatile (Winterhalter and Rouseff, 2001). While all norisoprenoids derive from carotenoid breakdown, two possible formation routes are possible (figure 5): 1. the direct degradation from carotenoids present in the grape must or in the wine, as seems to be the case of β-ionone (Winterhalter and Rouseff, 2001) 2. the cleavage and further rearrangement of non-volatile glycosides of C13norisoprenoids intermediates formed from carotenoids (β-damascenone, TDN, vitispirane or Riesling acetal (Winterhalter, 1991; Winterhalter and Rouseff, 2001). Figure 5 Possible pathways to form volatile norisoprenoids compounds from carotenoid initial precursors involving multiple glycosylated intermediates and/or chemical, enzymatic or acid hydrolysis In MendesPinto, 2009. Introduction 26 One of the difficulties is that for all the important odorants, there are multiple precursors, whose structures are not well known. This was first observed by Peter Winterhalter in 1991 for TDN, vitispirane and Riesling acetal, but seems to be valid also for β-damascenone. To complicate more things, the same precursors can yield different molecules, and some of them, such as Riesling acetal, can be intermediates in the production of TDN (Gök, 2015). 2.3.3 Release and formation of volatile phenols Volatile phenols such as guaiacol, vanillin, cresols and eugenol can be extracted from wood (oak barrels, wood chips, etc) contributing with smoky, sweet or clove flavours to wine (Kennison et al., 2008). Small amounts of these volatiles are also present under the form of glycosides in grapes, so that these compounds can be also released by enzymatic or acid hydrolysis. Moreover, grapes contain a large variety of phenolic compounds including phenolic acids, flavonoids, anthocyanins or tannins (Kheir et al., 2013). Major differences exist between white and grape varieties, which precisely provides colour differences in wine. However, some of these phenolic compounds are actual aroma precursors of volatile phenols, such as vinyl phenols, which can be also found as glycosidic precursors. Vinyl phenols are more important in white wines since the biochemical reactions for the transformation of phenolic acids are inhibited by red wine components (Basha et al., 2004; Chatonnet et al., 1993). Phenolic acids in grape are divided into two groups benzoic and cinnamic acids and are present in larger amount in red grapes. Cinnamic acids exist in grape berries under different forms, since in addition to the free molecules, they can also be as esters of tartaric acid and as glycosides (Basha et al., 2004; Kheir et al., 2013). S. cerevisiae is able to form vinylphenols during alcoholic Introduction 27 fermentation from cinnamic acids, in particular ferulic and p-coumaric acid catalysed by the enzyme cinnamate decarboxylase (Chatonnet et al., 1993). The conversion of vinylphenols into ethyl phenols is rare in microorganisms and large amounts of these compounds have been linked with spoilage yeast Brettanomyces spp. (Kheir et al., 2013). 3. Selection of non-Saccharomyces yeast strains Non-Saccharomyces yeasts are rarely used as single inoculums since they are inhibited by high ethanol levels and their sugar metabolism is not as efficient as that of S. cerevisiae. In fact, non-Saccharomyces yeasts have been in the past associated with sluggish or stuck fermentations with an increased risk of spoilage by competitor microorganisms such as acetic acid bacteria (Fleet, 2003). These non-Saccharomyces are then used as mixed inoculum or following sequential inoculation procedures in which the final part of the fermentation is carried out by S. cerevisiae. In both cases, non-Saccharomyces yeasts can act as potential enhancers of wine aroma due to their potentially different enzymatic activity compared to S. cerevisiae, namely by their specific b-glucosidase and b-lyase activities (Mendes Ferreira et al., 2001; Zott et al., 2011), but there are many other aroma compounds whose levels are modulated by the presence of these yeasts. The modulation may take via different mechanisms not yet clear, such a direct interaction between yeast strains or competition. The reduction of nutrients, the early amino acid intake or even limitations in dissolved oxygen could be important modulators of yeast metabolism (Clemente-Jimenez et al., 2005; Fleet, 2003; Kapsopoulou et al., 2007; Moreno et al., 1991). On the other hand, some strains of non-Saccharomyces with abilities to secrete lipolytic enzymes to Introduction 28 the media, may degrade grape lipids into free fatty acids, compounds which can inhibit the growth of S. cerevisiae (Escribano et al., 2017). Three commercial strains, isolated from wine environments, are often used in wine making and have shown particular traits. 3.1 Torulaspora delbrueckii T. delbrueckii was one of the first non-Saccharomyces commercial strains. It is described as having medium fermentative power and to be a low producer of acetic acid. When co-inoculated with S. cerevisiae, it was shown to improve the sensory description of wine, however it is not clear how this was accomplished, since researchers described that co-inoculation induced the decrease of isoamyl acetate, of fatty acids with known role on fruity aromas, such as hexanoic acid and also of vinyl phenols (Azzolini et al., 2014; Jolly et al., 2014). Researchers have also suggested that this strain has relevant β-glycosidase and carbon-sulfur lyase activities (Escribano et al., 2017; Padilla et al., 2016). 3.2 Pichia kluyveri P. kluyveri was first associated with a higher release of volatile thiols (Anfang et al., 2009), but it has been also linked with the formation of acetate esters (Viana et al., 2008). Comparing to other yeast strains, this yeast has a quite specific metabolic characteristics including an oxidative metabolism characterized by the formation of biofilms (Barata et al., 2012). 3.3 Lachancea thermotolerans This yeast was first described by its ability to produce lactic acid and thus, contribute for the wine roundness (Kapsopoulou et al., 2007; Varela and Borneman, 2017). Besides, sensory descriptours such as spiciness where found to increase in wines fermented with this yeast (Gobbi et al., 2013). Bibliography 30 4. Bibliography Albers, E., Larsson, C., Lidén, G., Niklasson, C., and Gustafsson, L. (1996). Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation. Applied and Environmental Microbiology 62, 3187– 3195. Anfang, N., Brajkovich, M., and Goddard, M. R. (2009). Co-fermentation with Pichia kluyveri increases varietal thiol concentrations in sauvignon blanc. Australian Journal of Grape and Wine Research 15, 1–8. Azzolini, M., Tosi, E., Lorenzini, M., Finato, F., and Zapparoli, G. (2014). Contribution to the aroma of white wines by controlled Torulaspora delbrueckii cultures in association with Saccharomyces cerevisiae. World Journal of Microbiology and Biotechnology 31, 277–293. Baert, J. J., De Clippeleer, J., Hughes, P. S., De Cooman, L., and Aerts, G. (2012). On the origin of free and bound staling aldehydes in beer. Journal of Agricultural and Food Chemistry 60, 1149–11472. Barata, A., Malfeito-Ferreira, M., and Loureiro, V. (2012). The microbial ecology of wine grape berries. International Journal of Food Microbiology 153, 243–259. Basha, S. M., Musingo, M., and Colova, V. S. (2004). Compositional differences in the phenolics compounds of muscadine and bunch grape wines. African Journal of Biotechnology 3, 523–528. Benito, Á., Calderón, F., Palomero, F., and Benito, S. (2015). Combine Use of Selected Schizosaccharomyces pombe and Lachancea thermotolerans yeast strains as an alternative to the traditional malolactic fermentation in red wine production. Molecules 20, 9510–9523. Chatonnet, P., Dubourdieu, D., Boidron, J., and Lavigne, V. (1993). Synthesis of volatile phenols by Saccharomyces cerevisiae in wines. Journal of the Science of Food and Agriculture 62, 191–202. Clemente-Jimenez, J. M., Mingorance-Cazorla, L., Martínez-Rodríguez, S., Las Heras-Vázquez, F. J., and Rodríguez-Vico, F. (2005). Influence of sequential yeast mixtures on wine fermentation. International Journal of Food Microbiology 98, 301–308. Darriet, P., Tominaga, T., Demole, E., and Dubourdieu, D. (1993). Evidence of the presence of a 4-mercapto-4-methylpentan-2-one precursor in Vitis vinifera Bibliography 31 Sauvignon. Comptes Rendus de l’Academie des Sciences, Serie III: Sciences de la Vie 316, 1332–5. De Nicola, R., Hall, N., Bollag, T., Thermogiannis, G., and Walker, G. M. (2009). Zinc accumulation and utilization by wine yeasts. International Journal of Wine Research 1, 85–94. De Smidt, O., Du Preez, J. C., and Albertyn, J. (2008). The alcohol dehydrogenases of Saccharomyces cerevisiae : A comprehensive review. FEMS Yeast Research 8, 967–978. de-la-Fuente-Blanco, A., Sáenz-Navajas, M.-P., and Ferreira, V. (2016). On the effects of higher alcohols on red wine aroma. Food Chemistry 210, 107–114. Eden, A., Van Nedervelde, L., Drukker, M., Benvenisty, N., and Debourg, A. (2001). Involvement of branched-chain amino acid aminotransferases in the production of fusel alcohols during fermentation in yeast. Applied Microbiology and Biotechnology 55, 296–300. Ehrlich, F. (1904). Ueber das natürliche Isomere des Leucins. Berichte der deutschen chemischen Gesellschaft 37, 1809–1840. Escribano, R., González-Arenzana, L., Garijo, P., Berlanas, C., López-Alfaro, I., López, R., et al. (2017). Screening of enzymatic activities within different enological non-Saccharomyces yeasts. Journal of Food Science and Technology 54, 1555–1564. Fairbairn, S. (2018). Defining the chemical features of wine perception. Ferreira, V. (2010). “Volatile aroma compounds and wine sensory attributes,” in Managing Wine Quality Woodhead Publishing Series in Food Science, Technology and Nutrition., ed. A. G. Reynolds (Woodhead Publishing), 3–28. Ferreira, V., Carrascón, V., Bueno, M., Ugliano, M., and Fernandez-Zurbano, P. (2015). Oxygen consumption by red wines. Part I: consumption rates, relationship with chemical composition and role of SO2. Journal of agricultural and food chemistry. Ferreira, V., de-la-Fuente-Blanco, A., and Sáenz-Navajas, M.-P. (2019). “Volatile aroma compounds and wine sensory attributes,” in Managing wine quality Volume I: viticulture and wine quality, ed. A. G. Reynolds (Woodhead Publishing). Ferreira, V., Lopez, R., and Cacho, J. F. (2000). Quantitative determination of the odorants of young red wines from different grape varieties. Journal of the Science of Food and Agriculture. 80, 1659–1667. Bibliography 32 Ferreira, V., and San Juan, F. (2012). “Flavor of Wine,” in Food Flavors: Chemical, Sensory, and Technological Properties. Fischer, U., Roth, D., and Christmann, M. (1999). The impact of geographic origin, vintage and wine estate on sensory properties of Vitis vinifera cv. Riesling wines. Food Quality and Preference 10, 281–288. Fleet, G. (2003). Yeast interactions and wine flavour. International Journal of Food Microbiology 86, 11–22. Gobbi, M., Comitini, F., Domizio, P., Romani, C., Lencioni, L., Mannazzu, I., et al. (2013). Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: A strategy to enhance acidity and improve the overall quality of wine. Food Microbiology 33, 271–281. Gök, R. (2015). Einfluss von Hefen auf die glykosidisch gebundenen Aromavorstufen und Untersuchungen zur Vermeidung der TDN-Fehlnote in Riesling. Günata, Y. Z., Bayonove, C. L., Baumes, R. L., and Cordonnier, R. E. (1985). The aroma of grapes. Localisation and evolution of free and bound fractions of some grape aroma components cv. Muscat during first development and maturation. Journal of the Science of Food and Agriculture. 36(9):, 857-862. Gunata, Z., Bitteur, S., Brillouet, J.-M., Bayonove, C., and Cordonnier, R. (1988). Sequential enzymic hydrolysis of potentially aromatic glycosides from grape. Carbohydrate Research 184, 139–149. Hazelwood, L. A., Daran, J. M., van Maris, A. J., Pronk, J. T., and Dickinson, J. R. (2008). The Ehrlich pathway for fusel alcohol production: a century of research on Saccharomyces cerevisiae metabolism. Applied and Environmental Microbiology 74, 2259–2266. Henick-Kling, Edinger, Daniel, and Monk (1998). Selective effects of sulfur dioxide and yeast starter culture addition on indigenous yeast populations and sensory characteristics of wine. Journal of Applied Microbiology 84, 865–876. Hernandez-Orte, P., Bely, M., Cacho, J., and Ferreira, V. (2006). Impact of ammonium additions on volatile acidity, ethanol, and aromatic compound production by different Saccharomyces cerevisiae strains during fermentation in controlled synthetic media. Australian Journal of Grape and Wine Research 12, 150–160. Hernández-Orte, P., Cacho, J., and Ferreira, V. (2002). Relationship between varietal amino acid profile of grapes and wine aromatic composition. experiments with model solutions and chemometric Study. Journal of agricultural and food chemistry 50, 2891–2899. Bibliography 33 Hernández-Orte, P., Cersosimo, M., Loscos, N., Cacho, J., Garcia-Moruno, E., and Ferreira, V. (2008). The development of varietal aroma from non-floral grapes by yeasts of different genera. Food Chemistry 107, 1064–1077. Hirst, M. B., and Richter, C. L. (2016). Review of Aroma Formation through Metabolic Pathways of Saccharomyces cerevisiae in Beverage Fermentations. American Journal of Enology and Viticulture 67, 361–370. Hopfer, H., Nelson, J., Collins, T. S., Heymann, H., and Ebeler, S. E. (2015). The combined impact of vineyard origin and processing winery on the elemental profile of red wines. Food Chemistry 172, 486–496. Jiranek, V., Langridge, P., and A Henschke, P. (1995). Regulation of hydrogen sulfide liberation in wine-producing Saccharomyces cerevisiae strains by assimilable nitrogen. Applied and Environmental Microbiology 61, 461–467. Jolly, N. P., Varela, C., and Pretorius, I. S. (2014). Not your ordinary yeast: nonSaccharomyces yeasts in wine production uncovered. FEMS Yeast Research 14, 215–237. Kapsopoulou, K., Mourtzini, A., Anthoulas, M., and Nerantzis, E. (2007). Biological acidification during grape must fermentation using mixed cultures of Kluyveromyces thermotolerans and Saccharomyces cerevisiae. World Journal of Microbiology and Biotechnology 23, 735–739. Kennison, K. R., Gibberd, M. R., Pollnitz, A. P., and Wilkinson, K. L. (2008). Smoke-Derived Taint in Wine: The Release of Smoke-Derived Volatile Phenols during Fermentation of Merlot Juice following Grapevine Exposure to Smoke. Journal of Agricultural and Food Chemistry 56, 7379–7383. Kheir, J., Salameh, D., Strehaiano, P., Brandam, C., and Lt, B. (2013). Impact of volatile phenols and their precursors on wine quality and control measures of Brettanomyces/Dekkera yeasts. European Food Research and Technology 237, 655–671. Landaud, S., Helinck, S., and Bonnarme, P. (2008). Formation of volatile sulfur compounds and metabolism of methionine and other sulfur compounds in fermented food. Applied Microbiology and Biotechnology 77, 1191–1205. Liu, J., Zhu, X.-L., Ullah, N., and Tao, Y.-S. (2017). Aroma Glycosides in Grapes and Wine. Journal of Food Science 82, 248–259. Loscos, N., Hernandez-Orte, P., Cacho, J., and Ferreira, V. (2007). Release and formation of varietal aroma compounds during alcoholic fermentation from nonfloral grape odorless flavor precursors fractions. Journal of Agricultural and Food Chemistry 55, 6674–6684. Section I Strecker aldehydes are normal by-products of alcoholic fermentation linked to yeast sulphite metabolism Section I - Introduction 43 1. Introduction Strecker aldehydes (SAs), namely 2-methylpropanal, 3-methylbutanal, 2methylbutanal, methional and phenylacetaldehyde, are powerful aroma molecules playing relevant roles in the flavour of wine and beer. In beer, 3methylbutanal and 2-methylbutanal were first proposed as responsible for the malt flavour, note characteristic of some alcohol-free beers (Beal and Mottram, 1994). Other authors later demonstrated that methional was in fact more relevant in such off-odour (Perpete and Collin, 1999a). Furthermore, the implication of methional and phenylacetaldehyde in some negative odour characteristics of oxidized/aged beer was demonstrated in 2004 (Soares da Costa et al., 2004) and confirmed in more recent studies (Saison et al., 2010; Wietstock et al., 2016). In the case of wine, the involvement of methional in the “cooked vegetables” note of oxidized wines was stablished in 2000 (Escudero et al., 2000) and phenylacetaldehyde was identified by GC-O (GC-Olfactometry) as one of the key odorants of oxidized white wines in 2003 (Silva Ferreira et al., 2003). The main roles played by these compounds in the odour notes of oxidized wines were further confirmed in 2007 (Cullere et al., 2007). These ubiquitous and powerful smelling molecules are chemically or biochemically related to the so called Strecker amino acids: valine, leucine, isoleucine, methionine and phenylalanine. The oxidative deamination of these amino acids in the presence of tea polyphenols to form the corresponding aldehydes was observed as soon as 1954 and was confirmed in the 70’s when Japanese researchers demonstrated that the degradation involved the reaction of the amino acid with a quinone derived from a flavanol undergoing oxidation (Saijō and Takeo, 1970a). The chemical routes leading to the formation of these aldehydes in oxidation-related processes have been relatively well established Strecker aldehydes are normal by-products of alcoholic fermentation linked to yeast sulfite metabolism 44 (Baert et al., 2012; Bueno et al., 2018; Grant-Preece et al., 2013; Rizzi, 2006; Wietstock et al., 2016). The fermentative origin of these compounds was first suggested also by (Saijō and Takeo, 1970b) who observed that supplementing fermenting tea leaves with phenylalanine resulted in phenylacetaldehyde formation. Nowadays, it is known that SAs are normal intermediates of the Ehrlich pathway in the amino acid metabolism, but the current believes establish that they are mostly reduced to the corresponding alcohols, so that levels of SAs in fresh beer or wines are thought to be negligible. However, there are some evidences pointing out that in certain conditions, yeast cannot reduce all the SAs. This was first observed in cold fermentation conditions for the production of alcohol-free beer (Perpete and Collin, 2000a). Such inability was tentatively attributed to the presence of sulphite or flavonoids (Perpete and Collin, 2000b). Other researchers further confirmed that refermentation of aged beer reduced but was not able to completely eliminate SAs and that residual levels were strain dependent (Saison et al., 2010). I.e., these works show that researchers have been long aware of the fact that aldehydes can form stable and reversible non-volatile adducts with SO2 (Baert et al., 2012; de Azevedo et al., 2007), that such adducts could limit the efficiency of yeast reductases, and that adducts could also play some role in the ulterior development of oxidized notes. In spite of this evidence, the potential importance of alcoholic fermentation as a relevant source of SAs remains unexplored. Recently, we developed an analytical method able to measure free aldehydes and to estimate the bonded fraction (Bueno et al., 2014). Using such methodology, it was possible to confirm that non-oxidized wines may contain a large pool of SAs under the form of sulphite adducts. These adducts are progressively cleaved Section I - Introduction 45 during the first stages of wine oxidation as free SO2 is depleted, concomitantly releasing the free forms of the aldehydes (Bueno et al., 2016). The production of SAs from oxidative degradation of amino acids was found to take place only when levels of free SO2 become smaller than 4 mg/L, suggesting that oxidative odour notes developed by some wines during aging could be in fact due to the simple release of sulphite adducts, and not to the oxidation of amino acids, alcohols or other precursors. Furthermore, PLS modelling suggested that SAs present in normal non-oxidized wines as sulphite adducts, could have been formed in fermentation as a consequence of a failure in the action of alcohol dehydrogenases, possibly induced by a lack of zinc, and likely by the aldehydeprotecting action of SO2 (Bueno et al., 2016). In order to confirm those evidences, the research presented in this paper, studies the formation of SAs in alcoholic fermentation using synthetic media resembling grape must. The major objectives are to assess the effects of the strain of yeast and of the levels of zinc and SO2 of the initial must on the levels of SAs formed in the alcoholic fermentation. Strecker aldehydes are normal by-products of alcoholic fermentation linked to yeast sulfite metabolism 46 2. Methodology 2.1 Reagents and standards Sodium metabisulfite (97%), zinc chloride (97%), hydrogen peroxide 3% stabilized w/v, indicator 4.4 mixed (methyl red-methylene blue), sodium hydroxide 0.01 mol/L, ortho phosphoric acid (85%) of VINIKIT line were obtained from Panreac (Barcelona, Spain). Dichloromethane (DCM), ethanol and methanol (≥ 99%) with Distol-Pesticide residue grade were supplied by Merck (Darmstadt, Germany). Glyoxal solution 40 wt. % in H2O was purchased from Sigma-Aldrich (Madrid, Spain). The internal standards methyl 2-methylbutyrate (≥ 99%) and 2-butanol (≥ 99%) were obtained from Merck, while 4-methyl-2-pentanol (99%), 4-hydroxi-4methyl-2-pentanone (99%), ethyl heptanoate (99%) 2-octanol (99.5%) and heptanoic acid (99 %) were purchased from Sigma Aldrich. Water was purified using Milli-Q® system from Millipore (Merck). 2.2 Culture conditions: 2.2.1 Synthetic must composition Synthetic must: the synthetic must resembling grape must was adapted from Bely, Sablayrolles, & Barre, 1990 and had the following composition: Oligoelements: MnCl2.4H2O 4.7 mg/L, Co(NO3)2·6H2O 0.49 mg/L, NaMoO4·2H2O 0.19 mg/L, CuCl2 0.54 mg/L, KIO3 1.29 mg/L, H3BO3 1 mg/L, SO4Mg·7H2O 0.2 g/L, KH2PO4 2 g/L, CaCl2·2H2O 0.155 g/L; Acids: malic acid 0.3 g/L, tartaric acid 3 g/L, citric acid 0.3 g/L, with pH adjusted to 3.5 with HCl; Vitamins -all supplied from Merck (≥ 98%): pyridoxine hydrochloride 1 mg/L, nicotinic acid 1 mg/L, calcium pantothenate 1 mg/L, thiamine hydrochloride 1 mg/L, p-aminobenzoic acid 1 mg/L, riboflavin 0.2 mg/L, folic acid 0.2 mg/L, Section I - Methodology 47 biotin 0.04 mg/L; myo-inositol (≥ 99%) 0.3 g/L, ergosterol (≥ 75%) 15 mg/L; Sugars were from Panreac Applichem (Spain): glucose 100 g/L; fructose 100 g/L; tween 80® 0.05 % (v/v) (Sigma-Aldrich); nitrogen source: (NH4)2HPO4 0.2199 g/L; amino acids (Merck) (mg/L): GABA 44.37, alanine 58.51, tyrosine 14.34, valine 17.73, isoleucine 14.43, leucine 13.42, aspartate 34.82, glutamic acid 61.83, glutamine 104.83, serine 21.21, glycine 1.11, histidine 109.2, threonine 18.8, arginine 199.5, proline 241.46, methionine 29.85, phenylalanine 11.15, lysine 3.33. Zinc was added from a stock solution of ZnCl2, 162 mg/L. SO2 was added from a freshly prepared Na2S2O5 solution, 5000 mg/L. 2.2.2 Yeast culture and fermentation set-up Yeast culture: three commercial Saccharomyces cerevisiae yeast strains were selected: L1 - Q23 (Lallemand), L2 - Merit (Chr. Hansen); L3 - Fermicru AR2 (Oenobrands). The yeast cells were hydrated for 1 hour at 35°C followed by the addition of synthetic must to activate them at the same temperature for around 30 min to 1hour. The fermenters were inoculated with 106 cells/ml. Must manipulation: the synthetic must was sterilized by means of sterile cellulose nitrate membrane filters with 0.45 µm pores (Albet, A&S Filter Co., Ltd) after its preparation and pH adjustment. All media manipulations were made inside a vertical laminar flow chamber PV-100 (Telstar, S.A), to ensure working under aseptic conditions. Small volumes were sterilized using syringe filters with 0.2 µm HT Tuffryn® membrane from PALL (New York, USA). All glassware was sterilized using an autoclave AES-28 from Raypa (Barcelona, Spain). Fermentation set-up: The first experimental set-up was performed without SO2 and the concentrations of zinc tested were 10 mg/L, 5 mg/L and 1 mg/L. In the Strecker aldehydes are normal by-products of alcoholic fermentation linked to yeast sulfite metabolism 48 second set-up all samples contained 30 mg/L of SO2 and the concentrations of zinc were 10 mg/L, 1 mg/L and 0 mg/L. Two biological replicates containing 150 mL of the must were used for each condition. Fermentation: Fermentations were carried out in 250 mL blue cap glass flasks (Ilmabor TGI, Germany) closed with airlock valves and were kept at constant temperature of 20°C. The progress of fermentation was monitored by daily control of the weight. Fermentation was considered finished when the loss of weight between two consecutive days was smaller than 0.1 g. Once fermentation was considered finished, the fermenters were sealed and sonicated for 15 minutes and were then introduced in an anoxic chamber from Jacomex (Dagneux, France) where they were let to sediment for 5 hours and were then aliquoted. Aldehyde and SO2 analysis were performed on the following hours, while the aliquots for the remaining aroma compounds were preserved in vials in the fridge. 2.3 Analytical methods 2.3.1 Classical oenological characterization The wines were characterized according to their general enological parameters using the recommended methodologies by OIV (International Organization of Vine and Wine): reducing sugars, total acidity and volatile acidity (International Organisation of vine and wine, 2011). pH was measured using a pHmeter. Wine total acidity was measured by titration with NaOH 0.1 N. Volatile acidity was titration with NaOH 0.02 M of the volatile fraction obtained by steam distillation. Residual sugars were calculated using the Fehling procedure based on oxidation of reducing sugars with CuII in alkaline media by boiling the solution. Excess of cupper not oxidized by the Section I - Methodology 49 sugars, oxidases iodine added as KI. The solution is then titrated with sodium thiosulfate using starch as indicator to determine I2. Free sulphur dioxide was determined by HeadSpace Gas Chromatography Mass Spectrometry (HS-GC-MS) using a GCMS-QP2010 from Shimadzu (Kyoto, Japan) as described in the literature (Carrascón et al., 2017). A DB-WAX column was used (30 m x 0.25 mm i.d x 0.25 µm film thickness) from J&W Scientific (Agilent Technologies, Santa Clara, CA USA). This method is based on the displacement of SO2 equilibrium forms with orthophosphoric acid (85%), in which 4.5 mL of wine with 20 µL of 2-cloroethanol (internal standard) are carfully capped in a 10 mL headspace vial. Just before the analysis the sample is acidified with 500 µL of orthophosphoric acid (85%). Total SO2 was analysed using the aspiration/ titration method described by Rankine and recommended by the OIV (International Organisation of vine and wine, 2011). 3 mL of the hydrogen peroxide 3% (p/v) with 3 drops of methyl red-methylene blue indicator and 2-3 drops of NaOH 0.01 M, so that the solution turns from purple to green, are prepared in a heart-shape flask with a bubbler. The 10 mL of wine are transferred to a round flask with 5 mL of H3PO4 at 20% are secured in the water vacuum system with a bubbler tube. The round flask is heated and the aspiration system is activated for 15 minutes. Total SO2 is measured by titration with NaOH 0.01 M. 2.3.2 Quantification of total aldehydes Total aldehydes were analysed using a previously described method (Bueno et al., 2014). Aldehyde-sulphite adducts (hydroxyalkylsulfonates) were previously cleaved by incubating the wine in strict anoxic conditions with 6 g/L of glyoxal at 50°C during 6 hours. Released aldehydes are further analysed by HeadSpace Solid Phase MicroExtraction Gas Chromatography Mass Spectrometry (HS- Strecker aldehydes are normal by-products of alcoholic fermentation linked to yeast sulfite metabolism 56 added final yeast zinc yeast*zinc yeast zinc yeast*zinc SO2SO2 total SO20.000 0.033 - 0.000 - 0.040 0.000 0.002 aldehydes 2-methylpropanal - - - 0.003 - - - - 3-methylbutanal - - - 0.011 0.002 0.011 0.018 0.000 2-methylbutanal - - - 0.003 - - - - methional 0.000 - - - - - 0.01 - phenylacetaldehyde 0.007 - - 0.000 0.001 0.000 0.000 0.000 fusel alcohols isobutanol 0.000 - - 0.000 0.014 0.047 0.006 0.000 isoamyl alcohol 0.000 - - 0.000 0.025 - 0.001 0.000 methionol 0.001 - - 0.000 - - - - 2-phenylethanol 0.002 0.010 - 0.000 0.010 0.009 0.000 - iso-acids 2-methylpropanoic acid 0.000 - - 0.000 - - - 0.000 3-methylbutanoic acid 0.000 0.010 - 0.000 - - - 0.000 aldehyde/alcohol ratio 2-methylpropanal/ isobutanol - - - 0.003 - - - 0.000 3-methylbutanal/ isoamyl alcohol 0.019 - - 0.001 0.007 0.041 - 0.000 methional/ methionol 0.002 - - 0.001 0.021 - 0.002 - phenylacetaldehyde / 2phenylethanol 0.001 - - 0.000 0.000 0.000 0.000 0.000 aldehyde/acid ratio 2-methylpropanal/ 2methylpropanoic acid - - - 0.049 - - - 0.000 3-methylbutanal/ 3methylbutanoic acid - - - 0.000 0.002 0.003 - 0.000 isobutanol/2-methylpropanoic acid 0.000 - - - 0.042 - 0.006 - isoamyl alcohol/ 3methylbutanoic acid 0.008 - - - 0.043 - - - 2-methylbutanal/ isovaleraldeyde 0.000 - 0.046 0.015 - - 0.004 0.003 medium chain fatty acids hexanoic acid 0.000 - 0.019 0.000 0.008 - 0.019 - octanoic acid 0.000 0.027 - 0.000 - - - - decanoic acid 0.001 - 0.029 0.009 - - - - esters isoamyl acetate 0.000 - - 0.000 - - 0.000 - ethyl hexanoate - 0.000 - - 0.000 - 0.004 - ethyl octanoate 0.001 0.000 0.006 0.023 0.000 - - - ethyl decanoate 0.000 0.000 0.001 0.032 0.001 - 0.001 - ester/acid ratios ethyl hexanoate/ hexanoic acid 0.010 0.000 - - 0.000 - 0.000 - ethyl octanoate/ octanoic acid - 0.000 - - 0.000 - - - ethyl decanoate/ decanoic acid - 0.000 - - 0.000 - 0.000 - 2-way ANOVA correlation without SO2 with SO2 Table 1 Summary of the significance of the effects played by the factors yeast and zinc as well as their interaction on the levels of total SO2, SAs and major fermentation volatiles assessed by two way-ANOVAs carried out in the two data sets; addition of external SO2 and the levels of total SO2 found after fermentation given by significance of their correlation with compounds and ratios. Section I – Results and Discussion 57 The ability of S. cerevisiae to form and excrete SAs during cold contact fermentation in the production of low alcoholic beers was demonstrated time ago (Perpete and Collin, 2000a). Similar conclusions were reached studying the reduction of aldehydes by re-fermentation (Saison et al., 2010). It is, however, believed that the formation of aldehydes by fermenting yeast in beer is most likely limited and of scarce importance, at least in comparison with other sources linked to wort production (Baert et al., 2012). The experiments carried out by Saison et al., 2010, much in accordance with all observations regarding the ability of yeast to reduce aldehydes during fermentation (Peppard and Halsey, 1981), suggest that SAs produced during malting and boiling will be reduced to the corresponding alcohols during fermentation, but that there is a fraction of SAs which will remain and is dependent on the yeast strain and wort composition. Likewise, this work hereby confirms that, during alcoholic fermentation of grape must, a fraction of SAs at sensory relevant levels remains in the wine and this is highly depend on the yeast strain, as seen in Table 1. Regarding the particular effects of each strain on levels of SAs, data in Table 2 reveal that levels formed are specific for each case. L1 produces in general smaller levels, reaching smallest values for 2-methylpropanal and 2methylbutanal, but levels of phenylacetaldehyde produced by this strain were relatively large. On the other hand, L3 produced maxima values of 3methylbutanal and phenylacetaldehyde, but levels of methional were close to those produced by L1. In contrast, L2 produced maxima levels of methional and minima of phenylacetaldehyde. This complex pattern of dependence would have been expected, since these aldehydes are produced during the synthesis of amino acids and are further reduced to alcohols by a complex and heterogeneous enzymatic system which has to restore the cell redox cycle (Peppard and Halsey, 1981). This pool is integrated by alcohol dehydrogenases (ADHs), aldehyde Strecker aldehydes are normal by-products of alcoholic fermentation linked to yeast sulfite metabolism 58 dehydrogenases and aldoketoreductases using either NAD(H) of NADP(H) as cofactors (Perpete and Collin, 1999b; Van Iersel et al., 1997). Attending to the previous discussion, levels of SAs retained in the wines after fermentation, should then be related to the amount of higher alcohols produced by the strain, to the selectivity and effectivity of the ADH-system of the strain, and eventually, to the differential level of any molecular species able to protect the aldehyde from reduction or oxidation, such as SO2 (Perpete and Collin, 2000b). The fact that the ratios between the levels of aldehyde and those of the corresponding alcohol are also significantly related to the yeast strain (Tables 1 and 2), supports that the specific ability of each strain to reduce these aldehydes exerts a major role on the final level of remaining aldehydes. Wines made with L3 have maxima aldehyde/ alcohol ratios (except methional/ methionol) and very low levels of higher alcohols (except methionol). On the contrary, wines made with L2 contained maxima levels of higher alcohols (except 2phenylethanol) and lowest aldehyde/alcohol ratios (except methional/methionol), suggesting that this strain reduces aldehydes to alcohols efficiently. Section I – Results and Discussion 59 L1 L2 L3 total SO2*22.1 ± 2b11 ± 1a32.8 ± 3c aldehydes 2-methylpropanal 3.6 ± 0.4a7.2 ± 0.7b6.1 ± 0.8b 3-methylbutanal*23.8 ± 2.1a23 ± 1.8 a 30.4 ± 3.2b 2-methylbutanal 1.7 ± 0.1 a 2.6 ± 0.2b2.7 ± 0.3b methional 23.7 ± 0.6 a 28.8 ± 0.7b24.7 ± 1.4a phenylacetaldehyde*14.2 ± 2.9b8.2 ± 1.5a18.6 ± 4.3c fusel alcohols isobutanol*13.9 ± 0.5a41.1 ± 2.3c18.1 ± 0.9b isoamyl alcohol 163 ± 6b242 ± 9c135 ± 10a methionol 4 ± 0.2 a 6.1 ± 0.2b6.1 ± 0.3b 2-phenylethanol*27.1 ±1.5c23 ± 0.9b20 ± 1.5a iso-acids 2-methylpropanoic acid 0.97 ± 0.0a2.2 ± 0.1c1.2 ± 0.1b 3-methylbutanoic acid 1.2 ± 0.0a1.9 ± 0.1b1.2 ± 0.1a aldehyde/alcohol ratio isobutiraldehyde/ isobutanol 0.26 ± 0.0ab 0.2 ± 0.0a0.4 ± 0.0c 3-methylbutanal/ isoamyl alcohol*0.15 ± 0.01a0.1 ± 0.0a0.2 ± 0.0 b methional/ methionol 6.3 ± 0.5c4.8 ± 0.2b4.2 ± 0.2a phenylacetaldehyde/ 2-phenylethanol*0.5 ± 0.1a0.4 ± 0.1a0.88 b 2-methylpropanal/ 2-methylpropanoic acid - - - 3-methylbutanal/ 3-methylbutanoic acid*20.6 ± 1.8 b 12.3 ± 1.1a26.5 ± 3.8c isobutanol/ 2-methylpropanoic acid 14.8 ± 1a18.9 ± 1b15.4 ± 1.1a isoamyl alcohol/ 3-methylbutanoic acid 141 ± 7b129 ± 6ab 112.5 ± 8.5a 2-methylbutanal/ 3-methylbutanal•0.1 ± 0.0a0.1 ± 0.0b0.1 ± 0.0 a medium chain fatty acids hexanoic acid•1.9 ± 0.0c1.2± 0.0b1 ± 0.1a octanoic acid 3.7 ± 0.2b2.4 ± 0.1a2.3 ± 0.1a decanoic acid•0.9 ± 0.1c0.6 ± 0.0 b 0.4 ± 0.0a esters isoamyl acetate 0.3 ± 0.0b0.5 ± 0.0c0.2 ± 0.0a ethyl hexanoate - - - ethyl octanoate•3.7 ± 0.2b2.4 ± 0.1a2.3 ± 0.1a ethyl decanoate•0.3 ± 0.1b0.2 ± 0.0b0.1 ± 0.0a ratios ethyl hexanoate/ hexanoic acid 14.5 ± 2.4a23 ± 5.9b23.6 ± 5.4b ethyl octanoate/ octanoic acid - - - ethyl decanoate/ decanoic acid - - - Table 2 Average levels of total SO2, aroma compounds and of some relevant ratios attending to the yeast strain. Concentration data are in mg/L except aldehydes which are in µ g/L. Cases showing significant interactions yeast x zinc are marked with • or * for the experiments without or with SO2, respectively. Significant differences attending to Duncan test are indicated with letters a-c being “a” the lowest average value. Strecker aldehydes are normal by-products of alcoholic fermentation linked to yeast sulfite metabolism 60 3.2 Role of SO2 It is worth mentioning that the final levels of total SO2 found in the fermenting media were in fact more related with the yeast strain than with the initial level of SO2 added to the must, as can be seen in Table 2 and in Figure 7. Table 2 shows that the average levels of SO2 remaining in samples fermented with L3 were the highest, with 32.8 mg/L, whereas those remaining in samples fermented with L2 were the lowest, with 11.03 mg/L. Figure 7 further illustrates that all samples fermented with L2 had final levels of total SO2 below 15 mg/L, even if the initial must contained 30 mg/L of this antioxidant. On the other hand, samples fermented with L3 without external SO2 contained 20-25 mg/L at the end of fermentation, and the external addition brought about an extra increase of nearly 20 mg/L. It is obvious that each yeast strain metabolizes SO2 differently, using it either as source of sulphur or, on the contrary, producing it from other sulphur sources, most likely to take advantage of its toxic effect on competing microorganisms. In fact, the resistance to SO2 is a genetically determined characteristic of yeasts linked with an interesting molecular mechanism only observed in wine strains (Perez-Ortin et al., 2002) which has received some attention for its potential industrial interest (Divol et al., 2012). Moreover, Nadai et al., 2016 have recently found that strains showing higher resistance to SO2, produced higher levels of SO2 in comparison to those sensitive to this molecule, which suggests that SO2 resistance and production are related. Furthermore, resistant strains were shown to have much higher basal gene expression level of SSU1, the gene considered the main responsible for sulphite tolerance by regulating the transport of this molecule through the plasmatic membrane (Avram and Bakalinsky, 1997), and in some strains, also of those genes related to sulphur metabolism. Section I – Results and Discussion 61 In any case, those data suggest that each strain of yeast has to contain a certain level of SO2 within the cell, and it can be postulated that such internal SO2 level will be correlated with the final level of SO2 remaining in the media after fermentation. As one of the most obvious reasons limiting the efficiency of yeast reductases would be that part of the aldehydes were protected by SO2, it can be further postulated that the ratios aldehyde/ alcohol and aldehyde/ acid should be significantly correlated to the final level of SO2 remaining in the media after fermentation. This seems to be the case, as shown in Table 1 and particularly in Figure 8, which highlights three examples. In this Figure, the average ratios of the different samples (means of two biological replicates) are segregated by yeast and represented versus the final content in total SO2 of the fermented media. Figure 8a corresponds to phenylacetaldehyde/ 2-phenylethanol ratio; Figure 8b to 3-methylbutanal/ 3-methylbutanoic acid ratio and Figure 8c to methional/ methionol ratio. It can be observed that in the first two cases the three strains followed a similar dependency, so that the fraction of aldehyde remaining 0 5 10 15 20 25 30 35 40 45 50 L1 L2 L3 total SO2, mg/L Total SO2formed by different yeast strains at the end of fermentation No SO₂ 30 mg/L SO₂ Figure 7 – Effects of yeast and of external SO2 on the final levels of SO2: Average final SO2 levels found in samples fermented with three types of strain and with or without addition of external SO2 (30 mg/L). Error bars are standard errors of the mean. Strecker aldehydes are normal by-products of alcoholic fermentation linked to yeast sulfite metabolism 62 per unit of alcohol (Figure 8a) or acid formed (Figure 8b) is directly proportional to the level of SO2, being the proportionality constant roughly independent of the strain of yeast. However, in the third case, the proportionality constant between the fraction of aldehyde remaining per unit of alcohol formed in yeast 3 is much smaller than those of yeast strains 1 and 2. This explains why the overall correlation coefficient between these parameters was not significant for methional (Table 1). As previously mentioned, this would be consistent with the hypothesis that SO2 present within the yeast cell (in both cytoplasm and mitochondria) is the main factor determining the final levels of SAs after fermentation. The strong strain dependency could, therefore, be primarily due to the yeast intrinsic metabolism of SO2. Figure 8 Aldehyde/alcohol or aldehyde/acid ratios and final SO2 content: Plots showing the relationship between important aldehyde/alcohol or aldehyde/acid ratios and the final SO2 levels found in samples fermented with three different yeast strains. A) phenylacetaldehyde/2-phenylethanol ratio; B) 3methylbutanal/3-methylbutanoic acid ratio; C) methional/ methionol ratio 0 0,5 1 1,5 2 2,5 010 20 30 40 50 60 Ratio total SO2 A. phenylacetaldehyde/ phenylethanol 0 10 20 30 40 50 60 010 20 30 40 50 60 Ratio total SO2 B. isovaleraldehyde/ isovaleric acid 0 2 4 6 8 10 12 010 20 30 40 50 60 Ratio total SO2 C. methional/ methionol L1 L2 L3 Section I – Results and Discussion 63 3.3 Role of Zn on SA formation As previously mentioned, the effects of Zn on the formation of SAs seems to be secondary (Table 1) only being significant in the cases of 3-methylbutanal and phenylacetaldehyde in the experiment in which the 0 level of zinc was considered. As shown in Table 3, the levels of those aldehydes were minimum when levels of Zn are 1 mg/L, becoming maxima at 10 and 0 mg/L. As in this experiment, levels of isobutanol, isoamyl alcohol and 2-phenylethanol were significantly smaller in conditions of Zn starvation, some of the ratios aldehyde/ alcohol become at Zn starvation significantly higher than those observed at 1 mg/L. The same is observed in the aldehyde/ acid ratio 3-methylbutanal/ 3methylbutanoic acid. Alcohol/ acid ratios were also significantly affected by Zn levels, significantly increasing with Zn levels (Table 3). These results suggest Zn levels may have an effect on the ADH activities of yeast which may have an indirect effect on SAs formation. In fact, Zn is an essential component of many dehydrogenases (De Smidt et al., 2008) which contain a zinc-containing active site (Persson et al., 1993). Our data suggest that the overall ADH efficiency of yeasts largely decreases in conditions of complete zinc starvation. These observations are consistent with the known fact that under low zinc condition, a regulatory metal-responsive protein alters several S. cerevisiae metabolic pathways including repressing some of the genes that express yeast ADH (Eide, 2009). 3.4 Fatty acids and their ethyl esters As expected, levels of fatty acids and of their ethyl esters were significantly influenced by the yeast strain, as shown in Table 1. As seen in Table 2, yeast strain L1 produced maxima levels of the three acids and of two of the ethyl esters, while L3 produced in most cases the smallest levels. Effects were not Strecker aldehydes are normal by-products of alcoholic fermentation linked to yeast sulfite metabolism 64 significant for ethyl hexanoate, for which the esterification ratio in L1 was minimum. However, the most remarkable effect on levels of fatty acids, their ethyl esters and their esterification rates are played by Zn content. As seen in Tables 1 and 3, Zn levels have a significant, but not very important effect on the absolute levels of hexanoic and octanoic acids, but quite intense effects on the levels of esters and on the esterification ratios. As seen in Table 3, levels of ethyl hexanoate, octanoate and decanoate reached maxima values in conditions of Zn starvation or of low Zn levels in the first experiment. Maximum levels were more than 3-4 times higher than the minimum levels. The effects on the esterification ratios followed a similar trend, with maxima levels in conditions of Zn starvation or of low Zn levels. To the best of our knowledge, the relevant effects of Zn on ethyl esters and particularly, on esterification ratios, have not been previously reported. By using genomic and transcriptomic analysis it has been known for a time that the activities of all the enzymes of the cytidine diphosphate diacylglycerol (CDPDAG) pathway, the major route in the synthesis of phospholipids, are decreased in Zn-limited cells (Iwanyshyn et al., 2004) while a different set of enzymes related to an alternative route of synthesis known as Kennedy pathway, displays more activity (Kersting and Carman, 2006; Soto and Carman, 2008). I.e., it is well established that in low Zn conditions there is a metabolic remodelling in the synthesis of phospholipids (Eide, 2009), and therefore it should not be surprising that the levels of fatty acids and their ethyl esters, which are by-products of such synthesis, change. Section I – Results and Discussion 65 10 mg/L 5 mg/L 1 mg/L 10 mg/L 1 mg/L 0 mg/L total SO2*20.3 ± 3.7b13.6 ± 3.5a13.8 ± 2.5 a --- aldehydes 2-methylpropanal - - - - - - 3-methylbutanal*---33 ± 1.8b24.5 ± 1.6a32.8 ± 3.4b 2-methylbutanal - - - - - - methional - - - - - - phenylacetaldehyde*---25.5 ± 5b17 ± 2a24.1 ± 4.9b fusel alcohols isobutanol*---30 ± 8b26.6 ± 5.4b21.6 ± 4.8a isoamyl alcohol - - - 207 ± 29b202 ± 16b163 ±17a methionol - - - - - - 2-phenylethanol*17.7 ± 2a20.6 ± 1.4ab 23.1 ± 1.2b27.2 ± 2.7b27.9 ± 1.4b23.8 ± 1a iso-acids 2-methylpropanoic acid - - - - - - 3-methylbutanoic acid 1.2 ± 0.1a1.5 ± 0.1b1.5 ± 0.2 b --- aldehyde/ alcohol ratio 2-methylpropanal/isobutanol - - - - - - 3-methylbutanal/isoamyl alcohol*---0.2 ± 0.0b0.1 ± 0.0a0.2 ± 0.0b methional/ methionol - - - 5.1 ± 0.3a5.6 ± 0.5a6.7 ± 1b phenylacetaldehyde/2-phenylethanol*---1 ± 0.3b0.6 ± 0.0a1 ± 0.2b aldehyde/ acid ratio 2-methylpropanal/2-methylpropanoic acid - - - - - - 3-methylbutanal/3-methylbutanoic acid*---26.9 ± 4.2b17.3 ± 2.8a23.6 ± 4.4b isobutanol/2-methylpropanoic acid - - - 20.4 ± 1.4b18.8 ± 1.9ab 13.6 ± 1.2a isoamyl alcohol/3-methylbutanoic acid - - - 151 ± 8b136 ± 13ab 108 ± 8a 2-methylbutanal/3-methylbutanal• - - - - - - medium chain fatty acids hexanoic acid• - - - 1.5 ± 0.2b1.4 ± 0.2ab 1.3 ± 0.2a octanoic acid 2.7 ± 0.2b2.3 ± 0.2a2.9 ± 0.4b--- decanoic acid• - - - - - - esters isoamyl acetate - - - - - - ethyl hexanoate 0.1 ± 0.0a0.1 ± 0.0a0.4 ± 0.0b0.2 ± 0.0a0.2 ± 0.0a0.7 ± 0.1b ethyl octanoate• 0.2 ± 0.0a0.1 ± 0.0a0.5 ± 0.1b0.3 ± 0.1a0.3 ± 0.1a1 ± 0.1b ethyl decanoate• 0.1 ± 0.0a0.1 ± 0.0a0.3 ± 0.1b0.1 ± 0.0a0.1 ± 0.0a0.4 ± 0.1b ratios ethyl hexanoate/hexanoic acid 8.7 ± 0.8a9.2 ± 0.6a31.9 ± 4b12.7 ± 1.1a12.4 ± 1a47.5 ± 7.7b ethyl octanoate/octanoic acid 5.0 ± 0.7a4.8 ± 0.4a14.8 ± 0.7b8.2 ± 1.1a8.2 ± 1.2a29.3 ± 3.6b ethyl decanoate/decanoic acid 15.7 ± 1.5a11.7 ± 0.8a44.8 ± 5.2b11.4 ± 1.2a12.7 ± 0.6a42 ± 3.8b No SO2 30ppm SO2 Table 3 Average levels of total SO2, aroma compounds and of some relevant ratios attending to the Zn levels of the must in the two experiments. Concentration data are in mg/L except aldehydes which are in µ g/L. Cases showing significant interactions Zn x yeast are marked with • or * for the experiments without or with SO2, respectively. Significant differences attending to Duncan test are indicated with letters a-c being “a” the lowest average value. Section II Roles of yeast on the formation and evolution of the aroma of Riesling and Garnacha wines Introduction and Methodology Chapter 1 Effects of sequential fermentation with different non-Saccharomyces on the formation and further evolution of Riesling aroma Chapter 2 The roles of yeasts on the formation and evolution of Garnacha wine aroma Chapter 3 Observations, questions and conclusions derived from the comparison between varieties Introduction and Methodology Section II - Introduction 77 1. Section II – Introduction For long, the aroma of wine has been known to have different origins and has been traditionally divided into grape-derived compounds, fermentative or aged related compounds. Nonetheless, aroma formation is an interactive and complex process, which implies that these three groups are not necessarily segregated. In fact, grape composition deeply modulates the activity of microorganisms carrying out fermentation so that fermentative profiles can be strongly dependent on the variety of grape (Ferreira et al., 1996; Hernández-Orte et al., 2002)). Similarly, the development and evolution of wine aroma with time and hence, its aging potential is related to a complex array of chemical processes acting on compounds derived from the grape or formed during fermentation (Ferreira and San Juan, 2012). The majority of aroma compounds in neutral grapes are present as non-volatile precursors. Many of them are conjugates of an aroma molecule and a nonvolatile and water-soluble molecule, such as a glycoside. They represent a fundamental source of precursors of varietal aroma compounds in wine. Glycosidic precursors are formed by one or more sugar moieties (glycones) linked to an aglycone which can originate a volatile odorant upon release. To date, several aglycones have been identified and vary from straight chain alcohols to terpenoids, shikimic acid metabolites or norisoprenoids. Glycosidic precursors are linked to the formation of important varietal compounds such as linalool, geraniol, b-damascenone, a-ionone and b-ionone or 1,1,6-trimethyl1,2-dihydronaphthalene (TDN) (Hjelmeland and Ebeler, 2015; Winterhalter and Rouseff, 2001; Winterhalter and Skouroumounis, 1997; Zoecklein et al., 1999). Studies with glycosidic precursors were first carried out with aromatic varieties such as Muscat, but also with Riesling for its high content in several of the Section II - Introduction 78 compounds previously mentioned, and for the particular interest aroused by the development with time of descriptors such as floral, citrus or kerosene (Fischer, 2007; Simpson, 1978; Simpson and Miller, 1983; Winterhalter et al., 1990; Zoecklein et al., 1999). On the other hand, little is known about the relevance of glycosidic precursors to form the aroma of red varieties, specially Garnacha. This variety has been described with black fruit, chocolate and even flowery notes however the compounds involved in the formation of these descriptors are not fully known (Lopez et al., 2004). In order to form an odorant, the aglycone has to be released from the glycoside either by slow acid hydrolysis at wine pH or by enzymatic hydrolysis. This last can be carried out by enzymes from the plant, but it takes mainly place by the action of the different microorganisms carrying out fermentation. Alcohols and monoterpenes have been identified as compounds that could be directly released from glycosidic precursors (Waterhouse et al., 2016; Williams et al., 1980, 1993). Nonetheless, some relevant aroma compounds are not formed by straight hydrolysis of the glycosidic bound between the sugar and the aglycone but are formed after further spontaneous chemical rearrangements of the aglycone. This is the case of some relevant aroma compounds derived from carotenoids like norisoprenoids (Fischer, 2007; Mendes-Pinto, 2009; Waterhouse et al., 2016; Winterhalter et al., 1990; Winterhalter and Rouseff, 2001; Winterhalter and Skouroumounis, 1997). Difficulties arise since the hydrolysis of one particular precursor can originate different compounds and one specific odorant can be often formed from different precursors. This makes that linking aroma compounds with specific precursors is rather difficult task (Waldmann and Winterhalter, 1992; Winterhalter and Skouroumounis, 1997; Zoecklein et al., 1999). Moreover, there is also evidence that most glycosides are not hydrolysed during winemaking or that they are Section II - Introduction 79 hydrolysed yielding non-volatile compounds like polyols, which by further chemical rearrangement will yield the aroma molecule (Williams et al., 1980; Zoecklein et al., 1999). This implies that fermentation can have a quite complex set of effects on the aroma potential of wine, and that many of these effects will not be identified but after long time. Furthermore, those effects can be further influenced by storage conditions, oxygen contact and presence of lees (Zoecklein et al., 1998, 1999), which adds more difficulties in the rationalization of the effects of fermentation on varietal aroma. The efficiency of the hydrolysis due to enzymatic activity is highly strain dependent, since the glycosidase activities of different strains can differ both in intensity and in the range of active substrates. Additional activities can be found in non-Saccharomyces genera, which has leaded to the development of fermentations combining cultures of non-Saccharomyces and Saccharomyces cerevisiae sequentially inoculated. These strategies have been shown to modulate aroma formation and to have potential to produce wines of higher quality and complexity, which has been attributed to the different abilities to release volatiles from grape precursors (Benito et al., 2015; Escribano et al., 2017; Padilla et al., 2016). However, few studies have actually been carried out with extensive analysis of the aroma compounds formed, and few less have taken into consideration the effects of aging, hence there are yet many open questions regarding the role of yeast in wine aroma formation and evolution, especially in what concerns their action on grape derived precursors. Section II - Introduction 80 2. Goals The present set of studies intends to bring some light into the roles played by yeasts, in the formation and development of varietal and fermentative aroma of wine. For this, a specific research involving sequential fermentations with different yeast strains, synthetic must containing real fractions of precursors from two grape varieties and different aging times has been carried out. The aims of the study are: 1. To determine the hierarchy of factors (yeast, precursors, time) affecting wine aroma profile. 2. To assess the specific effects linked to the presence of aroma precursors on wine aroma and on its evolution with time. 3. To assess the effects of yeast on varietal and fermentative aromas and on the evolution of aroma with time. 4. To derive general practical conclusions about the possibilities to modulate wine aroma using sequential fermentations. Section II - Methodology 81 3. Section II – Methodology 3.1 Reagents and standards Dichloromethane (DCM), ethanol and methanol (≥ 99%) Disto-Pesticide residue grade were supplied by Merck (Darmstadt, Germany). Milli-Q® system from Millipore (Merck, Germany). 2-butanol (≥ 99%), 4-methyl-2-pentanol (99%), 4-hydroxi-4-methyl-2pentanone (99%), ethyl heptanoate (99%) and heptanoic acid (99%) were used as internal standards for major compounds analysis and 2-octanol (99.5%), 3octanone (99%) and 3,4-dimethylphenol (99%) were used as internal standards for minor and trace compounds analysis and were purchased from Merck. The chemical standards used in this study were supplied by Merck with purity ³ 98%. TDN was synthesised by Synchem UG & Co with a purity of 80%. An alkane solution in dichloromethane (C7-C28) was used to calculate approximate linear retention index of analytes. 3.2 Glycosidic precursors extraction 3.2.1 Grape processing The glycosidic precursor fractions were obtained during harvest 2016 and approximately 23 Kg of grapes were obtained for each variety. Riesling grapes were obtained in Neustadt an der Weinstrasse, Germany and Garnacha grapes were given by Bodegas Román from D.O. Campo de Borja, Spain. The grapes were crushed by feet and cold macerated for 24 hours in the case of Riesling and 48 hours in the case of Garnacha, in the presence of LafazymâCL (Laffort, France). The differences of maceration time relate with the fact that white and red winemaking have originally different maceration periods, and thus this approximates real winemaking to the small-scale experiments. To protect Section II - Methodology 88 Table 4 Mass spectra ions selected to quantify minor and trace compounds using GC-MS. Compounds RT m/z Ethyl esters and acetates Ethyl isobutyrate 7.5 71a, 116 Ethyl 2-methylbutyrate 12.0 57a, 102 Ethyl 3-methylbutyrate 13.10 88a, 115, 70 Ethyl 4-methylpentanoate 24.15 88a, 101 Ethyl cyclohexanoate 45.55 83a, 101, 156 Isobutyl acetate 9.71 56a, 73 Phenylethyl acetate 79.90 91a Norisoprenoids Rose oxide 39.73/ 40.93 139a, 154 Vitispirane*52.8/ 53.08 192a, 93, 121, 171 Riesling acetal*59.9 138a, 125, 133 b-damascenone 79.86 69a, 190 a-ionone 72,4 121a, 93, 192 b-ionone 77.08 177a, 192 1,1,6-Trimethyl-1,2-dihydronaphthalene (TDN) 66.48 157a, 142, 172 Monoterpenes Linalool 55.01 71a, 93, 121 a-terpineol 64.05 93a, 121, 136 Geraniol 72.63 69a, 123 b-citronellol 68.13 69a, 81, 123 Lactones d-nonalactone 81.81 85a, 100 d-decalactone 87.36 85a, 100 Whiskylactone 74.56/ 78.18 99a, 114 Cinnamates Ethyl dihydrocinnamate 74.54 178a, 133 Ethyl cinnamate 86.80 131a, 176 Volatile phenols Guaiacol 73.5 109a, 124 o-cresol 81.16 108a, 79 m-cresol 85.35 108a, 79 4-ethylguaiacol 82.17 137a, 152 Eugenol 88.73 164a, 149 E-isoeugenol 96.83 164a, 149 4-ethylphenol 89.33 107a, 122 4-propylguaiacol 85.96 137a, 166 4-vinylguaiacol 90.14 150a, 135 4-vinylphenol 99.04 120a, 91 2.6-dimethoxyphenol 93.27 154a, 139 4-allyl-2,6-dimethoxyphenol 104.87 194a, 119 Vanillin derivates Vanillin 105.85 151a, 152, 123 Acetovanillone 108.83 166a, 123 Syringaldehyde 127.15 182a, 181, 167 *Compounds tentatively quantified using alkanes to determine the retention index; a Quantitative fragments m/z Section II - Methodology 89 For Vitispirane and Riesling acetal a commercial standard was not available thus, their identification was made using m/z and retention index from bibliography references (Loscos et al., 2007) in SCAN mode as well as injection of alkanes to calculate retention index in a DB-wax column. 3.5 Data treatment Relative areas were obtained by dividing the ion peak area of the analyte by the area of the corresponding internal standard. Those areas were transformed into concentrations by interpolation in the calibration graphs built by the analysis of calibrated samples. Data processing was made using Microsoft Excel Visual Basic for application (VBA) simple coding. Analysis of variance (ANOVA) was made on the compounds with area above the limit of quantification, assessing the factors presence of precursor fraction, yeast strain and accelerated aging time as well as the binary interactions (presence of precursors x yeast strain and yeast strain x aging time). Principal Component Analysis and Scatter plots were used to analyse the data. These analyses were performed using XLSTAT (Addinsoft, 2018 version). The complete data set for each grape variety was also analyzed by Principal Component Analysis (PCA) to assess the hierarchy of factors affecting the aroma formation. All graphics were made using Microsoft Excel, 2016 version. The data obtained for each variety were analysed individually in chapters I and II and a further comparative analysis was performed in Chapter III. Bibliography 90 4. Bibliography Bely, M., Sablayrolles, J.-M., and Barre, P. (1990). Automatic detection of assimilable nitrogen deficiencies during alcoholic fermentation in oenological conditions. Journal of Fermentation and Bioengineering 70, 246–252. Benito, S., Hofmann, T., Laier, M., Lochbühler, B., Schüttler, A., Ebert, K., et al. (2015). Effect on quality and composition of Riesling wines fermented by sequential inoculation with non-Saccharomyces and Saccharomyces cerevisiae. European Food Research and Technology 241, 707–717. Escribano, R., González-Arenzana, L., Garijo, P., Berlanas, C., López-Alfaro, I., López, R., et al. (2017). Screening of enzymatic activities within different enological non-Saccharomyces yeasts. Journal of Food Science and Technology 54, 1555–1564. Ferreira, V., Fernández, P., and Cacho, J. F. (1996). A Study of factors affecting wine volatile composition and its application in discriminant analysis. LWT - Food Science and Technology 29, 251–259. Ferreira, V., and San Juan, F. (2012). “Flavor of Wine,” in Food Flavors: Chemical, Sensory, and Technological Properties. Fischer, U. (2007). “Wine Aroma,” in Flavours and Fragrances: Chemistry, Bioprocessing and Sustainability, ed. R. G. Berger (Berlin, Heidelberg: Springer Berlin Heidelberg), 241–267. Hernández-Orte, P., Cacho, J., and Ferreira, V. (2002). Relationship between varietal amino acid profile of grapes and wine aromatic composition. experiments with model solutions and chemometric Study. Journal of agricultural and food chemistry 50, 2891–2899. Hjelmeland, A. K., and Ebeler, S. E. (2015). Glycosidically Bound Volatile Aroma Compounds in Grapes and Wine: A Review. American Journal of Enology and Viticulture 66. Lopez, R., Aznar, M., Cacho, J., and Ferreira, V. (2002). Determination of minor and trace volatile compounds in wine by solid-phase extraction and gas chromatography with mass spectrometric detection. Journal of Chromatography A 966, 167–177. Lopez, R., Ezpeleta, E., Sánchez, I., Cacho, J., and Ferreira, V. (2004). Analysis of the aroma intensities of volatile compounds released from mild acid hydrolysates of odourless precursors extracted from Tempranillo and Grenache Bibliography 91 grapes using gas chromatography-olfactometry. Food Chemistry 88, 95–103. Loscos, N., Hernandez-Orte, P., Cacho, J., and Ferreira, V. (2007). Release and formation of varietal aroma compounds during alcoholic fermentation from nonfloral grape odorless flavor precursors fractions. Journal of Agricultural and Food Chemistry 55, 6674–6684. Mendes-Pinto, M. M. (2009). Carotenoid breakdown products the— norisoprenoids—in wine aroma. Archives of Biochemistry and Biophysics 483, 236–245. OIV Compendium of International Methods of Analysis of Wines and Musts (2 vol.). oiv.int. Available at: http://www.oiv.int/en/technical-standards-anddocuments/methods-of-analysis/compendium-of-international-methods-ofanalysis-of-wines-and-musts-2-vol. Ortega, C., Lopez, R., Cacho, J., and Ferreira, V. (2001). Fast analysis of important wine volatile compounds Development and validation of a new method based on gas chromatographic – flame ionisation detection analysis of dichloromethane microextracts. 923, 205–214. Padilla, B., Gil, J. V., and Manzanares, P. (2016). Past and future of nonSaccharomyces yeasts: from spoilage microorganisms to biotechnological tools for improving wine aroma complexity. Frontiers in Microbiology 7, 1–20. Simpson, R. F. (1978). Aroma and compositional changes in wine with oxidation, storage and ageing. Vitis, 274–287. Simpson, R. F., and Miller, G. C. (1983). Aroma composition of aged Riesling wine. Vitis 22, 51–63. Waldmann, D., and Winterhalter, P. (1992). Identification of a novel vistispirane precursor in Riesling wine. Vitis 31, 169–174. Waterhouse, A. L., Sacks, G. L., and Jeffery, D. W. (2016). Understanding wine chemistry. Wiley. Williams, P. J., Sefton, M. A., and Marinos, V. A. (1993). Hydrolytic flavor release from non-volatile precursors in fruits, wines and some other plantderived foods. in, 283–290. Williams, P. J., Strauss, C. R., and Wilson, B. (1980). Hydroxylated linalool derivatives as precursors of volatile monoterpenes of muscat grapes. Journal of Agricultural and Food Chemistry 28, 766–771. Winterhalter, P., and Rouseff, R. (2001). “Carotenoid-Derived Aroma Compounds: An Introduction,” in Carotenoid-Derived Aroma Compounds, eds. P. Winterhalter and R. L. Rouseff (Washington, DC: American Chemical Bibliography 92 Society), 1–17. Winterhalter, P., Sefton, M. A., and Williams, P. J. (1990). Volatile C13 - norisoprenoid compounds, in Riesling wine are generated from multiple precursors. American Journal of Enology and Viticulture 41, 277–283. Winterhalter, P., and Skouroumounis, G. K. (1997). “Glycoconjugated aroma compounds: occurrence, role and biotechnological transformation,” in Advances in Biochemical Engineering/ Biotechnology (Berlin, Heidelberg: Springer Berlin Heidelberg), 73–105. Zoecklein, B. W., Hackney, C. H., Duncan, S. E., and Marcy, J. E. (1999). Effect of fermentation, aging and thermal storage on total glycosides, phenol-free glycosides and volatile compounds of White Riesling (Vitis vinifera L.) wines. Journal of Industrial Microbiology and Biotechnology 22, 100–107. Zoecklein, B. W., Jasinski, Y., and McMahon, H. (1998). Effect of fermentation, aging, and aging sur lie on total and phenol-free Riesling (Vitis vinifera L.) glycosides. Journal of Food Composition and Analysis 11, 240–248. Chapter 1 Effects of sequential inoculation with different nonSaccharomyces on the formation and further evolution of Riesling wine aroma Section II – Chapter 1 97 Chapter 1 - Effects of sequential fermentation with different non-Saccharomyces on the formation and evolution of Riesling aroma 1. Results and discussion The experimental approach followed in this work makes it possible to identify the aroma compounds that are exclusively formed from components present in the grape glycosidic precursor fraction, differencing them from those which are formed exclusively due to yeast metabolism. The former will be only found in samples containing precursors, fermented or non-fermented -acid-hydrolysis controls. The latter will be found in all the fermented samples, regardless of the presence of glycosidic precursor fraction. The approach also allows the identification of differential effects of yeasts on the formation of varietal compounds, first by comparing fermented samples with the unfermented controls containing just precursors from which, aroma compounds are formed by acid hydrolysis and second by comparing the samples fermented with different yeasts. Finally, the approach adds a time variable, since wines have been submitted to accelerated aging allowing the identification of different aging patterns linked to the presence of yeast. The wines were firstly characterized according to their classical oenological parameters and then a comparative analysis of the volatile composition among the different controls and wines spiked with glycosidic precursors was made. Effects of sequential fermentation with different non-Saccharomyces on the formation and further evolution of Riesling aroma 104 samples made only with S. cerevisiae are found in the middle of the plane and not clearly separated from those made with T. delbrueckii, which are at their left side. Finally, all samples fermented with L. Thermotolerans have negative scores in the first component. The influence of aging time is also seen in the figure, with aged samples having higher scores in the second component. However, the presence of precursors has no clear effect in the representation, meaning that the contents in fermentative volatiles are not highly affected by the presence of precursors in the fermenting must. The variable loading plot, given in the upper part of the figure, shows that nearly all components have positive loadings in the first component, which is particularly correlated with volatile fatty acids, their ethyl esters and with the acetates of fusel alcohols. Only 1-butanol, ethyl lactate and 1-hexanol, keep a negative correlation with the first component. This, certainly, indicates that samples fermented with P. kluyveri have the highest levels of most volatile compounds, notably of fatty acids, their ethyl esters and of the acetates of higher alcohols. As for the second component, it is positively correlated with the ethyl esters of branched acids, with g-butyrolactone and b-phenylethanol and negatively correlated with the acetates of fusel alcohols and with butyric acid. Figures 11 to 13 include a selection of plots showing the evolution with time of the different compounds in the wines fermented with different yeasts, in order to facilitate the interpretation of results. Figure 11, gives the plots with the evolution of acetates and other esters and acids. As can be seen, isoamyl acetate and phenylethyl acetate are found at much higher levels in samples fermented with P. kluyveri. It is also obvious that levels of acetates are slightly, but significantly, higher in samples not containing Section II – Chapter 1 105 precursors, suggesting that the acetyl transferase activity has been negatively influenced by the fraction of precursors. Nevertheless, recently fermented samples contain more than 2.5 mg/L of phenylethyl acetate, an amount exceeding, by far, the odour threshold of this compound. Levels of isoamyl acetate are, however, not particularly large. Both compounds follow a decreasing trend with time, since the acid-alcohol/ester equilibrium is displaced towards the dissociated form. Yet, levels of phenylethyl acetate after 5 weeks of aging are high enough to have high sensory implications. Hexanoic and decanoic acids, as well as their corresponding ethyl esters are also illustrated in Figure 11. Also, in this case, it is evident that samples fermented by P. kluyveri have the highest levels, although differences are not as marked as for acetates. Nonetheless, levels followed the order P. kluyveri < Saccharomyces < T. delbrueckii < L. Thermotolerans. Levels of the ethyl esters are not particularly high, but this can be partly attributed to the large evaporation rate of these compounds when fermentation is carried out in small volumes. Nevertheless, levels of the corresponding fatty acids are normal-high, promoting the ethyl esters content to remain constant with time, contrarily to the case of acetates. Effects of sequential fermentation with different non-Saccharomyces on the formation and further evolution of Riesling aroma 106 The most relevant higher alcohols in wine can be seen in Figure 12. In all cases, yeast strains had a significant effect, but the outcome is compound dependent. In the case of isoamyl alcohol, wines fermented with S. cerevisiae contained significantly higher levels, while wines fermented with P. kluyveri had the 0 100 200 300 400 500 600 0 1 2 5 Concentration ( ! g/L) Measurement time (week) Isoamyl acetate PR S. cerevisiae PR P. kluyveri PR T. delbrueckii PR L. thermotolerans CTL S. cerevisiae CTL P. kluyveri CTL T. delbrueckii CTL L. thermotolerans 0 500 1000 1500 2000 2500 3000 3500 4000 0 1 2 5 Concentration ( ! g/L) Measurement time (week) Phenylehtyl acetate S. cerevisiae P. kluyveri T. delbrueckii L. thermotolerans CTL S. cerevisiae CTL P. kluyveri CTL T. delbrueckii CTL L. thermotolerans 0 50 100 150 200 250 300 0 1 2 5 Concentration ( ! g/L) Measurement time (week) Ethyl hexanoate PR S. cerevisiae PR P. kluyveri PR T. delbrueckii PR L. thermotolerans CTL S. cerevisiae CTL P. kluyveri CTL T. delbrueckii CTL L. thermotolerans 0 500 1000 1500 2000 2500 3000 0 1 2 5 Concentration ( ! g/L) Measurement time (week) Hexanoic acid PR S. cerevisiae PR P. kluyveri PR T. delbrueckii PR L. thermotolerans CTL S. cerevisiae CTL P. kluyveri CTL T. delbrueckii CTL L. thermotolerans 0 50 100 150 200 250 0 1 2 5 Concentration ( ! g/L) Measurement time (week) Ethyl decanoate PR S. cerevisiae PR P. kluyveri PR T. delbrueckii PR L. thermotolerans CTL S. cerevisiae CTL P. kluyveri CTL T. delbrueckii CTL L. thermotolerans 0 500 1000 1500 2000 2500 0 1 2 5 Concentration (mg/L) Measurement time (week) Decanoic acid PR S. cerevisiae PR P. kluyveri PR T. delbrueckii PR L. thermotolerans CTL S. cerevisiae CTL P. kluyveri CTL T. delbrueckii CTL L. thermotolerans Figure 11 Yeast and aging effects on acetate esters, fatty acids and they ethyl esters – evolutions with time of two acetates, two ethyl esters and their corresponding fatty acids (µg/L) according to the presence (PR) or absence (CTL) of precursors and to the yeast genera that carried out fermentation; samples were taken after fermentation – 0 and 1, 2 and 5 weeks of accelerated aging. Acetates and ethyl esters show decreasing tendencies while acids show increasing tendencies with time. P. kluyveri outstands in the production of esters and of hexanoic acid. Section II – Chapter 1 107 smallest levels. On the contrary, for methionol and b-phenylethanol the levels follow the order P. kluyveri > T. delbrueckii > S. cerevisiae > L. Thermotolerans; In the case of isobutanol, the pattern for all yeast is similar to methionol and bphenylethanol with the exception of S. cerevisiae which can produce equivalent levels to those of P. kluyveri. Levels of these compounds remain fairly stable during aging, as can be seen in the figure. Regarding ethyl esters of branched acids, these compounds are formed by slow esterification of their corresponding acids. Accordingly, levels of the ethyl esters are close to 0 in the recently fermented samples, increasing with time, as shown in Figure 13. Levels of isobutyric acid are fairly stable with time. Regarding yeast aptitude to form these two compounds, the order was T. delbrueckii > P. kluyveri > S. cerevisiae > L. Thermotolerans and, as observed in the case of acetates, samples fermented without precursors have higher contents than those spiked with Riesling precursors. Fermentative compounds are by-products of yeast secondary metabolisms. Several aroma compounds are formed in routes related to yeast amino acids metabolism and a second group to yeast lipid metabolism. A third group, the acetates, are related with both metabolic routes since their production is formed by acetyl-CoA transferases acting on higher alcohols which are produced during the synthesis of amino acids. Thus, it is evident that these routes are highly strain-dependent and are also influenced by the interaction between non-Saccharomyces and S. cerevisiae (Fleet, 2003). In fact, and as shown in Figure 9 and 10, despite that all wines were sequentially inoculated with S. cerevisiae, non-Saccharomyces introduced a major source of aroma variability on all main metabolic outcomes. Effects of sequential fermentation with different non-Saccharomyces on the formation and further evolution of Riesling aroma 108 0 100 200 300 400 500 600 700 800 0 1 2 5 Concentration (µg/L) Measurement (week) Isobutyric acid PR S. cerevisiae PR P. kluyveri PR T. delbrueckii PR L. thermotolerans CTL S. cerevisiae CTL P. kluyveri CTL T. delbrueckii CTL L. thermotolerans 0 20 40 60 80 100 120 0 1 2 5 Concentration ( ! g/L) Measurement time (week) Ethyl isobutyrate PR S. cerevisiae PR P. kluyveri PR T. delbrueckii PR L. thermotolerans CTL S. cerevisiae CTL P. kluyveri CTL T. delbrueckii CTL L. thermotolerans Figure 12 Yeast and aging effects on branched acids and ethyl esters - one of the branched acids and its corresponding ethyl ester evolutions with time (µg/L) according to the presence (PR) or absence (CTL) of precursors and to the yeast genera that carried out fermentation; samples were taken after fermentation and after 1, 2 and 5 weeks of accelerated aging. Each yeast strains shows similar formation patterns for both compounds. The ester increases continuously and the acid is stable with time. 0 10000 20000 30000 40000 50000 60000 0 1 2 5 Concentration ( ! g/L) Measurement time (week) Isoamyl alcohol PR S. cerevisiae PR P. kluyveri PR T. delbrueckii PR L. thermotolerans CTL S. cerevisiae CTL P. kluyveri CTL T. delbrueckii CTL L. thermotolerans 0 1000 2000 3000 4000 5000 6000 7000 0 1 2 5 Concentration (µg/L) Measurement (week) Methionol PR S. cerevisiae PR P. kluyveri PR T. delbrueckii PR L. thermotolerans CTL S. cerevisiae CTL P. kluyveri CTL T. delbrueckii CTL L. thermotolerans 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 0 1 2 5 Concentration (µg/L) Measurement (week) β-phenylethanol PR S. cerevisiae PR P. kluyveri PR T. delbrueckii PR L. thermotolerans CTL S. cerevisiae CTL P. kluyveri CTL T. delbrueckii CTL L. thermotolerans 0 2000 4000 6000 8000 10000 12000 0 1 2 5 Concentration (µg/L) Measurement (week) Isobutanol PR S. cerevisiae PR P. kluyveri PR T. delbrueckii PR L. thermotolerans CTL S. cerevisiae CTL P. kluyveri CTL T. delbrueckii CTL L. thermotolerans Figure 13 Yeast and aging effects on fusel alcohols - major fusel alcohols evolution with time (µg/L) according to the presence (PR) or absence (CTL) of precursors and to the yeast genera that carried out fermentation; samples were taken after fermentation and after 1, 2 and 5 weeks of accelerated aging. L. thermotolerans produces lowest levels of these compounds. Fusel alcohols are quite stable during time. Section II – Chapter 1 109 The effects of P. kluyveri on the levels of fatty acids, their ethyl esters and particularly on the acetates of higher alcohols, clearly show that these metabolic routes have been much promoted in the presence of this yeast, although S. cerevisiae has carried out most part of the fermentation (Padilla et al., 2016). On the contrary, the presence of L. thermotolerans has the opposite effect, strongly limiting the number of esters, acetates and fatty acids produced during fermentation. On the other hand, L. thermotolerans has an outstanding capacity to produce ethyl lactate as can be seen in the variables plot of Figure 9. That is most likely linked to this yeast reported aptitude to form lactic acid during alcoholic fermentation (Benito et al., 2015; Gobbi et al., 2013; Kapsopoulou et al., 2007). Samples fermented with T. delbrueckii on its side, show fermentative volatile profiles closer to those of S. cerevisiae. Remarkably, the three nonSaccharomyces resulted in wines with smaller levels of isoamyl alcohol compared with fermentations carried entirely out by S. cerevisiae. This reduction may have sensory relevance since this compound is a strong suppressor of wine fruity and woody notes (de-la-Fuente-Blanco et al., 2016). Furthermore, these data suggest that sequential inoculation of non-Saccharomyces yeast strains leads to wines with higher aroma complexity (Escribano et al., 2017; Jolly et al., 2014). Indeed, all samples where non-Saccharomyces yeast were inoculated had final aroma content fairly different from S. cerevisiae revealing that this methodology has repercussions on the final wine profile. The fact that wines fermented with P. kluyveri have overall higher ester content and above their odour threshold is likely to result in more fruity and flowery-like wines. On the other hand, L. thermotolerans strongly limits the levels of fermentative odorants formed, suggesting that it can be an important modulator of wine tactile properties, due to its ability to produce lactic acid and its derivatives (Benito et al., 2015; Swiegers et al., 2005) and also that is able to produce wines in which non-fermentative notes will be more easily perceived. Effects of sequential fermentation with different non-Saccharomyces on the formation and further evolution of Riesling aroma 110 1.4 Varietal compounds in Riesling Figure 14 summarizes the PCA carried out on varietal aroma compounds found in the data set. Only samples containing precursors, fermented or not, were included in the analysis. The two first components retain nearly a 60% of the original variance. As can be seen in the plot, the factor most influential in the location of samples in the plane is aging time, followed by fermentation and the yeast strain. The freshly fermented samples have the most negative scores in the first component, with a single exception for S. cerevisiae. On the contrary, samples with 5 weeks of accelerated aging have highest scores in the first component. Additionally, unfermented samples have the highest scores of the second component contrarily to samples fermented with S. cerevisiae with equivalent aging times, which have the smallest scores on the same axis. The plot suggests that in this case, samples fermented with S. cerevisiae are the most different to the pure varietal aroma obtained by simple acid hydrolysis, while the intervention of non-Saccharomyces yeasts creates varietal wine profiles more similar to those observed by simple acid hydrolysis. A look at the sample loading plot of Figure 6, reveals that aging is related to geraniol and linalool decreases and with TDN and vinylphenols increases. Moreover, the existence of fermentation leads to increased levels of acetovanillone and minima of linalool, a-terpineol and b-damascenone. Figures 15-17 represent the evolution with time of varietal compounds in the wines fermented with the different yeast strains throughout time. Looking at the different plots, it should be noted that in some relevant cases, notably those of linalool, a-terpineol, Riesling acetal, b-damascenone and geraniol, unfermented controls contained always higher levels than fermented samples (Figures 15 and 16). Section II – Chapter 1 111 TDN β-damascenone Linalool α-terpineol β-citronellol Geraniol Guaiacol 4-vinylguaiacol 2-6dimethoxyphenol E-isoeugenol 4-vinylphenol vanillin acetovanillone β-ionone Ethyl cinnamate -1 -0,75 -0,5 -0,25 0 0,25 0,5 0,75 1 -1 -0,75 -0,5 -0,25 00,25 0,5 0,75 1 F2 (25,43 %) F1 (34,31 %) Variables (axes F1 and F2: 59,74 %) 0 1 2 5 0 1 25 0 1 2 5 0 12 5 0 1 2 5 -5 -4 -3 -2 -1 0 1 2 3 4 5 -4 -3 -2 -1 01234567 F2 (25,43 %) F1 (34,31 %) Observations (axes F1 and F2: 59,74 %) AH L. thermotolerans P. kluyveri S. cerevisiae T. delbrueckii Figure 14 Principal Component Analysis on varietal compounds quantified in fermented samples with Riesling precursor fraction, as well as unfermented controls spiked with precursors. The plot shows the projection of variables (top plot) or samples (bottom) showing the two biological replicatesin the plane formed by the first two components, which retained 59.74 % of the original variance. Numbers in the samples refer to the weeks of anoxic storage at 50°C. Effects of sequential fermentation with different non-Saccharomyces on the formation and further evolution of Riesling aroma 112 This pattern was not really expected and suggests that a large portion of these compounds’ precursors were not really glycosides, but different polyols, which by simple rearrangement in acid media, yielded the aroma compounds within the few weeks between the preparation of the synthetic musts and the time of analysis after fermentation. Although this might affect equally the fermented and unfermented samples, since the fraction is the same, the volatile compounds already present during fermentation will inevitably be partially co-evaporated with CO2 produced during fermentation, which helps explaining why levels in fermented samples are consistently smaller. Figure 15 Evolution with time of the levels of the main monoterpenes, linalool, geraniol and α-terpineol, in fermented samples and unfermented controls containing precursors extracted from Riesling grapes. Samples were taken at the end of fermentation (0) and after 1, 2 and 5 weeks of accelerated aging. Wines fermented exclusively with S. cerevisiae were used as controls and fermentations with non-Saccharomyces strains of P. kluyveri. T. delbrueckii and L. thermotolerans were sequentially inoculated with S. cerevisiae. AH (acid hydrolysis) was used as unfermented control of synthetic wine spiked with the glycosidic precursor fraction. Data of geraniol content in wines fermented by P. Kluyveri are given by a single sample. 0 20 40 60 80 100 120 140 160 0 1 2 5 Concentration ( ! g/L) Measurement time (week) Linalool AH S. cerevisiae P. kluyveri T. delbrueckii L. thermotolerans 0 20 40 60 80 100 120 140 160 180 200 0 1 2 5 Concentration ( ! g/L) Measurement time (week) " -terpineol AH S. cerevisiae P. kluyveri T. delbrueckii L. thermotolerans 0 10 20 30 40 50 60 0 1 2 5 Concentration ( ! g/L) Measurement time (week) Geraniol AH S. cerevisiae P. kluyveri T. delbrueckii L. thermotolerans Section II – Chapter 1 113 The different cases will be briefly analysed and discussed. Linalool and geraniol are quite unstable compounds at wine pH and have a general tendency to decrease during aging (Figure 15). The decreasing rate is mitigated since new molecules released from precursors replace the decomposed ones. As aforementioned, the lower levels in fermented controls could be attributed to the partial evaporation of early formed aroma compounds during fermentation. Differences between yeast strains were only moderately significant (see supplementary data) showing that wines fermented with P. kluyveri seem to have the lowest content of linalool, a-terpineol and geraniol. The action of such strain is not limited to a low efficiency in the hydrolysis of the precursors, but to the fact that precursors were probably transformed into different compounds. Otherwise, a slower rate of decrease should have been observed. Data also show that wines from L. thermotolerans have significantly higher levels of the three aroma compounds after 1 week of aging. This suggests that either the enzymes excreted by this strain during fermentation or upon cell autolysis were still active during accelerated aging, or that enzymes from this strain were particularly efficient at avoiding transforming precursors into molecules different to the targeted odorants. Most surprisingly, levels of geraniol were found to increase in P. kluyveri wines after 2 weeks of aging. Levels in these wines were above 50 µg/L, the highest of this compound observed. This peculiar result needs further experimental checking, since the analytical results of one biological replicate for geraniol was lost. The trend followed by a-terpineol is different, since the compound increases to a maximum level before starting to decrease, which is consistent with the fact Effects of sequential fermentation with different non-Saccharomyces on the formation and further evolution of Riesling aroma 120 Saccharomyces strains have similar levels between them and to those found in the unfermented control, may indicate low or even absent hydroxycinnamate decarboxylase activity in these yeast strains, suggesting that a large fraction is produced by simple acid hydrolysis of the glycoside. Nonetheless, normally and especially in red wines, vinyl phenols have decreasing tendencies with time due to their reactivity with anthocyanins. The formation of adducts with ethanol has also been described as potential cause for volatile phenols decreases in wine (Kennison et al., 2008; Waterhouse et al., 2016). Section II – Chapter 1 121 2. Conclusions The sequential inoculation fermentation approach used in this study introduces a large variability in the pattern of fermentative compounds and in the wine aroma profile. Wines obtained by fermentation with P. kluyveri were the most distinct, containing highest levels of fusel alcohol acetates, of fatty acids and of their ethyl esters. On the other hand, samples fermented with L. thermotolerans contained minima levels of those compounds. Remarkably, all wines obtained by sequential inoculation fermentation contained smaller levels of isoamyl alcohol, which is a strong odour suppressor. Levels of some relevant varietal aroma compounds were found at higher levels in unfermented controls, suggesting the existence of a pool of easily hydrolysable precursors, such as polyols, among Riesling precursors. Among fermented samples, those made with L. thermotolerans contained the highest levels of monoterpenes while those fermented with P. kluyveri contined minima levels of monoterpenes and norisoprenoids. The formation of TDN is timedependent but it is also strongly enhanced by fermentation, suggesting that hydrolytic activities of yeasts are essential to produce acid-hydrolysable precursors of this molecule. In comparison with S. cerevisiae, wines made with P. kluyveri have a lower ability to accumulate TDN. Most remarkably, samples fermented with the three non-Saccharomyces yeasts had much smaller levels of vinylphenols. In summary, results presented here further support that a sequential inoculation fermentation approach can be successfully used not only to modulate wine aroma but to control its evolution with time. Bibliography 122 3. Bibliography Benito, Á., Calderón, F., Palomero, F., and Benito, S. (2015). Combine Use of Selected Schizosaccharomyces pombe and Lachancea thermotolerans yeast strains as an alternative to the traditional malolactic fermentation in red wine production. Molecules 20, 9510–9523. Capone, D. L., and Jeffery, D. W. (2011). Effects of Transporting and Processing Sauvignon blanc Grapes on 3-Mercaptohexan-1-ol Precursor Concentrations. Journal of Agricultural and Food Chemistry 59, 4659–4667. Carrau, F. M., Medina, K., Boido, E., Farina, L., Gaggero, C., Dellacassa, E., et al. (2005). De novo synthesis of monoterpenes by Saccharomyces cerevisiae wine yeasts. FEMS Microbiology Letters 243, 107–115. Daniel, M. A., Capone, D. L., Sefton, M. A., and Elsey, G. M. (2009). Riesling acetal is precursor to 1,1,6-trimethyl-1,2-dihydronapthalene (TDN) in wine. Australian Journal of Grape and Wine Research 15, 93–96. de-la-Fuente-Blanco, A., Sáenz-Navajas, M.-P., and Ferreira, V. (2016). On the effects of higher alcohols on red wine aroma. Food Chemistry 210, 107–114. Escribano, R., González-Arenzana, L., Garijo, P., Berlanas, C., López-Alfaro, I., López, R., et al. (2017). Screening of enzymatic activities within different enological non-Saccharomyces yeasts. Journal of Food Science and Technology 54, 1555–1564. Fischer, U. (2007). “Wine Aroma,” in Flavours and Fragrances: Chemistry, Bioprocessing and Sustainability, ed. R. G. Berger (Berlin, Heidelberg: Springer Berlin Heidelberg), 241–267. Fleet, G. (2003). Yeast interactions and wine flavour. International Journal of Food Microbiology 86, 11–22. Gobbi, M., Comitini, F., Domizio, P., Romani, C., Lencioni, L., Mannazzu, I., et al. (2013). Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: A strategy to enhance acidity and improve the overall quality of wine. Food Microbiology 33, 271–281. Gök, R. (2015). Einfluss von Hefen auf die glykosidisch gebundenen Aromavorstufen und Untersuchungen zur Vermeidung der TDN-Fehlnote in Riesling. Hixson, J. L., Hayasaka, Y., Curtin, C. D., Sefton, M. A., and Taylor, D. K. (2016). Hydroxycinnamoyl glucose and tartrate esters and their role in the Bibliography 123 formation of ethylphenols in wine. Journal of Agricultural and Food Chemistry 64, 9401–9411. Jolly, N. P., Varela, C., and Pretorius, I. S. (2014). Not your ordinary yeast: nonSaccharomyces yeasts in wine production uncovered. FEMS Yeast Research 14, 215–237. Kapsopoulou, K., Mourtzini, A., Anthoulas, M., and Nerantzis, E. (2007). Biological acidification during grape must fermentation using mixed cultures of Kluyveromyces thermotolerans and Saccharomyces cerevisiae. World Journal of Microbiology and Biotechnology 23, 735–739. Kennison, K. R., Gibberd, M. R., Pollnitz, A. P., and Wilkinson, K. L. (2008). Smoke-Derived Taint in Wine: The Release of Smoke-Derived Volatile Phenols during Fermentation of Merlot Juice following Grapevine Exposure to Smoke. Journal of Agricultural and Food Chemistry 56, 7379–7383. Loscos, N., Hernández-Orte, P., Cacho, J., and Ferreira, V. (2009). Fate of grape flavor precursors during ptorage on yeast lees. Journal of Agricultural and Food Chemistry 57, 5468–5479. Marais, J. (1992). 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN): a possible degradation product of lutein and beta-carotene. South African Journal of Enology and Viticulture 13, 52–55. Mateo, J., and Jiménez, M. (2000). Monoterpenes in grape juice and wines. Journal of Chromatography A 881, 557–567. Mendes-Pinto, M. M. (2009). Carotenoid breakdown products the— norisoprenoids—in wine aroma. Archives of Biochemistry and Biophysics 483, 236–245. Padilla, B., Gil, J. V., and Manzanares, P. (2016). Past and future of nonSaccharomyces yeasts: from spoilage microorganisms to biotechnological tools for improving wine aroma complexity. Frontiers in Microbiology 7, 1–20. Simpson, R. F. (1978). Aroma and compositional changes in wine with oxidation, storage and ageing. Vitis, 274–287. Simpson, R. F., and Miller, G. C. (1983). Aroma composition of aged Riesling wine. Vitis 22, 51–63. Swiegers, J. H., Bartowsky, E. J., Henschke, P. a., and Pretorius, I. S. (2005). Yeast and bacterial modulation of wine aroma and flavour. Australian Journal of Grape and Wine Research 11, 139–173. Waldmann, D., and Winterhalter, P. (1992). Identification of a novel vistispirane precursor in Riesling wine. Vitis 31, 169–174. Bibliography 124 Waterhouse, A. L., Sacks, G. L., and Jeffery, D. W. (2016). Understanding wine chemistry. Wiley. Williams, P. J., Strauss, C. R., and Wilson, B. (1980). Hydroxylated linalool derivatives as precursors of volatile monoterpenes of Muscat grapes. Journal of Agricultural and Food Chemistry 28, 766–771. Wilson, B., Strauss, C. R., and Williams, P. J. (1984). Changes in free and glycosidically bound monoterpenes in developing Muscat grapes. Journal of Agricultural and Food Chemistry, 919–924. Winterhalter, P., and Rouseff, R. (2001). “Carotenoid-Derived Aroma Compounds: An Introduction,” in Carotenoid-Derived Aroma Compounds, eds. P. Winterhalter and R. L. Rouseff (Washington, DC: American Chemical Society), 1–17. Zoecklein, B. W., Hackney, C. H., Duncan, S. E., and Marcy, J. E. (1999). Effect of fermentation, aging and thermal storage on total glycosides, phenol-free glycosides and volatile compounds of White Riesling (Vitis vinifera L.) wines. Journal of Industrial Microbiology and Biotechnology 22, 100–107. Zoecklein, B. W., Jasinski, Y., and McMahon, H. (1998). Effect of fermentation, aging, and aging sur lie on total and phenol-free Riesling (Vitis vinifera L.) glycosides. Journal of Food Composition and Analysis 11, 240–248. Chapter 2 Effects of sequential inoculation with different nonSaccharomyces on the formation and further evolution of Garnacha wine aroma Section II - Chapter 2 129 Chapter 2 - The roles of yeasts on the formation and evolution of Garnacha wine aroma 1. Results and discussion Results presented in this chapter refer to fermentations carried out with synthetic must containing or not glycosidic precursors extracted from Garnacha grapes. The global goal of the chapter is to assess the roles of yeasts on the formation and evolution with time of aroma compounds in Spanish Garnacha wines. Specific goals are to better define what is varietal aroma of Garnacha wine and to assess the influence of yeast on its development during wine aging. A sequential inoculation protocol was followed, meaning that the sterile synthetic musts were inoculated first with a non-Saccharomyces strain and, after 4 days, with S. cerevisiae to complete the fermentation, except for one trial exclusively fermented with S. cerevisiae, which was kept as control. Half of the samples contained only synthetic must with all the necessary nutrients and elements to normal yeast metabolism and the second half were additionally spiked with the fraction of glycosidic precursors. Besides, unfermented control samples of synthetic wine spiked with precursors fraction were included in order to assess the role of acid hydrolysis and, particularly, enable a quantitative comparison between the efficiencies of acid versus enzymatic hydrolysis on the varietal aroma formation. A time variable, in which wine was aged in a complete anoxic environment for up to five weeks at 50ºC was also included. This set-up aims to further understand the origin and fate of the volatile compounds quantified in the wines, the role of different yeast genera and of slow hydrolytic processes, right after fermentation and during aging time. Impact of vineyard versus cellar microbiota from different harvests on the distinction of different Riesling vineyards 232 glycosidic precursors) not only in the formation of varietal character, but also and most importantly in the construction of specific terroir nuances in wine. Figure 48 Varietal aroma compounds in wines fermented aseptically and in the cellar from grapes from 5 different vineyards in 2016 harvest – Wines fermented from grapes handpicked aseptically from 7 different Pfalz regions and fermented in aseptic condition (vineyards 1-7) or in cellar (wg 1-7). Results are expressed in µ g/L. vineyard 1 vineyard 2 vineyard 3 vineyard 4 vineyard 5 vineyard 6 vineyard 7 wg1 wg2 wg4 wg5 wg6-202 wg6-77 wg6-775 wg7-209 wg7-30 20 30 40 50 60 70 80 90 100 110 120 Linalool Box plots (Linalool) vineyard 1 vineyard 2 vineyard 3 vineyard 4 vineyard 5 vineyard 6 vineyard 7 wg1 wg2 wg4 wg5 wg6-202 wg6-77 wg6-775 wg7-209 wg7-30 2 4 6 8 10 12 14 16 Geraniol Box plots (Geraniol) vineyard 1 vineyard 2 vineyard 3 vineyard 4 vineyard 5 vineyard 6 vineyard 7 wg1 wg2 wg4 wg5 wg6-202 wg6-77 wg6-775 wg7-209 wg7-30 0 2 4 6 8 10 12 Guaiacol Box plots (Guaiacol) vineyard 1 vineyard 2 vineyard 3 vineyard 4 vineyard 5 vineyard 6 vineyard 7 wg1 wg2 wg4 wg5 wg6-202 wg6-77 wg6-775 wg7-209 wg7-30 100 200 300 400 500 600 700 4-Vinylguaiacol Box plots (4-Vinylguaiacol) vineyard 1 vineyard 2 vineyard 3 vineyard 4 vineyard 5 vineyard 6 vineyard 7 wg1 wg2 wg4 wg5 wg6-202 wg6-77 wg6-775 wg7-209 wg7-30 0 200 400 600 800 1000 1200 4-Vinylphenol Box plots (4-Vinylphenol) Section III – Chapter 5 233 vineyard 1 vineyard 2 vineyard 3 vineyard 4 vineyard 5 vineyard 6 vineyard 7 wg1 wg2 wg4 wg5 wg6-202 wg6-77 wg6-775 wg7-209 wg7-30 0 500 1000 1500 2000 2500 3000 Isoamyl acetate Box plots (Isoamyl acetate) vineyard 1vineyard 2 vineyard 3 vineyard 4 vineyard 5 vineyard 6 vineyard 7 wg1 wg2 wg4 wg5 wg6-202 wg6-77 wg6-775 wg7-209 wg7-30 0 5 10 15 20 25 30 35 40 45 c-3-Hexenol Box plots (c-3-Hexenol) vineyard 1 vineyard 2 vineyard 3 vineyard 4 vineyard 5 vineyard 6 vineyard 7 wg1 wg2 wg4 wg5 wg6-202 wg6-77 wg6-775 wg7-209 wg7-30 0 100 200 300 400 500 600 700 800 900 Benzylic alcohol Box plots (Benzylic alcohol) vineyard 1 vineyard 2 vineyard 3 vineyard 4 vineyard 5 vineyard 6 vineyard 7 wg1 wg2 wg4 wg5 wg6-202 wg6-77 wg6-775 wg7-209 wg7-30 400 600 800 1000 1200 1400 1600 1800 Butyric acid Box plots (Butyric acid) vineyard 1 vineyard 2 vineyard 3 vineyard 4 vineyard 5 vineyard 6 vineyard 7 wg1 wg2 wg4 wg5 wg6-202 wg6-77 wg6-775 wg7-209 wg7-30 100 200 300 400 500 600 700 800 900 1000 Isobutyric acid Box plots (Isobutyric acid) vineyard 1 vineyard 2 vineyard 3 vineyard 4 vineyard 5 vineyard 6 vineyard 7 wg1 wg2 wg4 wg5 wg6-202 wg6-77 wg6-775 wg7-209 wg7-30 5 10 15 20 25 30 35 40 Isovaleraldehyde Box plots (Isovaleraldehyde) Figure 49 Fermentative aroma compounds in wines fermented aseptically and in the cellar from grapes from 5 different vineyards in 2016 harvest – Wines fermented from grapes handpicked aseptically from 5 different Pfalz regions and fermented in aseptic condition (vineyards 1-7) or in cellar (wg 1-7). Results are expressed in µ g/L. Impact of vineyard versus cellar microbiota from different harvests on the distinction of different Riesling vineyards 234 Figure 50 Varietal compounds in wines fermented aseptically and in the cellar from grapes from 5 different vineyards in 2015 harvest – Wines fermented from grapes handpicked aseptically from 5 different Pfalz regions and fermented in aseptic condition (vineyard 1-5) or in cellar (wg 1-5). Results are expressed in µ g/L. vineyard 1 vineyard 2 vineyard 3 vineyard 4 vineyard 5 wg1 wg2 wg3 wg4 wg5 0 20 40 60 80 100 120 Linalool Box plots (Linalool) vineyard 1 vineyard 2 vineyard 3 vineyard 4 vineyard 5 wg1 wg2 wg3 wg4 wg5 2 4 6 8 10 12 14 16 18 20 Geraniol Box plots (Geraniol) vineyard 1 vineyard 2 vineyard 3 vineyard 4 vineyard 5 wg1 wg2 wg3 wg4 wg5 0 2 4 6 8 10 12 14 16 18 Guaiacol Box plots (Guaiacol) vineyard 1 vineyard 2 vineyard 3 vineyard 4 vineyard 5 wg1 wg2 wg3 wg4 wg5 0 100 200 300 400 500 600 700 4-Vinylguaiacol Box plots (4-Vinylguaiacol) vineyard 1 vineyard 2 vineyard 3 vineyard 4 vineyard 5 wg1 wg2 wg3 wg4 wg5 0 200 400 600 800 1000 1200 4-Vinylphenol Box plots (4-Vinylphenol) Section III – Chapter 5 235 vineyard 1 vineyard 2 vineyard 3 vineyard 4 vineyard 5 vineyard 6 vineyard 7 wg1 wg2 wg4 wg5 wg6-202 wg6-77 wg6-775 wg7-209 wg7-30 20 30 40 50 60 70 80 90 100 110 120 Linalool Box plots (Linalool) vineyard 1 vineyard 2 vineyard 3 vineyard 4 vineyard 5 vineyard 6 vineyard 7 wg1 wg2 wg4 wg5 wg6-202 wg6-77 wg6-775 wg7-209 wg7-30 2 4 6 8 10 12 14 16 Geraniol Box plots (Geraniol) vineyard 1 vineyard 2 vineyard 3 vineyard 4 vineyard 5 vineyard 6 vineyard 7 wg1 wg2 wg4 wg5 wg6-202 wg6-77 wg6-775 wg7-209 wg7-30 0 2 4 6 8 10 12 Guaiacol Box plots (Guaiacol) vineyard 1 vineyard 2 vineyard 3 vineyard 4 vineyard 5 vineyard 6 vineyard 7 wg1 wg2 wg4 wg5 wg6-202 wg6-77 wg6-775 wg7-209 wg7-30 100 200 300 400 500 600 700 4-Vinylguaiacol Box plots (4-Vinylguaiacol) vineyard 1 vineyard 2 vineyard 3 vineyard 4 vineyard 5 vineyard 6 vineyard 7 wg1 wg2 wg4 wg5 wg6-202 wg6-77 wg6-775 wg7-209 wg7-30 0 200 400 600 800 1000 1200 4-Vinylphenol Box plots (4-Vinylphenol) Figure 51 Varietal compounds in wines fermented aseptically and in the cellar from grapes from 5 different vineyards in 2016 harvest – Wines fermented from grapes handpicked aseptically from 5 different Pfalz regions and fermented in aseptic condition (vineyard 1-5) or in cellar (wg 1-5). Results are expressed in µ g/L. Impact of vineyard versus cellar microbiota from different harvests on the distinction of different Riesling vineyards 236 2. Conclusions Wines made from the same grape variety and vineyard in two consecutive harvests show a completely different aroma composition. These differences are the likely result of specific climate conditions of each year, which closely relate to the vine management, including the degree of maturity of the grapes achieved in each year and also, the little changes in the winemaking practices introduce as a response to the specific conditions at which the grapes arrive to the cellar each harvest. These differences should translate into both changes in vineyard microflora and changes in grape berry composition, both of which can be potentially important aroma modulators. Although in this specific work, specifically related to the preliminary study of volatile compounds, it is not possible to make a definitive assessment of whether the observed changes are due to chemical differences or to differences in the microbiota, the study shows a relevant influence of the terroir within each vintage. Fermentative compounds such as esters highly reflect the harvest effect, whereas varietal compounds formed from grape glycosidic precursors show similar formation patterns in wines from 2015 and 216 harvest. On the contrary, polyfunctional mercaptans seem to be highly affected and their formation changes significantly from harvest to harvest. Grapes picked from the same vineyards and fermented under aseptic conditions or in commercial cellar environment originate different wines in which cellar microflora should have a major effect. Bibliography 238 3. Bibliography Capone, D. L., and Jeffery, D. W. (2011). Effects of transporting and processing Sauvignon blanc grapes on 3-mercaptohexan-1-ol precursor concentrations. Journal of Agricultural and Food Chemistry 59, 4659–4667. Parker, M., Capone, D. L., Francis, I. L., and Herderich, M. J. (2017). Aroma Precursors in Grapes and Wine: Flavor Release during Wine Production and Consumption. Journal of Agricultural and Food Chemistry. Peyrot des Gachons, C., Tominaga, T., and Dubourdieu, D. (2000). Measuring the aromatic potential of Vitis vinifera L. Cv. Sauvignon Blanc grapes by assaying S -cysteine conjugates, precursors of the volatile thiols responsible for their varietal aroma. Journal of Agricultural and Food Chemistry 48, 3387– 3391. Peyrot des Gachons, C., Tominaga, T., and Dubourdieu, D. (2002). Localization of S-cysteine conjugates in the berry: effect of skin contact on aromatic potential of Vitis vinifera L. cv. Sauvignon blanc must. American Journal of Enology and Viticulture 53, 144–146. Ribéreau-Gayon, P., Glories, Y., Maujean, A., and Dubourdieu, D. (2006). Handbook of Enology - Chemistry of wine, stabilization and treatments. 2nd ed. Tominaga, T., Baltenweck-Guyot, R., Des Gachons, C. P., and Dubourdieu, D. (2000). Contribution of volatile thiols to the aromas of white wines made from several Vitis vinifera grape varieties. Am. J. Enol. Vitic. 51, 178–181. Tominaga, T., Peyrot des Gachons, C., and Dubourdieu, D. (1998). A New Type of Flavor Precursors in Vitis v inifera L. cv. Sauvignon Blanc: S -Cysteine Conjugates. Journal of Agricultural and Food Chemistry 46, 5215–5219. Supplementary data Supplementary data 248 3. Supplementary data from Section II: Chapter 1 S.d. Table 4 3-way ANOVA assessing the effect of the factors: presence or absence of precursors, yeast strain, aging and their interaction on the volatile composition of Riesling synthetic wine. F and significance are indicated to each factor. Significance is expressed as *: <0.0001-0.001***; 0.001-0.01**; 0.01-0.05*. Ethyl acetate 18.0 *** 12.6 *** 1.3 n.s. 3.6 * 0.7 n.s. Isoamyl acetate 6.8 * 401.1 *** 24.2 *** 8.5 *** 21.6 *** Ethyl hexanoate 0.0 n.s. 96.7 *** 1.7 n.s. 6.8 *** 0.5 n.s. Ethyl octanoate 0.0 n.s. 81.9 *** 1.8 n.s. 4.8 ** 1.2 n.s. Ethyl decanoate 2.4 n.s. 28.8 *** 2.2 n.s. 2.6 * 1.1 n.s. Isobutanol 0.1 n.s. 128.7 *** 0.5 n.s. 2.1 n.s. 0.4 n.s. Isoamyl alcohol 0.7 n.s. 136.2 *** 0.1 n.s. 1.4 n.s. 0.1 n.s. Metionol 12.6 *** 221.4 *** 5.4 ** 1.5 n.s. 2.7 n.s. β-Phenylethanol 0.9 n.s. 111.8 *** 3.7 * 1.0 n.s. 1.5 n.s. Ethyl lactate 0.5 n.s. 16.1 *** 5.6 ** 1.0 n.s. 1.0 n.s. γ-Butyrolactone 1.7 n.s. 34.9 *** 45.2 *** 0.4 n.s. 2.6 n.s. Butyric acid 0.6 n.s. 2.1 n.s. 1.1 n.s. 0.8 n.s. 0.7 n.s. Isobutyric acid 28.5 *** 141.5 *** 1.2 n.s. 6.2 *** 1.7 n.s. Hexanoic acid 0.6 n.s. 367.6 *** 0.7 n.s. 12.5 *** 0.6 n.s. Octanoic acid 0.8 n.s. 340.0 *** 1.1 n.s. 9.6 *** 0.9 n.s. Decanoic acid 1.0 n.s. 18.9 *** 1.1 n.s. 0.1 n.s. 0.9 n.s. Ethyl isobutyrate 4.4 * 48.4 *** 76.7 *** 2.1 n.s. 9.8 *** Isobutyl acetate 19.6 *** 916.5 *** 57.6 *** 23.4 *** 61.2 *** Ethyl 2-methylbutyrate 0.2 n.s. 43.4 *** 91.1 *** 1.8 n.s. 10.2 *** Phenylethyl acetate 27.9 *** 1197.0 *** 61.7 *** 33.2 *** 68.9 *** γ-nonalactone 10.9 ** 145.0 *** 3.6 * 2.1 n.s. 0.8 n.s. γ-decalactone 1.1 n.s. 328.5 *** 7.6 *** 2.1 n.s. 1.9 n.s. TDN 34.2 *** 1.2 n.s. 11.1 *** 1.2 n.s. 0.4 n.s. β-damascenone 1100.1 *** 24.4 *** 8.2 *** 34.6 *** 0.8 n.s. Linalool 78.0 *** 4.8 ** 15.3 *** 5.0 ** 0.5 n.s. α-terpineol 186.8 *** 4.4 ** 12.0 *** 4.4 ** 0.4 n.s. β-citronellol 26.4 *** 3.8 ** 23.8 *** 1.0 n.s. 1.6 n.s. Geraniol 46.6 *** 3.7 * 1.4 n.s. 4.1 ** 2.1 n.s. 4-vinylguaiacol 122.7 *** 1.8 n.s. 14.3 *** 1.9 n.s. 0.7 n.s. 4-vinylphenol 166.9 *** 8.3 *** 6.4 *** 8.3 *** 2.7 ** vanillin 52.4 *** 7.3 *** 6.3 *** 8.0 *** 2.3 * acetovanillone 1102.0 *** 50.3 *** 1.5 n.s. 47.3 *** 1.5 n.s. Aging Yeast Precursors Precursors*yeast Yeast*Aging Supplementary data 249 Mosto CTL CTL CTL CTL Levadura Aging 0 1 2 5 0 1 2 5 0 1 2 5 0 1 2 5 0 1 2 5 0 1 2 5 Ethyl acetate 0 0 0 0 0 0 0 0 5625 ± 103 9936 ± 1171 13483 ± 1107 16193 ± 1708 30264 ± 262 29553 ± 1016 13523 ± 13523 23211 ± 778 28147 ± 996 29245 ± 131 27402 ± 1792 20699 ± 950 23011 ± 22656 22606 ± 21452 32724 ± 2401 24590 ± 592 Isoamyl acetate 0 0 0 0 0 0 0 0 30.6 ± 2.9 19.0 ± 3.8 14.8 ± 0.7 14.9 ± 4.5 32.4 ± 1.2 24.1 ± 0.0 17.1 ± 5.9 15.3 ± 1.0 492 ± 35 385 ± 38 416 ± 69 120 ± 19 395 ± 27 295 ± 65 283 ± 1 95.7 ± 13.9 Isobutyl acetate 1.75 1.50 1.18 1.26 2.39 1.60 1.79 1.39 17.2 ± 1.2 16.3 ± 1.1 16.9 ± 1.5 18.1 ± 1.2 20.5 ± 1.6 20.9 ± 0.3 20.7 ± 0.1 21.1 ± 1.0 263 ± 1 211 ± 3 165 ± 6 59.0 ± 5.0 194 ± 4 148 ± 12 128 ± 6 51.5 ± 2.4 Phenylethyl acetate 0.00 0.75 0.00 0.00 4.84 4.54 4.92 3.75 22.7 ± 1.1 17.1 ± 0.6 13.1 ± 0.6 9.4 ± 0.5 23.6 ± 0.6 18.7 ± 0.2 15.0 ± 0.2 11.4 ± 0.1 3727 ± 55 3078 ± 53 2477 ± 119 864 ± 55 2713 ± 62 2112 ± 215 1780 ± 16 671 ± 12 Ethyl hexanoate 0 0 0 0 0 0 0 0 70.1 ± 4.3 71.4 ± 31.0 65.1 ± 1.5 66.2 ± 12.0 94.7 ± 11.9 96.7 ± 10.1 86.8 ± 14.7 86.3 ± 3.9 123 ± 5 167 ± 14 129 ± 20 131 ± 3 162 ± 25 180 ± 62 171 ± 30 143 ± 28 Ethyl octanoate 0 0 0 0 0 0 0 0 104 ± 5 73.1 ± 30.8 99.3 ± 28.9 0.0 ± 0.0 138 ± 20 103 ± 4 111 ± 8 118 ± 10 168 ± 13 160 ± 17 189 ± 30 150 ± 1 146 ± 28 144 ± 13 148 ± 10 172 ± 33 Ethyl decanoate 0 0 0 0 0 0 0 0 34.2 ± 1.2 66.1 ± 11.4 54.3 ± 5.8 46.2 ± 27.5 47.9 ± 6.4 62.3 ± 13.6 48.0 ± 9.7 51.3 ± 3.4 87.9 ± 6.4 144 ± 47 92.1 ± 10.1 52.8 ± 4.7 141 ± 25 130 ± 83 73.8 ± 1.1 72.1 ± 17.7 Ethyl isobutyrate 0 0 0 0 0 0 0 0 1.1 ± 1.1 10.0 ± 0.5 21.9 ± 1.9 38.4 ± 6.1 1.4 ± 0.0 6.2 ± 0.4 13.6 ± 0.6 25.1 ± 2.7 4.2 ± 0.7 26.9 ± 1.6 48.8 ± 2.3 57.1 ± 1.2 2.7 ± 0.0 16.5 ± 0.8 33.3 ± 0.7 51.2 ± 17.0 Ethyl 2-methylbutyrate 0 0 0 0 0 0 0 0 0.0 ± 0.0 1.0 ± 0.1 2.3 ± 0.0 3.6 ± 0.5 0.6 ± 0.1 0.8 ± 0.0 2.0 ± 0.0 3.0 ± 0.5 0.0 ± 0.0 1.2 ± 0.1 2.4 ± 0.1 5.5 ± 0.1 0.6 ± 0.1 1.0 ± 0.1 2.5 ± 1.0 4.4 ± 0.9 Ethyl isovalerate 0 0 0 0 0 0 0 0 0.0 ± 0.0 0.9 ± 0.1 1.8 ± 0.2 3.4 ± 0.3 0.1 ± 0.0 0.7 ± 0.0 1.4 ± 0.1 2.8 ± 0.1 0.1 ± 0.0 0.9 ± 0.0 1.4 ± 0.1 3.9 ± 0.2 0.1 ± 0.0 0.6 ± 0.0 1.3 ± 0.1 3.1 ± 0.1 Ethyl lactate 0 0 0 0 0 0 0 0 100 ± 9 437 ± 9 668 ± 27 867 ± 68 110 ± 3 474 ± 18 841 ± 29 1158 ± 14 105 ± 24 413 ± 110 618 ± 136 1032 ± 311 117 ± 11 424 ± 19 785 ± 146 1226 ± 105 Isobutanol 0 0 0 0 0 0 0 0 8115 ± 946 7823 ± 1038 8226 ± 1900 7602 ± 966 8645 ± 441 8753 ± 201 9917 ± 197 9198 ± 159 8251 ± 510 9662 ± 714 8694 ± 186 7136 ± 552 9407 ± 3 9009 ± 1814 8744 ± 372 8409 ± 30 1-Butanol 0 0 0 0 0 0 0 0 60.0 ± 0.5 55.5 ± 4.0 55.2 ± 3.8 61.4 ± 2.4 63.8 ± 0.1 57.3 ± 6.6 58.4 ± 7.8 57.4 ± 8.8 47.1 ± 1.6 58.8 ± 13.3 63.6 ± 8.5 55.7 ± 0.7 54.0 ± 2.4 52.2 ± 0.7 53.8 ± 0.9 73.0 ± 18.4 Isoamyl alcohol 0 0 0 0 0 0 0 0 42922 ± 5794 41979 ± 6773 43341 ± 8181 41487 ± 5854 44279 ± 2897 44837 ± 1801 47300 ± 1647 45515 ± 2382 31823 ± 1978 33373 ± 3472 31011 ± 2881 31604 ± 2823 34994 ± 266 33848 ± 3496 35941 ± 1470 36035 ± 874 1-Hexanol 0 0 0 0 0 0 0 0 11.7 ± 0.1 7.5 ± 7.5 12.3 ± 0.1 9.9 ± 9.9 128 ± 4 137 ± 1 132 ± 3 141 ± 1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 5.4 ± 5.4 27.8 ± 0.4 31.3 ± 2.0 35.6 ± 0.5 39.1 ± 0.7 Metionol 0 0 0 0 0 0 0 0 2960 ± 112 3319 ± 360 3110 ± 439 3192 ± 220 3390 ± 166 3555 ± 64 3604 ± 38 3630 ± 107 3490 ± 75 4190 ± 251 4436 ± 217 4855 ± 23 3940 ± 29 4165 ± 860 5577 ± 733 5002 ± 90 β-Phenylethanol 0 0 0 0 0 0 0 0 4913 ± 521 5379 ± 717 5172 ± 788 5336 ± 643 5206 ± 139 5633 ± 257 5533 ± 40 5601 ± 215 5157 ± 53 5652 ± 391 6235 ± 352 7316 ± 42 5364 ± 188 5663 ± 580 7835 ± 983 7353 ± 46 γ-Butyrolactone 0 0 0 0 0 0 0 0 215 ± 41 932 ± 148 959 ± 98 965 ± 248 192 ± 8 870 ± 68 1025 ± 108 1050 ± 58 128 ± 86 875 ± 16 1021 ± 157 979 ± 50 240 ± 22 850 ± 99 1288 ± 233 1169 ± 124 γ-nonalactone 0.65 0 0 0.47 0 0 0 0 2.6 ± 1.6 4.7 ± 0.1 4.6 ± 0.0 4.9 ± 0.3 4.9 ± 0.1 4.9 ± 0.2 5.0 ± 0.1 5.3 ± 0.2 4.4 ± 0.1 5.0 ± 0.0 5.2 ± 0.1 5.2 ± 0.1 5.0 ± 0.3 5.0 ± 0.1 5.4 ± 0.4 5.5 ± 0.5 γ-decalactone 0 0 0 0 0 0 0 0 2.8 ± 0.4 2.9 ± 0.2 3.1 ± 0.1 3.0 ± 0.1 2.3 ± 0.1 2.8 ± 0.0 2.7 ± 0.2 2.9 ± 0.1 3.3 ± 0.3 3.9 ± 0.1 3.7 ± 0.2 4.2 ± 0.2 3.2 ± 0.1 3.7 ± 0.2 3.9 ± 0.3 4.3 ± 0.3 Butyric acid 0 0 0 0 0 0 0 0 285 ± 39 161 ± 27 104 ± 30 133 ± 42 360 ± 150 185 ± 15 210 ± 74 248 ± 3 2395 ± 2151 217 ± 6 442 ± 73 230 ± 82 278 ± 114 348 ± 186 262 ± 38 239 ± 12 Isobutyric acid 0 0 0 0 0 0 0 0 198 ± 16 219 ± 19 184 ± 25 233 ± 20 111 ± 2 98.7 ± 20.7 133 ± 5 141 ± 13 493 ± 24 500 ± 6 638 ± 54 332 ± 111 309 ± 9 272 ± 7 339 ± 1 294 ± 20 Hexanoic acid 0 0 0 0 0 0 0 0 1037 ± 113 1049 ± 63 1055 ± 15 1060 ± 78 1203 ± 61 1193 ± 18 1245 ± 58 1255 ± 43 2030 ± 48 2017 ± 179 2100 ± 146 2069 ± 9 2208 ± 134 2169 ± 222 2609 ± 37 2380 ± 51 Octanoic acid 0 0 0 0 0 0 0 0 2321 ± 122 2223 ± 200 2077 ± 97 2574 ± 69 2614 ± 95 2728 ± 33 2605 ± 155 2936 ± 113 3664 ± 374 3559 ± 229 3948 ± 460 4032 ± 134 3960 ± 214 3754 ± 358 4185 ± 171 4602 ± 213 Decanoic acid 0 0 0 0 0 0 0 0 960 ± 126 606 ± 63 610 ± 18 1067 ± 234 852 ± 149 741 ± 114 893 ± 105 1033 ± 17 1035 ± 131 796 ± 45 1271 ± 308 1417 ± 18 982 ± 58 1177 ± 414 1327 ± 5 1736 ± 226 TDN 0.84 0.27 0.27 0.20 1.61 5.06 10.4 37 1.5 ± 0.2 0.2 ± 0.0 0.3 ± 0.0 0.5 ± 0.5 1.4 ± 0.0 14.3 ± 0.7 47.4 ± 0.3 96.6 ± 0.4 0.5 ± 0.0 0.7 ± 0.0 0.0 ± 0.0 0.4 ± 0.0 0.5 ± 0.0 5.3 ± 0.8 20.1 ± 2.9 46.9 ± 6.4 β-damascenone 0 0 0 0 6.28 6.72 7.37 5.92 0.0 ± 0.0 0.1 ± 0.0 0.3 ± 0.0 0.3 ± 0.1 2.5 ± 0.0 3.4 ± 0.2 3.8 ± 0.0 4.5 ± 0.1 0.4 ± 0.0 0.5 ± 0.0 0.4 ± 0.0 0.3 ± 0.0 1.6 ± 0.0 2.6 ± 0.2 2.4 ± 0.2 3.0 ± 0.3 β-ionone 0.28 0.56 0.57 0.08 0.31 0.33 0.71 0.10 0.5 ± 0.0 0.2 ± 0.0 0.6 ± 0.1 0.5 ± 0.0 0.4 ± 0.0 0.2 ± 0.0 0.5 ± 0.0 0.4 ± 0.0 0.8 ± 0.0 0.3 ± 0.0 0.4 ± 0.0 0.1 ± 0.0 0.8 ± 0.1 0.3 ± 0.0 0.6 ± 0.2 0.3 ± 0.4 Ethyl cinnamate 00.13 0.00 0.02 0.05 0.09 0.10 0.15 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Linalool 0.76 2.10 1.98 0.29 150 114 76.7 9.37 4.2 ± 0.3 1.2 ± 0.1 2.8 ± 0.1 2.1 ± 0.1 62.9 ± 0.7 42.8 ± 1.3 7.1 ± 0.1 2.5 ± 0.0 2.2 ± 0.1 1.4 ± 0.1 1.9 ± 0.0 0.5 ± 0.0 46.3 ± 0.7 31.5 ± 4.9 9.4 ± 1.7 1.9 ± 1.4 α-terpineol 0.65 0.92 1.07 0.46 78.5 148 178 158 1.1 ± 0.1 1.5 ± 0.0 0.9 ± 0.5 1.0 ± 0.2 12.5 ± 12.0 139 ± 4 111 ± 1 50.1 ± 1.2 1.0 ± 0.0 0.9 ± 0.0 0.9 ± 0.1 0.3 ± 0.0 20.2 ± 0.4 73.1 ± 1.1 73.1 ± 2.7 26.1 ± 0.7 β-citronellol 0.23 0.71 0.63 00.43 0.60 0.86 02.3 ± 0.2 0.5 ± 0.0 0.8 ± 0.1 0.8 ± 0.2 6.9 ± 0.1 2.1 ± 0.0 1.1 ± 0.1 0.7 ± 0.1 1.3 ± 0.0 0.5 ± 0.0 0.6 ± 0.0 0.0 ± 0.0 4.5 ± 0.3 1.6 ± 0.1 1.1 ± 0.2 0.3 ± 0.5 Geraniol 01.75 0 0 38.7 36.2 26.6 1.88 2.6 ± 1.4 0.0 ± 0.0 0.4 ± 0.0 0.3 ± 0.3 9.6 ± 0.1 12.1 ± 0.0 1.7 ± 0.0 0.3 ± 0.3 0.4 ± 0.1 0.0 ± 0.0 0.5 ± 0.0 0.0 ± 0.0 5.5 ± 0.0 8.9 ± 1.6 51.2 38.5 ± 9.5 Guaiacol 0.06 0.04 0.06 0.00 0.41 0.38 0.58 1.86 0.3 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.3 ± 0.1 2.2 ± 0.1 2.1 ± 0.1 0.9 ± 0.3 5.3 ± 3.8 0.2 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.4 ± 0.1 0.9 ± 0.1 0.7 ± 0.1 1.0 ± 0.5 2.4 ± 0.6 4-vinylguaiacol 27.3 44.5 28.6 55.5 348 746 1404 5335 16.0 ± 9.9 27.1 ± 7.3 11.3 ± 4.4 67.0 ± 44.9 1902 ± 283 2016 ± 38 2207 ± 100 3496 ± 618 61.4 ± 2.1 24.0 ± 0.3 34.4 ± 8.1 106 ± 28 656 ± 11 682 ± 7 1506 ± 382 2705 ± 16 2-6-dimethoxyphenol 0.16 0 0 0.09 0 0 0 0 0.6 ± 0.2 0.1 ± 0.1 0.0 ± 0.0 0.5 ± 0.5 1.5 ± 1.5 0.0 ± 0.0 0.0 ± 0.0 4.1 ± 0.7 0.2 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.3 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 1.4 ± 0.1 E-isoeugenol 0 0 0 0 0 0.19 0 0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.4 ± 0.0 0.0 ± 0.0 0.2 ± 0.2 0.5 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.1 4-vinylphenol 14.6 15.9 14 36.1 364 944 1318 4299 149 ± 23 26.0 ± 1.5 16.7 ± 3.6 54.0 ± 36.8 4599 ± 237 2255 ± 78 2079 ± 214 2791 ± 523 70.7 ± 0.5 18.4 ± 1.3 35.2 ± 13.6 124 ± 26 1165 ± 85 825 ± 9 1065 ± 51 1774 ± 153 vanillin 1.58 0.67 00.78 13.16 14.3 41.9 91 1.8 ± 0.7 2.5 ± 1.2 1.0 ± 0.0 3.7 ± 2.5 9.8 ± 1.6 9.5 ± 0.0 10.0 ± 0.8 23.5 ± 9.5 1.6 ± 0.2 0.9 ± 0.1 1.1 ± 0.1 1.8 ± 0.4 7.2 ± 0.4 7.4 ± 1.4 6.6 ± 0.3 8.7 ± 1.4 acetovanillone 0.76 0 0 0 5.02 5.75 8.30 13.9 0.0 ± 0.0 1.0 ± 0.7 0.0 ± 0.0 2.3 ± 1.8 58.4 ± 0.8 60.7 ± 0.1 58.8 ± 4.7 58.2 ± 2.2 1.5 ± 1.5 0.3 ± 0.3 0.2 ± 0.2 0.0 ± 0.0 32.9 ± 0.9 29.4 ± 1.4 30.8 ± 1.4 32.1 ± 1.7 Control PR AH PR S. cerevisiae P. kluyveri CTL PR S.d. Table 5 Average concentration of volatiles measured above the limit of quantification in control wines and those spiked with glycosidic precursors from Riesling grapes in unfermented controls (Acid Hydrolysis) and in wines fermented with S. cerevisiae. P. kluyveri. T. delbrueckii and L. thermotolerans. Supplementary data 251 Mosto Levadura Aging 0 1 2 5 0 1 2 5 0 1 2 5 0 1 2 5 Ethyl acetate 15005 ± 370 14768 ± 1645 13958 ± 244 6059 ± 5986 43321 ± 1201 38412 ± 772 28832 ± 84 17938 ± 1208 13398 ± 2392 12579 ± 2117 11574 ± 1377 10963 ± 479 26460 ± 12517 33153 ± 3367 26992 ± 3771 17095 ± 3153 Isoamyl acetate 17.2 ± 2.2 16.9 ± 1.5 11.1 ± 1.4 0.0 ± 0.0 10.8 ± 1.9 11.0 ± 3.5 11.5 ± 1.9 0.0 ± 0.0 16.9 ± 2.4 12.3 ± 1.1 13.0 ± 3.3 0.0 ± 0.0 11.0 ± 1.4 16.3 ± 3.6 13.8 ± 2.0 12.5 ± 0.1 Isobutyl acetate 22.4 ± 0.2 21.2 ± 1.0 18.1 ± 0.9 15.6 ± 0.6 20.2 ± 5.4 18.0 ± 4.8 10.0 ± 1.6 13.8 ± 3.3 9.2 ± 0.4 8.0 ± 0.8 7.5 ± 0.3 7.5 ± 0.3 5.9 ± 1.8 6.4 ± 1.3 11.9 ± 5.5 7.5 ± 1.3 Phenylethyl acetate 48.9 ± 3.1 39.4 ± 3.0 32.7 ± 2.1 20.6 ± 1.1 48.0 ± 5.2 36.1 ± 4.0 16.6 ± 10.7 17.3 ± 1.2 3.6 ± 0.0 2.9 ± 0.1 2.6 ± 0.0 2.5 ± 0.1 5.3 ± 1.3 4.4 ± 0.9 18.7 ± 14.4 5.0 ± 0.5 Ethyl hexanoate 76.3 ± 6.1 67.1 ± 7.9 80.2 ± 7.6 59.6 ± 1.9 37.8 ± 21.0 39.7 ± 11.5 34.7 ± 5.9 0.0 ± 0.0 39.9 ± 1.5 37.9 ± 5.2 31.5 ± 2.4 32.3 ± 9.8 26.0 ± 5.6 43.7 ± 17.8 35.1 ± 6.0 21.5 ± 1.1 Ethyl octanoate 77.5 ± 10.3 66.7 ± 12.1 26.5 ± 26.5 0.0 ± 0.0 0.0 ± 0.0 27.5 ± 27.5 49.7 ± 19.8 0.0 ± 0.0 37.3 ± 6.1 27.0 ± 27.0 40.9 ± 4.7 34.7 ± 5.4 0.0 ± 0.0 20.9 ± 0.7 26.5 ± 9.4 25.1 ± 3.3 Ethyl decanoate 62.1 ± 0.2 53.8 ± 11.0 60.4 ± 1.5 74.8 ± 15.2 0.0 ± 0.0 48.0 ± 1.1 26.6 ± 1.7 0.0 ± 0.0 19.0 ± 19.0 43.2 ± 10.4 0.0 ± 0.0 27.5 ± 1.1 0.0 ± 0.0 0.0 ± 0.0 16.5 ± 1.1 0.0 ± 0.0 Ethyl isobutyrate 4.0 ± 0.3 25.7 ± 3.3 44.2 ± 2.0 93.2 ± 4.3 4.9 ± 1.2 27.0 ± 6.3 25.8 ± 9.8 77.9 ± 9.3 2.2 ± 0.4 5.2 ± 5.2 7.9 ± 7.9 31.2 ± 1.6 1.1 ± 1.1 6.4 ± 1.5 28.2 ± 17.9 26.6 ± 5.0 Ethyl 2-methylbutyrate 0.6 ± 0.0 1.7 ± 0.0 2.7 ± 0.2 5.9 ± 0.5 0.6 ± 0.0 1.8 ± 0.2 1.4 ± 0.5 5.2 ± 0.7 0.3 ± 0.3 0.3 ± 0.3 1.0 ± 0.2 1.1 ± 0.1 0.5 ± 0.0 0.4 ± 0.0 1.5 ± 0.8 2.3 ± 0.4 Ethyl isovalerate 0.0 ± 0.0 0.6 ± 0.0 1.1 ± 0.1 2.4 ± 0.2 0.0 ± 0.0 0.6 ± 0.1 0.6 ± 0.1 2.1 ± 0.5 0.0 ± 0.0 0.1 ± 0.1 0.2 ± 0.2 0.6 ± 0.1 0.0 ± 0.0 0.1 ± 0.1 0.7 ± 0.4 0.4 ± 0.4 Ethyl lactate 93.9 ± 10.8 335 ± 5 602 ± 49 1067 ± 184 119 ± 34 363 ± 93 588 ± 169 944 ± 176 364 ± 179 2313 ± 1097 3475 ± 1413 5141 ± 2459 1852 ± 1664 1060 ± 156 1839 ± 399 2910 ± 606 Isobutanol 7668 ± 225 7857 ± 293 8389 ± 482 8074 ± 654 7332 ± 1567 7865 ± 1237 7408 ± 1272 7344 ± 1344 5254 ± 632 5136 ± 244 5104 ± 197 5394 ± 277 4451 ± 452 4663 ± 962 4894 ± 917 4227 ± 675 1-Butanol 93.2 ± 1.8 105 ± 10 113 ± 2 110 ± 0 104 ± 6 102 ± 4 92.1 ± 13.5 93.5 ± 14.9 110 ± 2 115 ± 3 102 ± 16 96.9 ± 10.8 109 ± 1 110 ± 0 106 ± 4 91.3 ± 10.1 Isoamyl alcohol 35329 ± 1164 36262 ± 1071 37246 ± 957 39467 ± 1418 33266 ± 5029 34338 ± 4879 33867 ± 4951 34361 ± 4040 33012 ± 1798 33355 ± 102 32447 ± 744 32648 ± 730 34520 ± 5166 33537 ± 4542 35077 ± 5092 34306 ± 5380 1-Hexanol 12.1 ± 0.3 13.7 ± 0.8 14.0 ± 0.8 19.4 ± 2.0 130 ± 4 129 ± 1 137 ± 5 138 ± 3 12.0 ± 0.4 17.6 ± 2.1 14.4 ± 2.5 16.1 ± 0.5 159 ± 10 156 ± 2 156 ± 3 164 ± 5 Metionol 4114 ± 1 3871 ± 346 4315 ± 146 5400 ± 930 4355 ± 71 4220 ± 38 4446 ± 79 5587 ± 952 1483 ± 54 1616 ± 90 1307 ± 18 1616 ± 12 2404 ± 114 2188 ± 84 2291 ± 185 2389 ± 129 β-Phenylethanol 6538 ± 155 6240 ± 257 6571 ± 210 8521 ± 2431 6276 ± 304 5947 ± 347 6607 ± 431 7334 ± 677 3834 ± 148 4351 ± 22 4111 ± 133 4192 ± 320 4861 ± 911 4465 ± 567 4680 ± 938 4894 ± 879 γ-Butyrolactone 173 ± 6 657 ± 56 823 ± 31 1089 ± 204 237 ± 23 716 ± 38 811 ± 54 1076 ± 141 95.2 ± 49.5 959 ± 20 1022 ± 138 1115 ± 9 682 ± 503 819 ± 41 977 ± 65 1149 ± 36 γ-nonalactone 4.2 ± 0.0 4.2 ± 0.0 4.6 ± 0.0 4.5 ± 0.1 4.8 ± 0.1 4.8 ± 0.1 4.7 ± 0.5 5.3 ± 0.2 3.2 ± 0.1 3.1 ± 0.2 3.2 ± 0.2 3.6 ± 0.0 3.6 ± 0.3 3.7 ± 0.0 4.6 ± 0.8 4.2 ± 0.2 γ-decalactone 2.9 ± 0.0 2.9 ± 0.0 3.1 ± 0.1 3.2 ± 0.2 2.6 ± 0.1 2.8 ± 0.1 2.6 ± 0.2 3.0 ± 0.1 1.7 ± 0.2 2.1 ± 0.0 2.2 ± 0.0 1.9 ± 0.2 1.9 ± 0.1 2.1 ± 0.2 2.5 ± 0.6 2.1 ± 0.2 Butyric acid 102 ± 39 172 ± 3 89.0 ± 27.3 113 ± 63 116 ± 64 78.2 ± 23.3 168 ± 38 0.0 ± 0.0 494 ± 59 150 ± 21 356 ± 226 160 ± 59 229 ± 114 317 ± 225 242 ± 102 240 ± 104 Isobutyric acid 479 ± 13 479 ± 13 489 ± 11 413 ± 82 387 ± 59 410 ± 49 409 ± 26 446 ± 13 98.5 ± 44.8 166 ± 2 203 ± 37 227 ± 47 155 ± 6 144 ± 11 140 ± 22 143 ± 18 Hexanoic acid 1007 ± 59 948 ± 4 996 ± 8 1187 ± 263 603 ± 265 578 ± 256 650 ± 279 637 ± 249 488 ± 51 540 ± 54 342 ± 142 556 ± 12 301 ± 8 264 ± 1 291 ± 18 279 ± 14 Octanoic acid 1824 ± 31 1884 ± 207 1766 ± 172 1680 ± 386 1073 ± 513 1072 ± 535 1183 ± 481 1024 ± 502 984 ± 144 885 ± 55 895 ± 151 1002 ± 95 549 ± 116 505 ± 85 549 ± 60 602 ± 64 Decanoic acid 531 ± 36 500 ± 63 508 ± 98 559 ± 211 364 ± 178 1099 ± 929 509 ± 211 433 ± 197 628 ± 56 434 ± 32 762 ± 361 689 ± 73 814 ± 596 530 ± 318 559 ± 264 954 ± 457 TDN 0.5 ± 0.0 0.2 ± 0.0 0.7 ± 0.0 0.8 ± 0.3 0.5 ± 0.0 15.9 ± 1.2 35.2 ± 3.3 105 ± 5 1.3 ± 0.0 0.1 ± 0.1 0.1 ± 0.1 0.1 ± 0.1 0.5 ± 0.0 10.9 ± 0.7 38.8 ± 11.7 85.4 ± 10.5 β-damascenone 0.2 ± 0.1 0.3 ± 0.0 0.3 ± 0.0 0.1 ± 0.1 2.3 ± 0.3 3.1 ± 0.3 4.0 ± 0.5 4.4 ± 0.2 0.3 ± 0.0 0.1 ± 0.1 0.1 ± 0.1 0.2 ± 0.0 2.3 ± 0.3 3.2 ± 0.1 3.9 ± 0.2 4.0 ± 0.1 β-ionone 0.6 ± 0.0 0.5 ± 0.0 0.3 ± 0.0 0.6 ± 0.3 0.7 ± 0.0 0.5 ± 0.0 0.3 ± 0.0 0.4 ± 0.0 0.4 ± 0.0 0.3 ± 0.1 0.5 ± 0.0 0.4 ± 0.0 0.6 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0.3 ± 0.0 Ethyl cinnamate 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.0 0.2 ± 0.0 Linalool 2.4 ± 0.1 3.0 ± 0.0 1.5 ± 0.0 2.5 ± 1.2 73.8 ± 1.7 40.6 ± 1.1 10.2 ± 0.7 2.1 ± 0.0 3.5 ± 0.0 1.2 ± 0.1 1.7 ± 0.6 1.8 ± 0.1 65.3 ± 1.6 57.3 ± 2.5 11.2 ± 4.5 2.8 ± 0.5 α-terpineol 1.0 ± 0.0 1.6 ± 0.0 1.2 ± 0.0 1.0 ± 0.3 30.8 ± 1.9 138 ± 4 116 ± 3 48.4 ± 3.8 1.1 ± 0.0 1.2 ± 0.2 0.9 ± 0.3 0.7 ± 0.1 26.6 ± 1.6 134 ± 6 121 ± 11 66.8 ± 9.0 β-citronellol 1.2 ± 0.0 0.8 ± 0.0 0.5 ± 0.0 0.7 ± 0.3 5.6 ± 0.7 2.2 ± 0.2 0.7 ± 0.0 0.6 ± 0.1 1.4 ± 0.1 0.4 ± 0.1 0.3 ± 0.3 0.5 ± 0.1 4.4 ± 0.1 1.7 ± 0.2 0.8 ± 0.1 0.7 ± 0.1 Geraniol 1.1 ± 0.1 0.0 ± 0.0 0.3 ± 0.1 0.5 ± 0.6 13.3 ± 0.9 12.5 ± 1.4 2.9 ± 0.1 0.0 ± 0.0 1.1 ± 0.0 1.3 ± 1.0 1.4 ± 1.0 0.4 ± 0.1 12.4 ± 0.0 17.8 ± 1.1 3.5 ± 1.2 0.0 ± 0.0 Guaiacol 0.1 ± 0.1 0.1 ± 0.0 0.1 ± 0.0 0.2 ± 0.2 0.9 ± 0.3 0.4 ± 0.0 0.8 ± 0.0 1.1 ± 0.0 0.3 ± 0.1 0.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.7 ± 0.1 0.7 ± 0.0 0.9 ± 0.3 0.9 ± 0.0 4-vinylguaiacol 17.6 ± 5.1 13.0 ± 6.0 23.0 ± 3.0 60.3 ± 35.7 525 ± 156 1114 ± 65 1719 ± 117 2827 ± 25 45.9 ± 15.0 13.9 ± 10.9 28.8 ± 24.8 22.2 ± 1.0 399 ± 58 1055 ± 29 1748 ± 186 2458 ± 56 2-6-dimethoxyphenol 0.9 ± 0.9 0.0 ± 0.0 0.1 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 1.9 ± 1.9 0.0 ± 0.0 0.3 ± 0.1 0.2 ± 0.0 0.1 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 E-isoeugenol 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 4-vinylphenol 22.1 ± 2.8 21.4 ± 6.5 15.8 ± 1.6 30.4 ± 23.8 643 ± 230 1094 ± 169 1383 ± 96 2243 ± 41 134 ± 71 75.7 ± 58.4 80.9 ± 57.4 10.2 ± 10.2 469 ± 126 793 ± 82 1463 ± 164 1874 ± 21 vanillin 8.1 ± 7.1 1.0 ± 0.1 0.9 ± 0.0 1.6 ± 0.5 5.5 ± 0.6 7.4 ± 0.5 7.4 ± 0.1 16.5 ± 10.0 1.1 ± 0.3 0.7 ± 0.4 0.4 ± 0.4 1.3 ± 0.1 5.4 ± 0.4 7.9 ± 0.5 8.2 ± 0.3 5.2 ± 0.2 acetovanillone 5.8 ± 5.8 0.4 ± 0.4 0.2 ± 0.2 0.6 ± 0.1 49.6 ± 4.5 43.6 ± 3.9 41.5 ± 2.3 36.6 ± 9.7 0.5 ± 0.5 0.0 ± 0.0 0.4 ± 0.4 0.0 ± 0.0 48.8 ± 3.5 47.2 ± 7.1 42.8 ± 3.2 27.8 ± 2.3 PR L. thermotolerans PR CTL T. delbrueckii CTL S.d. Table 5 (cont) Average concentration of volatiles measured above the limit of quantification in control wines and those spiked with glycosidic precursors from Riesling grapes in unfermented controls (Acid Hydrolysis) and in wines fermented with S. cerevisiae. P. kluyveri. T. delbrueckii and L. thermotolerans. 252 S.d. Table 6 Average values of compounds above the limit of quantification according the yeast strain that carried fermentation and acidic hydrolysis (AH) of wines with or without Riesling precursors. The letters a-d express the ducan post-hoc test being a the highest value. AH P. kluyveri S. cerevisiae T. delbrueckii L. thermotolerans ethyl acetate 0 c 26053 a 17723 b 22287 ab 19027 b isoamyl acetate 0 b 310 a 21 b 9.8 b 12 b isobutyl acetate 1.6 c 152 a 19 b 17.4 b 8 c phenylethyl acetate 2.3 b 2177 a 16.4 b 32.4 b 5.6 b ethyl hexanoate 0 e 151 a 79.7 b 49.4 c 33.5 d ethyl octanoate 0 d 160 a 93.3 b 31 c 26.5 c ethyl decanoate 0 c 99 a 51.3 b 40.7 b 13.2 c ethyl isobutyrate 0 d 29.7 b 14.5 c 37.8 a 13.6 c ethyl 2-methylbutyrate 0 d 2.2 a 1.7 b 2.5 a 0.9 c ethyl lactate 0 b 590 b 582 b 514 b 2369 a isobutanol 0 d 8664 a 8534 a 7742 b 4890 c isoamyl alcohol 0 c 33579 b 43957 a 35517 b 33612 b metionol 0 d 4457 a 3344 b 4538 a 1912 c β-phenylethanol 0 d 6322 a 5346 b 6754 a 4424 c ethyl lactate 0 b 590 b 582 b 514 b 2369 a γ-butyrolactone 0 c 819 ab 776 ab 698 b 852 a 𝛾-nonalactone 0 d 5.1 a 4.6 b 4.6 b 3.7 c 𝛾-decalactone 0 d 3.8 a 2.8 b 2.9 b 2.1 c butyric acid 0 b 551 a 210 ab 105 b 274 ab isobutyric acid 0 d 397 b 165 c 439 a 159 c hexanoic acid 0 e 2198 a 1137 b 826 c 383 d octanoic acid 0 e 3963 a 2510 b 1438 c 746 d decanoic acid 0 d 1218 a 845 b 563 c 671 bc TDN 7 a 9.2 a 20.3 a 19.9 a 17.1 a β-damascenone 3.3 a 1.4 c 1.9 b 1.8 b 1.8 b linalool 44.5 a 11.8 b 15.7 b 17 b 18.1 b α-terpineol 70.9 a 24.5 b 39.7 b 42.2 b 44 b β-citronellol 0.4 b 1.2 a 1.9 a 1.5 a 1.3 a Geraniol 13.1 a 10.003 ab 3.381 c 3.840 c 4.733 bc 4-vinylguaiacol 999 ab 722 b 1218 a 787.5 ab 721.2 b 4-vinylphenol 876 b 636 b 1496 a 681.8 b 612.3 b vanillin 20.4 a 4.4 b 7.7 b 6.1 b 3.8 b acetovanillone 4.2 d 15.9 c 29.9 a 22.3 b 20.9 b Supplementary data 253 4. Supplementary data from Section II: Chapter 2 S.d. Table 7 3-way ANOVA assessing the effect of the factors: presence or absence of precursors. yeast strain. aging and their interaction on the volatile composition of Garnacha synthetic wine. Pr > F Precursors Yeast Aging Precursors* Yeast Precursors* Aging Yeast* Aging Ethyl acetate < 0.0001 0.00 n.s n.s 0.02 n.s Isoamyl acetate n.sa < 0.0001 n.s n.s n.s 0.01 Ethyl hexanoate 0.00 < 0.0001 0.00 0.01 0.03 0.02 Ethyl octanoate 0.00 < 0.0001 0.01 0.01 n.s n.s Ethyl decanoate 0.01 0.00 n.s n.s n.s n.s Isobutanol n.s 0.01 0.01 n.s n.s n.s 1-Butanol n.s 0.00 0.05 n.s n.s n.s Isoamyl alcohol 0.01 < 0.0001 n.s n.s n.s n.s 1-Hexanol < 0.0001 < 0.0001 0.00 0.00 0.01 n.s Metionol 0.01 < 0.0001 < 0.0001 n.s n.s n.s β-Phenylethanol n.s < 0.0001 n.s 0.04 n.s n.s Ethyl lactate 0.04 < 0.0001 < 0.0001 0.02 n.s < 0.0001 γ-Butyrolactone 0.00 < 0.0001 < 0.0001 0.01 n.s < 0.0001 Butyric acid n.s < 0.0001 0.00 n.s n.s 0.00 Isobutyric acid 0.05 < 0.0001 n.s n.s n.s n.s Hexanoic acid n.s < 0.0001 0.01 n.s n.s n.s Octanoic acid n.s n.s n.s n.s n.s n.s Decanoic acid 0.01 < 0.0001 0.01 0.00 n.s 0.02 Ethyl isobutyrate 0.01 < 0.0001 < 0.0001 n.s n.s 0.00 Isobutyl acetate 0.05 < 0.0001 0.00 0.04 n.s 0.01 Ethyl 2-methylbutyrate 0.00 < 0.0001 < 0.0001 0.03 0.05 0.01 Ethyl isovalerate 0.00 < 0.0001 < 0.0001 0.01 0.05 < 0.0001 Phenylethyl acetate 0.01 < 0.0001 < 0.0001 0.00 n.s < 0.0001 γ-nonalactone 0.01 0.01 n.s n.s n.s 0.05 γ-decalactone n.s < 0.0001 n.s n.s n.s n.s TDN < 0.0001 n.s < 0.0001 n.s < 0.0001 n.s β-damascenone < 0.0001 n.s 0.00 n.s 0.00 n.s Linalool < 0.0001 n.s < 0.0001 n.s < 0.0001 n.s α-terpineol < 0.0001 n.s < 0.0001 n.s < 0.0001 n.s 254 Pr > F Precursors Yeast Aging Precursors* Yeast Precursors* Aging Yeast* Aging β-citronellol < 0.0001 n.s < 0.0001 n.s 0.03 n.s Geraniol < 0.0001 0.01 0.00 n.s n.s 0.03 Guaiacol < 0.0001 0.01 0.00 0.01 0.00 n.s 4-vinylguaiacol < 0.0001 0.04 < 0.0001 0.03 < 0.0001 n.s 2-6dimethoxyphenol < 0.0001 0.02 0.00 0.03 0.00 n.s E-isoeugenol < 0.0001 0.00 n.s 0.00 n.s n.s 4-vinylphenol < 0.0001 n.s 0.00 n.s 0.01 n.s vanillin < 0.0001 n.s n.s n.s n.s n.s acetovanillone < 0.0001 n.s n.s n.s n.s n.s a n.s – not significant Supplementary data 255 S.d. Table 8 Average concentration of volatiles measured above the limit of quantification in control wines and those spiked with glycosidic precursors from Garnacha grapes in unfermented controls (Acid Hydrolysis) and in wines fermented with S. cerevisiae. P. kluyveri. T. delbrueckii and L. thermotolerans. Precursors Yeast Aging 0 1 2 5 0 1 2 5 0 1 2 5 0 1 2 5 0 1 2 5 0 1 2 5 Ethyl acetate 0 0 0 0 159 81.4 113 89.8 47916 ± 25983 40643 ± 400 51373 ± 815 75038 ± 3552 98265 ± 815 103863 ± 1277 105766 ± 8826 111643 ± 2094 115396 ± 3465 53785 ± 53547 134323 ± 6716 126562 ± 6215 188802 ± 1965 181048 ± 5838 171344 ± 4821 154900 ± 482 Isoamyl acetate 0 0 0 0 0 0 0 0 226 ± 107 407 ± 18 325 ± 8 233 ± 5 377 ± 24 377 ± 1 319 ± 27 224 ± 2 3415 ± 147 2998 ± 332 2727 ± 279 1378 ± 17 2997 ± 128 2557 ± 10 2235 ± 100 1191 ± 60 Isobutyl acetate 0 0 0.66 0.63 7.3 4.2 6.1 5.3 94.6 88.6 ± 5.4 96.2 ± 7.0 87.6 ± 4.6 88.2 ± 0.3 83.1 ± 2.9 82.3 ± 0.7 80.6 ± 1.8 219 ± 12 195 ± 10 186 ± 5 171 ± 13 252 ± 5 205 ± 0 197 ± 3 176 ± 7 Phenylethyl acetate 0 0 0 0 0 0 0 0 133 96.9 ± 5.2 150 ± 67 54.5 ± 1.2 150 ± 3 103 ± 0 92.6 ± 4.3 59.9 ± 0.9 9454 ± 676 7253 ± 446 4765 ± 365 9850 ± 94 6837 ± 363 6056 ± 318 3570 ± 354 Ethyl hexanoate 0 0 0 0 0 0 0 0 49.5 ± 0.5 218 ± 20 192 ± 22 544 ± 4 0.0 ± 0.0 628 ± 40 602 ± 54 595 ± 35 165 ± 13 179 ± 12 236 ± 1 210 ± 39 359 ± 54 618 ± 120 535 ± 39 501 ± 2 Ethyl octanoate 0 0 0 0 0 0 0 0 123 ± 123 294 ± 124 369 ± 27 929 ± 13 382 ± 82 1038 ± 83 1046 ± 66 1028 ± 17 235 ± 38 198 ± 75 408 ± 44 308 ± 86 199 ± 37 694 ± 42 827 ± 47 793 ± 40 Ethyl decanoate 0 0 0 0 0 0 0 0 75.7 ± 75.7 104 ± 51 128 ± 11 234 ± 1 109 ± 9 493 ± 37 424 ± 40 128 ± 128 89.2 ± 11.4 50.6 ± 5.5 57.9 ± 57.9 77.5 ± 32.7 109 ± 13 265 ± 96 317 ± 15 155 ± 17 Ethyl isobutyrate 0 0 0 0 0 0 0 0 55.5 146 ± 9 250 ± 29 386 ± 6 30.8 ± 0.5 117 ± 10 157 ± 3 277 ± 0 35.0 ± 5.2 141 ± 3 216 ± 15 400 ± 44 40.2 ± 5.1 112 ± 11 177 ± 21 320 ± 7 Ethyl isovalerate 0 0 0 0 0 0 0 0 6.6 21.1 ± 1.5 34.5 ± 2.4 58.8 ± 4.1 4.7 ± 0.1 16.3 ± 1.1 23.8 ± 0.1 51.6 ± 3.4 4.4 ± 0.1 14.8 ± 0.3 26.0 ± 0.1 54.8 ± 4.5 3.6 ± 0.0 13.1 ± 0.8 20.3 ± 0.3 45.6 ± 1.1 Ethyl 2-methylbutyrate 0 0 0 0 0 0 0 0 5.0 14.9 ± 1.2 24.3 ± 0.1 43.4 ± 2.3 3.6 ± 0.0 11.8 ± 0.8 17.5 ± 0.4 37.5 ± 1.8 4.7 ± 0.3 15.2 ± 0.2 26.4 ± 0.7 53.3 ± 4.2 4.5 ± 0.9 11.1 ± 0.6 17.3 ± 0.6 40.2 ± 0.8 Ethyl lactate 0 0 0 0 0 0 0 0 597 ± 0 1973 ± 3 2929 ± 12 4638 ± 64 524 ± 20 2027 ± 58 2914 ± 42 5187 ± 84 724 ± 26 2895 ± 38 4954 ± 3 7909 ± 163 771 ± 12 3111 ± 55 4739 ± 220 8145 ± 156 Isobutanol 0 0 0 0 0 0 0 0 45325 ± 1531 43427 ± 6037 74678 ± 12438 41391 ± 4333 32287 ± 2788 33469 ± 1743 80738 ± 43781 34710 ± 62 50869 ± 6169 46562 ± 4653 72764 ± 21711 46414 ± 1564 45094 ± 1215 60932 ± 20067 86347 ± 11814 43866 ± 750 1-Butanol 0 0 0 0 0 0 0 0 282 ± 84 319 ± 28 568 ± 91 307 ± 36 361 ± 43 340 ± 8 811 ± 480 354 ± 34 205 ± 20 184 ± 1 290 ± 95 187 ± 14 186 ± 20 290 ± 102 401 ± 45 220 ± 3 Isoamyl alcohol 0 0 0 0 0 0 0 0 191149 ± 21601 213686 ± 16738 221999 ± 21432 220444 ± 17295 192907 ± 1604 187282 ± 1231 202726 ± 8881 197827 ± 7893 183988 ± 396 180723 ± 8648 188716 ± 4510 185446 ± 9139 182229 ± 2320 178591 ± 5591 184375 ± 10388 198478 ± 892 1-Hexanol 0 0 0 0 0 0 0 0 51.1 ± 2.2 31.0 ± 1.0 29.2 ± 3.6 27.0 ± 2.4 127 ± 14 101 ± 4 97.2 ± 2.4 99.6 ± 5.5 32.5 ± 0.6 28.5 ± 0.3 26.5 ± 0.3 24.9 ± 3.5 79.0 ± 10.3 55.0 ± 0.4 59.3 ± 2.0 57.4 ± 1.5 Metionol 0 0 0 0 0 0 0 0 6215 ± 63 6312 ± 500 7113 ± 223 7042 ± 105 6453 ± 209 7297 ± 18 7650 ± 703 6612 ± 209 9467 ± 187 9795 ± 574 11083 ± 103 11266 ± 208 10042 ± 207 11590 ± 696 11968 ± 402 12035 ± 82 β-Phenylethanol 0 0 0 0 0 0 0 0 43093 ± 6427 28107 ± 972 29620 ± 2488 32684 ± 5416 30153 ± 1324 33933 ± 112 36173 ± 4712 34833 ± 2229 30452 ± 1547 26911 ± 655 26205 ± 643 30113 ± 165 27534 ± 803 34197 ± 423 34468 ± 2013 30689 ± 422 γ-Butyrolactone 0 0 0 0 0 0 0 0 1128 ± 192 6520 ± 8 8341 ± 622 9626 ± 48 1384 ± 121 6994 ± 90 8923 ± 562 10098 ± 234 870 ± 38 4026 ± 76 5497 ± 197 6007 ± 123 972 ± 21 4820 ± 357 6143 ± 371 7403 ± 443 γ-nonalactone 0.79 0 0.67 0.56 0.97 0.95 1.43 1.54 7.6 8.1 ± 0.2 14.5 ± 6.9 7.6 ± 0.1 10.0 ± 0.3 9.1 ± 0.4 9.4 ± 0.3 9.0 ± 0.1 8.6 ± 0.1 7.7 ± 0.2 8.2 ± 0.1 8.3 ± 0.5 10.4 ± 0.1 10.0 ± 0.1 10.1 ± 0.1 9.9 ± 0.4 γ-decalactone 0 0 0 0 0 0 0 0 4.9 5.1 ± 0.0 9.5 ± 4.9 4.6 ± 0.1 5.1 ± 0.4 4.3 ± 0.1 4.1 ± 0.1 4.3 ± 0.0 6.7 ± 0.1 6.2 ± 0.2 6.2 ± 0.0 6.3 ± 0.4 6.8 ± 0.1 6.2 ± 0.0 6.0 ± 0.4 5.8 ± 0.0 Butyric acid 0 0 0 0 0 0 0 0 652 ± 107 520 ± 18 543 ± 70 504 ± 10 660 ± 26 541 ± 2 646 ± 52 675 ± 32 4020 ± 197 2777 ± 215 2718 ± 174 1887 ± 335 3421 ± 103 3180 ± 213 2767 ± 246 1476 ± 109 Isobutyric acid 0 0 0 0 0 0 0 0 4618 ± 2256 2467 ± 80 2467 ± 200 2548 ± 117 1831 ± 162 1796 ± 11 1902 ± 102 1905 ± 48 2732 ± 127 2480 ± 154 2588 ± 128 2701 ± 194 2113 ± 135 2145 ± 139 2232 ± 53 2032 ± 72 Hexanoic acid 0 0 0 0 0 0 0 0 1257 ± 826 1359 ± 5 1413 ± 86 1044 ± 40 1905 ± 83 1139 ± 26 1227 ± 65 1140 ± 28 1874 ± 31 1382 ± 109 1304 ± 155 1443 ± 170 1652 ± 6 1048 ± 39 1068 ± 51 1042 ± 12 Octanoic acid 0 0 0 0 0 0 0 0 3121 ± 2071 3658 ± 68 4202 ± 174 3590 ± 26 5384 ± 158 3851 ± 198 4451 ± 643 4107 ± 18 4965 ± 71 4112 ± 152 3942 ± 438 4264 ± 470 4853 ± 176 3847 ± 46 3946 ± 13 3235 ± 16 Decanoic acid 0 0 0 0 0 0 0 0 333 ± 333 752 ± 304 961 ± 82 1191 ± 12 727 ± 188 961 ± 237 991 ± 13 1349 ± 157 676 ± 12 479 ± 1 1146 ± 135 1087 ± 216 655 ± 8 632 ± 130 825 ± 10 908 ± 76 TDN 0 0 0 0 0 0.81 7.5 24 0.5 0.4 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.8 ± 0.0 8.4 ± 0.5 18.8 ± 1.6 75.9 ± 6.5 0.7 ± 0.1 0.3 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.8 ± 0.1 7.1 ± 1.6 19.1 ± 2.1 66.3 ± 4.9 β-damascenone 0 0 0 0 1 3.5 7.1 7.1 0.4 0.2 ± 0.0 0.1 ± 0.1 0.0 ± 0.0 2.1 ± 0.1 3.4 ± 0.1 3.9 ± 0.4 4.5 ± 0.3 0.2 ± 0.0 0.1 ± 0.1 0.0 ± 0.0 2.2 ± 0.1 4.1 ± 0.0 1.4 ± 1.4 4.1 ± 0.1 β-ionone 0 0 0 0 0 0 0 0 0.6 0.6 ± 0.1 0.3 ± 0.3 0.2 ± 0.0 0.5 ± 0.0 0.7 ± 0.1 0.2 ± 0.1 0.2 ± 0.0 0.8 ± 0.0 0.3 ± 0.0 0.5 ± 0.3 0.4 ± 0.3 0.5 ± 0.0 0.6 ± 0.2 0.5 ± 0.2 0.1 ± 0.0 Ethyl cinnamate 0 0 0 0 0 0 0 0 0.0 0.0 ± 0.0 8.6 ± 8.6 1.1 ± 0.1 0.0 ± 0.0 0.2 ± 0.0 0.6 ± 0.1 1.6 ± 0.2 0.0 ± 0.0 0.1 ± 0.0 0.5 ± 0.2 0.0 ± 0.0 0.2 ± 0.0 0.4 ± 0.0 0.7 ± 0.4 1.0 ± 0.4 Linalool 0 0 0 0 1.5 9.6 12.7 6.9 3.3 2.3 ± 0.1 1.0 ± 0.2 0.8 ± 0.0 8.2 ± 0.0 18.4 ± 1.2 12.8 ± 0.1 2.8 ± 0.1 2.4 ± 0.1 2.0 ± 0.1 1.7 ± 0.3 0.8 ± 0.5 7.1 ± 0.1 19.9 ± 0.2 13.1 ± 0.3 2.9 ± 0.1 α-terpineol 0 0 0 0 0 7.4 19.5 23.0 1.1 2.1 ± 0.1 2.4 ± 0.1 1.6 ± 0.2 3.0 ± 0.0 20.5 ± 0.8 25.1 ± 2.0 23.8 ± 0.5 0.7 ± 0.1 1.5 ± 0.0 2.0 ± 0.1 1.8 ± 0.1 2.7 ± 0.1 18.1 ± 0.3 25.2 ± 0.3 26.7 ± 0.3 β-citronellol 0 0 0 0 0 0 0 0 2.6 1.8 ± 0.1 1.1 ± 0.1 0.3 ± 0.0 4.9 ± 0.2 3.5 ± 0.2 2.6 ± 0.1 0.9 ± 0.1 2.2 ± 0.2 1.4 ± 0.1 1.1 ± 0.1 0.4 ± 0.2 3.4 ± 0.1 2.6 ± 0.1 1.9 ± 0.2 0.8 ± 0.0 Geraniol 1 0 1 1 2 3 5 3 1.8 0.9 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 3.5 ± 0.2 5.2 ± 0.3 3.6 ± 0.2 1.7 ± 0.0 2.5 ± 0.0 1.3 ± 0.1 1.1 ± 0.0 0.8 ± 0.8 4.4 ± 0.2 5.8 ± 0.2 4.2 ± 0.4 2.3 ± 0.2 Guaiacol 0 0 0 0 0 1 1 2 0.2 0.2 ± 0.0 0.2 ± 0.0 0.1 ± 0.0 4.3 ± 0.3 4.2 ± 0.1 5.1 ± 0.9 5.8 ± 0.6 0.1 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0.3 ± 0.1 3.2 ± 0.5 2.4 ± 0.2 4.0 ± 0.3 6.3 ± 0.5 4-vinylguaiacol 12.7 5.6 5.8 16.5 77.6 127 481 649 20.2 16.3 ± 3.1 18.8 ± 0.6 6.2 ± 1.3 720 ± 5 1588 ± 49 2045 ± 236 2516 ± 123 5.7 ± 1.3 13.7 ± 1.8 12.7 ± 4.3 21.9 ± 0.7 512 ± 59 1277 ± 87 1999 ± 5 2731 ± 75 2-6-dimethoxyphenol 2 0 0 0 0 0.9 3.1 7.0 0.2 0.3 ± 0.1 0.5 ± 0.3 1.0 ± 0.8 11.2 ± 2.6 13.8 ± 3.7 18.4 ± 7.7 19.8 ± 1.7 0.1 ± 0.0 0.2 ± 0.0 0.3 ± 0.1 0.8 ± 0.3 8.3 ± 0.9 4.9 ± 1.0 9.9 ± 0.7 20.2 ± 3.4 E-isoeugenol 0 0 0 0 0 0 0.6 0.7 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 2.5 ± 0.0 2.8 ± 0.3 2.7 ± 0.4 2.4 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 2.3 ± 0.3 2.2 ± 0.2 2.8 ± 0.0 2.8 ± 0.1 4-vinylphenol 9.7 4.1 5.3 8.9 38.6 123 264 324 21.5 9.7 ± 9.7 19.9 ± 0.7 9.3 ± 1.8 424 ± 5 569 ± 116 598 ± 174 525 ± 5 11.6 ± 2.7 17.8 ± 1.3 18.3 ± 4.6 22.0 ± 0.1 270 ± 36 346 ± 10 477 ± 24 578 ± 26 Vanillin 10.2 0.6 2.0 1.7 6.9 8.9 21.2 36.8 1.5 0.7 ± 0.1 2.3 ± 1.0 5.7 ± 3.7 10.8 ± 0.7 36.6 ± 23.8 21.5 ± 2.4 20.5 ± 1.2 1.0 ± 0.2 0.8 ± 0.1 1.3 ± 0.4 2.6 ± 1.5 9.4 ± 0.5 9.6 ± 0.2 13.9 ± 4.2 21.5 ± 0.6 Acetovanillone 7.5 1.2 1.1 0.9 53.2 4.2 6.4 12.9 2.5 1.2 ± 0.3 1.5 ± 0.8 3.3 ± 3.3 126 ± 45 194 ± 17 186 ± 6 166 ± 5 0.7 ± 0.1 1.4 ± 0.7 1.0 ± 0.3 2.6 ± 0.5 170 ± 10 131 ± 16 145 ± 34 185 ± 4 Control PG AH Control S. cerevisiae PG Control P. kluyveri PG Supplementary data 256 Precursors Yeast Aging 0 1 2 5 0 1 2 5 0 1 2 5 0 1 2 5 Ethyl acetate 47032 ± 24509 75440 ± 1213 91377 ± 61 92847 ± 577 166213 ± 2999 155637 ± 7184 148887 ± 15598 127574 ± 458 59220 ± 56 66765 ± 299 67184 ± 1116 75513 ± 265 140554 ± 504 135488 ± 307 131983 ± 1955 100561 ± 177 Isoamyl acetate 295 ± 182 132 ± 1 122 ± 1 109 ± 8 111 ± 18 144 ± 0 137 ± 3 133 ± 2 217 ± 13 181 ± 7 150 ± 0 113 ± 4 233 ± 33 207 ± 0 167 ± 9 1901 ± 1784 Isobutyl acetate 88.6 ± 1.6 90.5 ± 1.4 87.6 ± 0.3 111 ± 6 140 ± 45 92.5 ± 0.2 98.2 ± 7.8 118 ± 4 66.4 ± 0.6 61.5 ± 2.6 62.2 ± 3.2 57.9 ± 3.2 67.4 ± 0.4 59.9 ± 3.7 60.4 ± 6.9 68.4 ± 5.6 Phenylethyl acetate 320 ± 9 327 ± 83 188 ± 1 142 ± 3 398 ± 139 351 ± 158 168 ± 5 116 ± 5 57.0 ± 7.3 41.2 ± 2.2 37.3 ± 2.5 36.0 ± 11.9 48.2 ± 0.3 57.0 ± 17.7 34.8 ± 1.7 32.4 ± 0.7 Ethyl hexanoate 115 ± 115 95.5 ± 6.5 148 ± 25 103 ± 2 0.0 ± 0.0 135 ± 19 137 ± 41 107 ± 24 48.2 ± 4.7 55.6 ± 1.1 25.2 ± 25.2 54.2 ± 0.2 57.2 ± 57.2 159 ± 2 152 ± 33 144 ± 30 Ethyl octanoate 268 ± 210 167 ± 7 167 ± 19 177 ± 21 63.8 ± 16.8 154 ± 4 157 ± 33 135 ± 1 93.1 ± 53.2 51.2 ± 8.0 51.4 ± 5.4 63.6 ± 8.1 180 ± 132 181 ± 18 134 ± 6 193 ± 45 Ethyl decanoate 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 49.5 ± 4.9 30.8 ± 3.4 34.2 ± 0.6 61.9 ± 38.1 59.3 ± 14.5 137 ± 40 54.8 ± 54.8 118 ± 27 Ethyl isobutyrate 114 ± 4 357 ± 15 505 ± 24 1011 ± 149 132 ± 36 296 ± 0 435 ± 4 816 ± 4 47.9 ± 1.6 139 ± 11 175 ± 36 327 ± 21 38.9 ± 2.1 78.6 ± 64.1 154 ± 19 371 ± 88 Ethyl isovalerate 2.2 ± 2.2 10.7 ± 0.4 16.8 ± 0.9 34.8 ± 2.6 5.2 ± 1.6 9.9 ± 0.1 15.6 ± 0.2 32.9 ± 1.4 2.3 ± 0.2 8.1 ± 0.0 13.2 ± 0.0 26.7 ± 1.8 1.7 ± 0.0 7.2 ± 0.2 11.8 ± 0.7 27.4 ± 0.2 Ethyl 2-methylbutyrate 4.6 ± 0.0 14.1 ± 0.8 21.8 ± 1.1 45.6 ± 2.9 5.7 ± 1.6 12.3 ± 0.4 20.7 ± 0.1 41.6 ± 0.9 2.9 ± 0.2 8.5 ± 0.4 14.2 ± 0.0 28.8 ± 0.7 2.1 ± 0.0 9.2 ± 0.1 12.7 ± 0.2 28.6 ± 0.8 Ethyl lactate 545 ± 54 2442 ± 21 3745 ± 28 6199 ± 11 662 ± 74 2531 ± 94 3882 ± 141 6348 ± 83 16111 ± 817 75862 ± 685 115387 ± 5327 186688 ± 7348 11845 ± 769 63983 ± 1630 95974 ± 2571 146020 ± 9999 Isobutanol 46525 ± 2868 75502 ± 20250 42812 ± 1791 45437 ± 1610 47816 ± 456 45798 ± 1892 44777 ± 335 50035 ± 4681 28429 ± 918 46129 ± 19150 59208 ± 32857 45903 ± 20089 24362 ± 3697 28357 ± 3159 24301 ± 1321 28164 ± 1491 1-Butanol 314 ± 61 391 ± 108 200 ± 5 219 ± 16 300 ± 5 270 ± 8 275 ± 26 319 ± 27 400 ± 9 643 ± 245 862 ± 474 700 ± 299 436 ± 1 448 ± 90 389 ± 3 474 ± 41 Isoamyl alcohol 199904 ± 33337 167528 ± 3780 169586 ± 8688 165996 ± 3231 165229 ± 782 153452 ± 2168 160373 ± 1153 159103 ± 1830 152611 ± 307 149919 ± 4033 156226 ± 7191 152385 ± 2096 145238 ± 6798 140945 ± 8614 147328 ± 9539 146940 ± 3959 1-Hexanol 49.7 ± 15.2 49.0 ± 0.8 49.1 ± 2.6 47.8 ± 4.4 149 ± 27 107 ± 10 108 ± 0 110 ± 2 33.3 ± 0.4 40.0 ± 3.8 52.8 ± 11.5 38.0 ± 10.7 133 ± 1 113 ± 1 120 ± 11 131 ± 3 Metionol 6110 ± 9 8165 ± 167 8098 ± 203 8572 ± 224 6363 ± 4 8104 ± 292 9006 ± 325 8776 ± 48 4911 ± 411 6167 ± 11 6519 ± 67 5753 ± 56 5324 ± 485 5890 ± 394 6072 ± 49 6112 ± 39 β-Phenylethanol 39683 ± 6635 57655 ± 5942 52489 ± 1223 59920 ± 6367 35655 ± 1161 45883 ± 2002 47021 ± 1223 45122 ± 1393 28106 ± 1776 25719 ± 68 27636 ± 789 22502 ± 1023 26220 ± 566 31365 ± 1782 30121 ± 2571 30003 ± 393 γ-Butyrolactone 1061 ± 110 3325 ± 133 3987 ± 169 4650 ± 311 919 ± 37 3213 ± 79 4194 ± 144 4543 ± 66 1548 ± 14 5418 ± 513 6681 ± 371 6665 ± 12 1626 ± 40 5445 ± 307 6666 ± 25 7160 ± 127 γ-nonalactone 9.8 ± 0.2 13.1 ± 4.9 8.6 ± 0.3 8.3 ± 0.3 17.7 ± 6.0 20.9 ± 10.8 10.2 ± 0.3 10.4 ± 0.5 8.1 ± 0.1 7.1 ± 0.1 6.7 ± 0.2 9.9 ± 3.1 9.1 ± 0.0 13.5 ± 5.2 7.7 ± 0.2 8.2 ± 0.1 γ-decalactone 8.4 ± 0.3 10.9 ± 3.9 7.0 ± 0.1 7.1 ± 0.3 11.5 ± 3.8 12.7 ± 6.2 6.2 ± 0.1 6.7 ± 0.3 33.9 ± 3.0 29.4 ± 0.4 28.2 ± 0.3 38.1 ± 11.2 31.7 ± 0.2 44.1 ± 17.7 26.4 ± 0.3 27.8 ± 0.1 Butyric acid 639 ± 73 397 ± 32 362 ± 35 494 ± 95 540 ± 29 426 ± 68 499 ± 52 445 ± 54 373 ± 12 329 ± 13 381 ± 54 269 ± 15 423 ± 100 275 ± 22 339 ± 24 289 ± 2 Isobutyric acid 4645 ± 1870 7041 ± 179 6593 ± 288 7413 ± 767 6339 ± 283 5603 ± 340 6046 ± 374 5682 ± 189 2012 ± 55 2195 ± 92 2466 ± 35 2765 ± 193 2098 ± 174 2048 ± 137 2137 ± 221 2298 ± 225 Hexanoic acid 1110 ± 698 263 ± 2 255 ± 6 231 ± 0 395 ± 26 262 ± 7 258 ± 13 253 ± 8 498 ± 43 409 ± 2 407 ± 12 330 ± 26 509 ± 57 274 ± 30 295 ± 31 279 ± 8 Octanoic acid 2696 ± 1634 922 ± 37 732 ± 7 871 ± 4 888 ± 120 728 ± 123 731 ± 10 672 ± 26 1263 ± 134 1138 ± 35 1093 ± 64 909 ± 112 1227 ± 80 886 ± 56 827 ± 29 12884 ± 12019 Decanoic acid 419 ± 371 96.2 ± 1.5 98.3 ± 2.3 151 ± 11 19.9 ± 19.9 105 ± 0 110 ± 0 161 ± 16 505 ± 31 585 ± 99 563 ± 26 736 ± 120 1096 ± 115 1316 ± 393 824 ± 39 1254 ± 318 TDN 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.1 12.8 ± 5.3 21.4 ± 0.9 70.7 ± 10.9 0.1 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.0 20.7 ± 6.2 39.3 ± 0.4 136 ± 9 β-damascenone 0.2 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.9 ± 0.6 4.6 ± 2.1 2.9 ± 0.2 3.7 ± 0.1 0.3 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 2.1 ± 0.3 4.7 ± 1.4 3.9 ± 0.4 4.6 ± 0.1 β-ionone 0.6 ± 0.0 0.2 ± 0.1 0.2 ± 0.0 0.1 ± 0.1 0.8 ± 0.2 0.3 ± 0.3 0.2 ± 0.0 0.1 ± 0.0 0.8 ± 0.1 0.5 ± 0.3 0.1 ± 0.1 0.2 ± 0.0 0.4 ± 0.0 0.1 ± 0.1 0.2 ± 0.0 0.1 ± 0.0 Ethyl cinnamate 0.0 ± 0.0 1.2 ± 0.7 0.6 ± 0.1 0.0 ± 0.0 0.1 ± 0.0 3.0 ± 2.7 0.7 ± 0.2 1.2 ± 0.4 0.0 ± 0.0 0.1 ± 0.1 1.6 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 2.2 ± 2.0 1.0 ± 0.4 1.0 ± 0.3 Linalool 3.3 ± 0.0 3.6 ± 0.4 2.3 ± 0.1 0.8 ± 0.1 13.9 ± 4.6 20.2 ± 0.4 11.9 ± 0.5 2.9 ± 0.1 3.4 ± 0.1 2.6 ± 0.2 1.3 ± 0.1 0.5 ± 0.1 10.1 ± 0.3 15.3 ± 0.1 8.3 ± 1.3 1.7 ± 0.0 α-terpineol 1.2 ± 0.0 3.0 ± 0.2 3.5 ± 0.1 3.4 ± 0.0 4.7 ± 1.6 22.7 ± 1.3 26.9 ± 0.8 25.5 ± 0.1 1.2 ± 0.1 2.6 ± 0.5 3.0 ± 0.1 1.8 ± 0.1 3.8 ± 0.0 26.6 ± 2.6 27.6 ± 1.0 21.7 ± 0.2 β-citronellol 2.7 ± 0.1 2.0 ± 0.4 1.2 ± 0.1 0.3 ± 0.3 6.7 ± 2.3 3.4 ± 0.0 2.2 ± 0.0 0.9 ± 0.1 3.3 ± 0.3 1.9 ± 0.0 1.2 ± 0.0 0.2 ± 0.2 4.6 ± 0.1 3.1 ± 0.3 2.2 ± 0.3 0.7 ± 0.0 Geraniol 5.9 ± 0.5 2.2 ± 0.7 1.3 ± 0.4 0.6 ± 0.6 13.3 ± 4.4 7.0 ± 0.9 3.7 ± 0.7 1.4 ± 0.4 3.9 ± 0.0 1.2 ± 0.1 0.4 ± 0.4 0.0 ± 0.0 6.0 ± 0.4 5.0 ± 0.1 2.9 ± 0.3 0.5 ± 0.5 Guaiacol 0.2 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.2 ± 0.1 3.2 ± 0.4 1.9 ± 0.0 3.0 ± 0.5 4.4 ± 0.2 0.2 ± 0.0 0.2 ± 0.1 0.7 ± 0.1 0.2 ± 0.0 2.2 ± 0.7 2.8 ± 0.1 3.6 ± 0.9 5.6 ± 0.2 4-vinylguaiacol 19.2 ± 11.8 13.0 ± 2.4 10.0 ± 2.1 14.9 ± 5.3 787 ± 188 1453 ± 208 2133 ± 204 2657 ± 13 17.5 ± 0.3 19.9 ± 3.8 48.4 ± 25.2 10.7 ± 2.7 582 ± 5 1305 ± 32 1644 ± 54 2113 ± 120 2-6-dimethoxyphenol 0.2 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 9.6 ± 1.1 5.0 ± 0.8 10.6 ± 3.1 15.9 ± 0.1 0.8 ± 0.4 0.4 ± 0.4 2.4 ± 0.0 0.0 ± 0.0 8.3 ± 1.6 6.6 ± 1.0 13.5 ± 5.5 19.0 ± 0.3 E-isoeugenol 0.2 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 4.1 ± 1.3 3.1 ± 0.4 3.1 ± 0.2 3.2 ± 0.1 0.1 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 1.4 ± 0.0 1.8 ± 0.1 1.6 ± 0.1 2.0 ± 0.2 4-vinylphenol 23.0 ± 3.4 18.6 ± 2.9 11.5 ± 1.0 0.0 ± 0.0 403 ± 149 447 ± 128 770 ± 290 560 ± 17 20.5 ± 4.3 25.5 ± 7.9 206 ± 119 0.0 ± 0.0 343 ± 7 331 ± 5 698 ± 277 491 ± 12 Vanillin 1.0 ± 0.3 0.5 ± 0.3 3.2 ± 1.6 2.0 ± 0.0 13.0 ± 4.1 9.3 ± 0.8 25.8 ± 3.2 11.5 ± 11.5 2.7 ± 1.7 1.2 ± 0.3 1.1 ± 1.1 1.2 ± 0.6 22.4 ± 2.1 8.1 ± 0.3 15.4 ± 2.3 20.1 ± 3.2 Acetovanillone 1.5 ± 0.9 0.0 ± 0.0 2.0 ± 0.8 1.2 ± 1.2 212 ± 78 71.7 ± 40.1 172 ± 3 102 ± 60 2.9 ± 2.0 1.0 ± 0.1 0.9 ± 0.9 0.8 ± 0.8 163 ± 1 32.6 ± 2.5 165 ± 3 92.6 ± 60.1 Control PG T. delbrueckii Control L. thermotolerans PG S.d. Table 8 (cont) Average concentration of volatiles measured above the limit of quantification in control wines and those spiked with glycosidic precursors from Garnacha grapes in unfermented controls (Acid Hydrolysis) and in wines fermented with S. cerevisiae. P. kluyveri. T. delbrueckii and L. thermotolerans. Supplementary data 257 S.d. Table 9 Average concentration of volatile compounds according to the yeast strain or acidic hydrolysis controls of wines with and without Garnacha precursors. Letters a-d are the results of Duncan post-hoc test; compounds with different letters indicate significant differences AH P. kluyveri S. cerevisiae T. delbrueckii L. thermotolerans Ethyl acetate 55.4 d 144395 a 79313 c 113126 b 97159 bc Isoamyl acetate 0 c 2298 a 311 bc 148 bc 396 b Ethyl hexanoate 0 c 377 a 354 a 105 b 86.9 bc Ethyl octanoate 0 d 490 b 651 a 161 c 118 c Ethyl decanoate 0 c 147 ab 212 a 0 c 68.1 bc Ethyl isobutyrate 0.2 c 201 b 178 b 458 a 166 b Ethyl 2-methylbutyrate 0 d 24 a 19.8 b 20.8 b 13.4 c Ethyl isovalerate 0 d 25.5 a 27.2 a 16 b 12.3 c Isobutyl acetate 3 e 197 a 87.6 c 103 b 63 d Phenylethyl acetate 0.1 b 6826 a 105 b 251 b 42.8 b Isobutanol 0 d 57426 a 48253 b 49838 ab 35606 c 1-Butanol 0 d 251 c 418 b 286 c 544 a Isoamyl alcohol 0 e 185508 b 203502 a 167646 c 148949 d 1-Hexanol 0.01 d 47.3 c 70.3 b 83.8 a 82.7 a Metionol 0 e 11111 a 6837 c 7899 b 5844 d β-Phenylethanol 0 d 30017 c 33575 b 47929 a 27709 c Ethyl lactate 0 b 4646 b 2599 b 3294 b 88984 a Butyric acid 0 d 2604 a 593 b 475 b 335 c Isobutyric acid 0 c 2327 b 2442 b 6170 a 2252 b Hexanoic acid 0 c 1277 a 1311 a 378 b 375 b Octanoic acid 0c 4028 a 4046 a 1030 bc 2529 ab Decanoic acid 0 c 819 a 908 a 145 b 860 a γ-nonalactone 0.9 c 9.2 b 9.4 b 12.4 a 8.8 b γ-decalactone 0.1 c 6.2 b 5.2 b 8.8 b 32.4 a γ-Butyrolactone 0 e 4981 c 6627 a 3236 d 5151 b TDN 4 a 13.4 a 13.1 a 13.1 a 24.5 a β-damascenone 2.3 a 1.7 a 1.8 a 1.5 a 2 a β-ionone 0.2 b 0.4 a 0.4 a 0.3 ab 0.3 ab Ethyl cinnamate 0.1 a 0.4 a 1.5 a 0.8 a 0.7 a Linalool 3.9 a 6.8 a 6.2 a 7.4 a 5.4 a α-terpineol 6.3 a 11.1 a 9.9 a 11.4 a 11 a β-citronellol 0.1 c 1.7 b 2.2 ab 2.4 a 2.2 ab Supplementary data 264 Compound 21 A 22 A 30 A 33 34 so2 med A-16 element a-16 7f9a-16 Acetate Esters Ethyl acetate 58606 ± 9924 39129 ± 459 78116 ± 8044 52660,7 56444,1 26193 ± 3530 28778 ± 909 23785 ± 171 Isoamyl acetate 505 ± 12 617 ± 1 623 ± 170 414,1 502,3 414 ± 56 582 ± 19 223 ± 8 Hexyl acetate 105 ± 1 128 ± 28 109 ± 9 130,6 137,3 105 ± 27 69.4 ± 5.9 29.0 ± 3.8 Isobutyl acetate 11.3 ± 0.9 10.3 ± 0.6 10.3 ± 0.3 12,3 11,1 11.1 ± 5.6 8.2 ± 0.0 3.8 ± 0.2 Butyl acetate 3.4 ± 1.3 3.2 ± 0.4 4.7 ± 0.1 3,5 4,4 1.0 ± 1.5 2.1 ± 0.8 1.7 ± 0.1 Phenylethyl acetate 33.0 ± 1.9 39.7 ± 0.9 39.5 ± 8.2 33,1 38,9 35.9 ± 4.8 90.9 ± 2.5 34.5 ± 0.2 Ethyl esters Ethyl propanoate 69.4 ± 10.8 0.0 ± 0.0 26.0 ± 26.0 0,0 78,2 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Ethyl butyrate 177 ± 16 143 ± 3 226 ± 18 152,0 156,8 81.4 ± 9.8 60.3 ± 16.3 82.4 ± 16.2 Ethyl hexanoate 1481 ± 626 565 ± 18 640 ± 59 668,3 759,4 571 ± 105 428 ± 26 570 ± 18 Ethyl octanoate 1493 ± 274 749 ± 27 615 ± 135 883,6 884,4 729 ± 167 671 ± 1 529 ± 142 Ethyl decanoate 165 ± 29 98.4 ± 1.9 51.0 ± 16.6 119,8 114,5 75.5 ± 16.0 75.0 ± 1.5 67.3 ± 11.1 Ethyl isobutyrate 57.1 ± 4.8 121 ± 4 38.6 ± 2.5 23,6 24,8 25.0 ± 19.6 46.3 ± 1.9 44.5 ± 2.5 Ethyl 2-methylbutyrate 1.6 ± 0.2 2.7 ± 0.1 1.4 ± 0.1 0,9 0,9 1.4 ± 0.6 2.7 ± 1.2 4.6 ± 0.4 Ethyl isovalerate 5.4 ± 0.6 7.1 ± 0.1 4.8 ± 0.2 2,2 2,6 3.2 ± 1.7 11.0 ± 1.2 9.6 ± 1.5 Miscelaneous esters Ethyl lactate 6197 ± 66 6297 ± 149 3229 ± 447 13302,8 12613,4 4579 ± 819 4584 ± 137 4980 ± 238 Diethyl succinate 910 ± 37 834 ± 49 364 ± 14 329,8 385,2 466 ± 147 539 ± 38 501 ± 45 Fusel alcohols Isobutanol 14485 ± 47 25041 ± 186 20180 ± 280 9344,0 9874,8 28096 ± 12995 27662 ± 1455 21434 ± 2448 1-Butanol 410 ± 1 227 ± 11 586 ± 83 597,8 674,6 519 ± 111 366 ± 32 412 ± 58 Isoamyl alcohol 76299 ± 2091 128039 ± 4359 118899 ± 4508 79937,8 85136,5 124684 ± 20513 212095 ± 8332 170652 ± 2664 1-Hexanol 1254 ± 13 1274 ± 64 1367 ± 96 1014,2 1094,3 1032 ± 15 1222 ± 27 1385 ± 15 c-3-Hexenol 74.9 ± 2.1 75.1 ± 1.5 74.6 ± 1.1 65,3 71,0 7.9 ± 11.2 0.0 ± 0.0 0.0 ± 0.0 Metionol 455 ± 17 736 ± 28 200 ± 15 154,8 164,5 1293 ± 475 1622 ± 51 1109 ± 36 Benzylic alcohol 92.8 ± 19.2 569 ± 29 44.1 ± 1.6 27,4 37,0 28.9 ± 4.2 19.7 ± 3.5 27.1 ± 0.4 β-Phenylethanol 10059 ± 1719 13831 ± 92 8343 ± 103 7792,8 8373,5 19778 ± 2028 46768 ± 699 37981 ± 60 Acids Butyric acid 723 ± 6 545 ± 15 1041 ± 42 651,9 709,9 637 ± 65 575 ± 17 659 ± 3 Isobutyric acid 608 ± 19 1126 ± 25 550 ± 15 336,4 332,9 513 ± 327 860 ± 72 912 ± 65 Isovalerianic acid 421 ± 23 639 ± 25 440 ± 4 266,4 256,4 395 ± 83 1025 ± 11 1225 ± 1 Hexanoic acid 4515 ± 1024 4640 ± 197 4886 ± 117 5313,6 5712,8 3742 ± 138 2235 ± 60 2686 ± 36 Octanoic acid 12274 ± 193 11147 ± 22 8163 ± 366 13132,4 12382,0 11386 ± 810 7258 ± 174 8808 ± 181 Decanoic acid 2833 ± 370 2668 ± 225 1369 ± 258 2871,4 3068,8 1846 ± 516 1083 ± 81 1445 ± 53 Monoterpenes Linalool 84.5 ± 4.1 74.2 ± 3.5 67.4 ± 1.2 71,5 76,9 74.4 ± 10.1 79.6 ± 0.4 81.0 ± 1.1 Linalool acetate 0.7 ± 0.5 0.8 ± 0.2 0.9 ± 0.2 0,5 0,8 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 α-Terpineol 53.3 ± 2.7 50.6 ± 2.0 70.6 ± 1.9 53,6 60,2 63.5 ± 9.3 40.4 ± 0.4 40.0 ± 2.0 β-Citronelol 3.3 ± 1.1 1.4 ± 1.4 6.4 ± 1.1 4,8 0,0 0.4 ± 0.6 0.6 ± 0.6 0.0 ± 0.0 Geraniol 11.1 ± 0.1 9.9 ± 0.0 6.7 ± 0.9 10,0 9,4 5.6 ± 2.4 8.9 ± 0.6 10.3 ± 1.0 Norisoprenoids β-Damascenone 12.7 ± 1.5 13.6 ± 1.0 9.9 ± 0.6 15,6 13,2 1.2 ± 0.4 2.0 ± 1.0 2.5 ± 1.1 α-Ionone 0.4 ± 0.3 0.7 ± 0.0 0.5 ± 0.0 0,6 0,6 0.0 ± 0.0 0.0 ± 0.0 0.3 ± 0.3 β-Ionone 0.2 ± 0.2 0.3 ± 0.1 0.4 ± 0.1 0,3 0,3 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Phenols Guaiacol 5.6 ± 1.0 4.9 ± 1.2 5.3 ± 1.3 5,0 5,0 1.0 ± 0.3 1.0 ± 0.1 1.1 ± 0.6 o-Cresol 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0,0 0,0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 4-Ethylguaiacol 0.3 ± 0.0 0.6 ± 0.2 0.5 ± 0.1 0,2 0,4 0.2 ± 0.2 0.1 ± 0.1 0.1 ± 0.1 m-Cresol 0.3 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0,2 0,2 0.2 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 Eugenol 0.2 ± 0.2 0.0 ± 0.0 0.6 ± 0.0 0,4 0,5 0.5 ± 0.1 0.4 ± 0.2 0.6 ± 0.3 4-Ethylphenol 0.4 ± 0.1 0.4 ± 0.1 0.5 ± 0.1 0,3 0,5 0.1 ± 0.2 0.3 ± 0.2 0.3 ± 0.3 4-Vinylguaiacol 102 ± 6 251 ± 3 225 ± 24 130,4 119,3 414 ± 246 278 ± 21 271 ± 46 E-Isoeugenol 2.4 ± 0.2 2.1 ± 0.3 2.0 ± 0.2 2,3 2,3 2.3 ± 1.2 2.6 ± 1.6 3.7 ± 2.1 2,6-Dimethoxyphenol 4.6 ± 1.4 3.1 ± 1.3 3.1 ± 1.2 2,1 2,5 0.3 ± 0.4 0.0 ± 0.0 0.0 ± 0.0 4-Vinylphenol 72.8 ± 1.4 103 ± 4 208 ± 21 69,6 80,8 187 ± 41 123 ± 5 119 ± 1 4-Alyl-2,6-dimethoxyphenol 0.3 ± 0.3 0.0 ± 0.0 0.0 ± 0.0 0,0 0,0 0.1 ± 0.2 0.2 ± 0.2 0.7 ± 0.3 Cinamates Ethyl dihidrocinnamate 0.0 ± 0.0 0.0 ± 0.0 1.1 ± 0.0 0,5 0,8 0.1 ± 0.2 0.8 ± 0.2 1.8 ± 0.0 Ethyl cinnamate 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0,0 0,0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Lactones t-Whiskylactone 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0,0 0,0 0.0 ± 0.0 0.4 ± 0.4 0.0 ± 0.0 c-Whiskylactone 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0,0 0,0 2.2 ± 3.1 2.6 ± 2.6 0.0 ± 0.0 γ-Butyrolactone 6337 ± 222 6976 ± 156 3672 ± 143 5552,6 5938,0 3913 ± 712 5854 ± 90 4678 ± 20 γ-Nonalactone 1.4 ± 0.1 1.3 ± 0.1 2.6 ± 0.1 1,1 1,4 0.4 ± 0.6 0.7 ± 0.7 0.5 ± 0.5 γ-Decalactone 17.8 ± 17.8 12.7 ± 1.3 31.0 ± 0.8 19,6 22,4 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Vanillin derivates Vanillin 5.3 ± 1.3 6.5 ± 0.2 7.1 ± 1.4 5,3 6,1 4.7 ± 3.3 5.0 ± 2.9 3.7 ± 2.4 Methyl vanillinate 59.2 ± 4.5 49.1 ± 3.0 59.0 ± 2.8 41,3 47,8 62.5 ± 6.3 59.0 ± 0.2 60.2 ± 0.3 Ethyl vanillate 2.0 ± 2.0 1.7 ± 0.2 2.4 ± 0.2 1,7 2,1 0.4 ± 0.1 0.6 ± 0.0 0.5 ± 0.0 Acetovanillone 50.0 ± 6.5 44.8 ± 2.0 54.2 ± 3.3 40,0 44,7 25.0 ± 2.1 24.5 ± 0.9 21.3 ± 2.0 Aldehydes Benzaldehyde 1.3 ± 0.2 1.7 ± 0.2 2.2 ± 0.2 2,0 1,9 0.2 ± 0.3 0.0 ± 0.0 0.4 ± 0.4 Isobutyraldehyde 10.1 ± 2.1 12.9 ± 0.0 13.5 ± 0.5 Isovaleraldehyde 15.9 ± 2.7 20.1 ± 0.5 18.4 ± 2.6 2-methylbutanal 1.5 ± 0.2 2.6 ± 0.1 2.3 ± 0.0 Methional 11.4 ± 2.5 14.5 ± 0.2 12.5 ± 0.2 Phenylacetaldehyde 207 ± 11 240 ± 21 248 ± 18 Polyfunctional mercaptans 2-metyl-3-furanthiol 1.2 ± 0.4 1.4 ± 0.7 0.6 ± 0.2 1,362 0,466 0.4 ± 0.3 0.7 ± 0.2 0.6 ± 0.2 Furfurylthiol 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0,005 0,003 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 4-Mercapto-4-methyl-2-pentanona 0.1 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0,000 0,000 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 3-mercaptohexyl acetate 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0,000 0,000 0.1 ± 0.1 0.1 ± 0.1 0.0 ± 0.0 3-Mercaptohexanol 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0,031 0,029 0.1 ± 0.0 0.1 ± 0.0 0.0 ± 0.0 Benzylmercaptan 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0,002 0,002 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 2015 2016 S.d. Table 6 Average content of odorants fermented in wines from harvest 2015 and 2016with different additives