Volatile metabolites produced by different flor yeast strains during wine biological ageing

Journal Pre-proofs Volatile metabolites produced by different flor yeast strains during wine biological ageing M.L. Morales, M. Ochoa, M. Valdivia, C. Ubeda, S. Romero-Sanchez, J.I. Ibeas, E. Valero PII: DOI: Reference:

S0963-9969(19)30657-X https://doi.org/10.1016/j.foodres.2019.108771 FRIN 108771

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Food Research International

Received Date: Revised Date: Accepted Date:

27 February 2019 11 September 2019 26 October 2019

Please cite this article as: Morales, M.L., Ochoa, M., Valdivia, M., Ubeda, C., Romero-Sanchez, S., Ibeas, J.I., Valero, E., Volatile metabolites produced by different flor yeast strains during wine biological ageing, Food Research International (2019), doi: https://doi.org/10.1016/j.foodres.2019.108771

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TITLE: VOLATILE METABOLITES PRODUCED BY DIFFERENT FLOR YEAST STRAINS DURING WINE BIOLOGICAL AGEING AUTHORS: Morales M.L.1*, Ochoa M.2, Valdivia M.3, Ubeda C.4, Romero-Sanchez S.5, Ibeas, J.I.5, Valero, E.3 ADDRESSES: 1Área de Nutrición y Bromatología, Dpto. Nutrición y Bromatología, Toxicología y Medicina Legal, Facultad de Farmacia, Universidad de Sevilla, C/ P. García González nº 2, E- 41012, Sevilla. España. 2Royal Berries Company, Crta. Almonte-El Rocío Km 24.2, 21730, Almonte (Huelva), España. 3Departamento de Biología Molecular e Ingeniería Bioquímica, Universidad Pablo de Olavide, Carretera de Utrera Km. 1, 41013, Sevilla, España. 4Instituto de Ciencias Biomédicas, Facultad de Ciencias, Universidad Autónoma de Chile, C/ El Llano Subercaseaux 2801, Santiago, Chile 5Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide-Consejo Superior de Investigaciones Científicas-Junta de Andalucía, Sevilla, España. *Corresponding author: e-mail: [email protected]; Tel.: 34954-556758; Fax: 34-954-556110

ABSTRACT Sherry white wine called Fino is produced by dynamic biological ageing under the action of flor yeasts using traditional practices aimed at ensuring uniform quality and characteristics over time. These kinds of yeasts provide typical sensory properties to Fino wines. Although there are studies of the volatile composition of these wines submitted to biological ageing in wood barrels, there is a lack of knowledge on the particular volatile

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profile produced by different flor yeast strains from Sherry zone wineries. For this reason, the aim of this study was to analyse the volatile profiles produced by 15 pure culture flor velum yeasts, with the goal of observing their suitability for obtaining high quality Fino sherry wines. Volatile composition was determined by dual sequential stir bar sorptive extraction, followed by GC-MS analysis. All yeast strains studied produced the increase of most acetals, highlighting acetaldehyde diethylacetal which was the compound that most increased. Among terpenes, nerolidol and farnesol underwent remarkable increases. However, results showed that in a month of biological ageing, significant differences were observed among the volatile metabolites produced by flor yeast strains studied. Only some of them stood out for their high production of volatile compounds characteristic of Sherry Fino wines, which are good candidates for producing starter cultures. KEYWORDS: Sherry wine; volatile compound; flor yeast; GC-MS analysis; Heatmap. 1. Introduction Sherry wines, one of the most distinctive Spanish wines, are produced in a particular area of southern Spain (between the cities of Jerez, El Puerto de Santa María y Sanlúcar de Barrameda), using traditional practices aimed at ensuring uniform quality and characteristics over time. These wines are legally protected by the Denominations of Origin (PDO) "Jerez-Xérès-Sherry" and "Manzanilla-Sanlúcar de Barrameda" (Consejería de Agricultura y Pesca, 2010). Several types of Sherry wines are produced, depending on the winemaking conditions (Pozo-Bayón & Moreno-Arribas, 2011). Sherry-type white wine called Fino or Manzanilla, when is produced in Sanlúcar de Barrameda, is produced from Palomino Fino grape variety and is characterised by dynamic biological ageing under the action of flor yeasts. These yeasts grow aerobically, forming a biofilm (flor velum) on the surface of wines with a high ethanol content (15–

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15.5%) (Bravo, 1995; Suarez, 1997). In these conditions, their metabolisms become oxidative (Ibeas, Lozano, Perdigones, & Jiménez, 1997a; Mauricio, Moreno, & Ortega, 1997). The restrictive conditions of biological ageing (low pH, presence of sulphite, albeit low, high ethanol and acetaldehyde concentrations, scarcity of sugars, and low oxygen concentration) are compatible with only a few S. cerevisiae. Hence, more than 95% of the film’s microbiota usually consists of film-forming S. cerevisiae strains (Martinez de la Ossa, Caro, Bonat, Pérez, & Domecq, 1997; Mesa, Infante, Rebordinos, Sanchez, & Cantoral, 2000). These flor yeasts have different metabolic and genetic characteristics from typical fermentative yeasts, showing a heterogeneous genetic profile, characterised by considerable variability in the DNA content, mitochondrial DNA restriction analysis, and chromosomal profiles (Budroni, Zara, Zara, Pirino, & Mannazzu, 2005; EsteveZarzoso, Peris-Torán, García-Maiquez, Uruburu, & Querol 2001, Esteve-Zarzoso, Fernandez-Espinar, & Querol, 2004). The thick velum that develops on the surface protects the wine from oxidation and is the origin of complex biochemical reactions, resulting from metabolism of the flor yeast and the reducing environment created in the wine. Thus, Fino wine exhibits special sensory features, including a light, dry and delicate flavour, a pale yellow colour, and a complex aroma. This last is developed during biological ageing as a result of the action of the flor yeasts, as well as the contribution of volatile compounds extracted from the wood casks where the wine is aged. One of the most significant metabolic changes occurring during biological ageing is acetaldehyde production. It makes an important organoleptic contribution, together with a marked reduction in glycerol and acetic acid content and ethanol metabolism. Acetaldehyde is synthesised from ethanol by flor yeasts by means of the enzyme alcohol dehydrogenase in the presence of NAD+ (Garcia-Maiquez, 1988). As a result, it is well known that some commercial Fino wines have similar acetaldehyde

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contents but differ markedly in quality, mainly in relation to their sensory properties. Some authors have pointed out that flor yeasts increase the content of other aroma compounds, such as higher alcohols and acetates, ethyl esters, lactones and terpenes (Zea, Moreno, & Medina, 1995). Changes in flavour-related substances such as acetaldehyde and its derivative, ethanol, organic acids, higher alcohols, esters, lactones and nitrogen compounds resulting from the metabolism of flor yeasts and their associated sensory properties in Fino wines have been examined by some authors (Mauricio, Valero, Millán, & Ortega, 2001; Mesa, Infante, Rebordinos, Sanchez, & Cantoral, 2000; Muñoz, Peinado, Medina, & Moreno, 2006; Peinado & Mauricio, 2009; Villamiel, Polo, & MorenoArribas, 2008; Zea, Moyano, Moreno, & Medina, 2007). However, the volatile profiles of different flor yeast strains, in pure cultures, under biological ageing conditions, has scarcely been analysed until now. Rencently, research is mainly focused in revealing genetics features of these yeasts and their adaptation to biological ageing conditions (Coi et al., 2017; Eldarov, Beletsky, Tanashchuk, Kishkovskaya, Ravin, & Mardanov, 2018) yeast diversity (David-Vaizant, & Alexandre, 2018; Marin-Menguiano, Romero-Sanchez, Barrales, & Ibeas, 2017) or proteome characterization (Moreno-García, Mauricio, Moreno, & García-Martínez, 2017; Moreno-García, Ogawa, Joseph, Mauricio, Moreno, & García-Martínez, 2019). In recent years, the interest in using flor yeast is increasing due to other possible biotechnological applications. Recently, one of the most important negative effects of global warming for winemakers in warm regions has been the increase of ethanol content of wine, in contrast with current market trends that favour low ethanol content wines. Therefore, the flor yeasts growing on the surface of finished wine tolerate its high alcohol content and are able to decrease the ethanol content of wines by 2% over a period of 30 days (Moreno-García, García-Martinez, Moreno, Millán, & Mauricio, 2014). Moreno,

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Moreno-García, López-Muñoz, Mauricio, & García-Martínez (2016) suggest that the flor yeast helps to modulate the ethanol, astringency and colour of red wines and supports a new biotechnological perspective for red winemakers. Marin-Menguiano et al. (2017) showed that most barrels (84%) were pure culture of one strain in the wineries of the Montilla-Moriles D.O. region (these wineries have similar winemaking procedure to sherry wineries). This observation was previously described in "Jerez-Xérès-Sherry" and "Manzanilla-Sanlúcar de Barrameda" PDO wineries (Ibeas, Lozano, Perdigones, & Jiménez, 1997b), suggesting dominance characteristics for the major strains. Hence, wines from wineries that are geographically very close may differ markedly in quality, owing to the influence of the most prominent flor velum yeast strain present in the wine. However, there is no scientific evidence about the production of different volatile profiles by distinct flor yeast strains in pure culture at present. For these reasons, and as a result of increased interest in flor yeast for other types of wines, the aim of this study was to analyse the volatile profiles produced by 15 pure culture flor velum yeasts, with the goal of observing their suitability for obtaining high quality Fino sherry wines. Hence, the novelty of this work is to provide data of volatile compounds production by this kind of yeast in pure culture, which has not yet been explored. These data will enable us to define the yeast strains that are suitable for the possible production of starter cultures of flor yeasts, which are not yet available on the market, and which are useful for Fino wines and other wine types. 2. Materials and methods 2.1. Yeast strains and biological ageing trials Yeast strains used in this work were isolated from flor yeast growing on the surface of Fino sherry wine or Manzanilla, from six different wineries from D.O. Jerez-XérèzSherry and Manzanilla-Sanlúcar de Barrameda. Different genotypes, named A-J of

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Saccharomyces cerevisiae were obtained by mtDNA pattern (Querol et al., 1992) and analysis of polymorphic microsatellite loci (Vaduano et al. 2008) (data not shown) and included in the yeast culture collection of Pablo de Olavide University. Two biological ageing trials were carried out in triplicate, the first with 10 flor yeast strains called A, B, C, D, E, F, G, H, I, and J, and the second with 5 flor yeast strains, K, L, M, N and O. The base wines were obtained from the same winery where yeast strains were isolated, showing similar pH values, 3.2 and 3.3; titratable acidity, 5.2 and 5.3; and both with a sulphur content of 60 mg/L. The ethanol contents were of 10.8% v/v and 13.3 % v/v respectively. Both base wines were fortified with wine alcohol to 15% v/v, before being centrifuged and sterilised by filtration (0.22 µm filter). Subsequently, the sterile base wine was distributed in 15 flasks of 500 ml refills each measuring 250 ml. The fermentation flaks were closed with cotton plugs. The flasks were inoculated with 1x106 viable cells/mL of each strain grown in YPD medium (0.5% yeast extract, 1% peptone and 2% dextrose) at 28ºC for 24 h, washed once with sterile water and re-suspended in the base wine. Total and viable cells were counted under an optic microscope using a Neubauer chamber. In order to form the flor biofilm, the inoculated flasks were placed in the dark at 20 ºC. After one week, a first control to check whether flor film was beginning to form was carried out. The parameter used to check the quality of velum was the thickness determined by the visual evaluation, as is usually performed in wineries. After one month of inoculation, all yeast strains had formed a continuous velum with suitable thickness, then, good quality velum (Table 1). After one month a sample of 50 mL of biologically aged wine was taken and conserved at -20 ºC until its analysis. 2.2. Volatile composition analysis by GC-MS Analyses were conducted using an Agilent 6890 GC system coupled up to an Agilent 5975 inert quadrupole mass spectrometer equipped with a Gerstel Thermo Desorption

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System (TDS2) and a Cooling Injector System CIS-4 PTV inlet (Gerstel, Müllheim an der Ruhr, Germany). A sequential extraction procedure was applied to analyse the wines’ volatile fraction by GC-MS. Two polydimethylsiloxane Twisters® were used in each sample extraction procedure, i.e., first in immersion (SBSE) and then in headspace (HSSE) (Ubeda et al., 2016). 7.5 mL of the sample were placed in a 20 ml vial, and 10 µL of the internal standard (IS) 4-methyl-2-pentanol (1,044 mg/L) plus 2.25 g of NaCl (30%) were added. A special stainless wire device, fixed to the stopper’s septum, was designed to keep the Twister® immersed. The SBSE was performed by placing the Twister in the special device and stirring the sample with a conventional magnetic stir bar at 200 rpm for one hour at room temperature. The HSSE was performed by placing a new twister in an open glass insert inside the vial and heating the sample in a water bath at 62ºC for one hour. In both cases, after extraction, the twister was removed with tweezers, rinsed with Milli-Q water, and dried with a lint-free tissue paper. Both twisters were then introduced into the same desorption tube and thermally simultaneously desorbed in a gas chromatograph/mass spectrometer (GC/MS). Compound identification was based on mass spectra matching using the standard NIST 98 mass spectral library and the linear retention index (LRI) of authentic reference standards. LRIs were calculated by injecting n-alkanes mixture (C10–C40) under identical conditions as the samples. Then, a compound was considered positively identified when the mass spectrum matched with that from NIST library and LRI value with that of real standards; tentatively identified (TI) when mass spectrum matched with that from library and LRI value with that from literature; with identification not confirmed when only the mass spectrum matched with that from NIST library, and unknown if the mass spectrum reached a low value of probability of right identification in library search report.

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2.3. Statistical analyses One-way ANOVA was performed to evaluate significant differences among yeast strains (significance levels p < 0.05). A principal component analysis (PCA) was carried out as an unsupervised method in order to ascertain the degree of differentiation between samples and which compounds were involved. ANOVA and PCA were performed using the Statistica (version 7.0) software package (Statsoft, Tulsa, USA).

Heatmap

visualisation of data was performed using the MetaboAnalyst 4.0 (web interface) (Chong and Xia, 2018). Data was normalised dividing the values of relative peak areas of each volatile compound by mean to perform PCA and heatmaps. Since we used two different wine substrates, to avoid some differences stemming from the wine substrate employed, we grouped the samples in two groups according to the base wine used, and then we calculated the mean of each compound for each group of wines aged and we normalised data using its corresponding mean value for each group. 3. Results and discussion A total of 137 volatile compounds were determined in the base wines and samples from biological ageing trials. Results were expressed as relative area values with respect to IS (Tables 2-4). The chemical group with the largest number of compounds was ethyl esters, followed by alcohols, acids, acetals and terpenes. Seventy-six of them were positively identified by mass spectrum according to the mass spectral data base and standard LRI and forty-three tentatively identified (TI) by mass spectrum and LRI according to data found in the literature. In general, during the biological ageing, most of the yeast strains studied produced an increase in the total content of volatile compounds, with a significant increase for 8 of them. The group of volatile compounds that increased the most was acetals, N and O yeast strains produced the highest values (Table 4). These compounds have been

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described as characteristic of wines aged under flor yeast velum by other authors (PozoBayón & Moreno-Arribas, 2011), flor yeast form these compounds from alcohols and aldehydes. Among them, acetaldehyde diethylacetal augmented in particular, followed by acetaldehyde ethyl amyl acetal and 2,4,5-trimethyl-1,3-dioxolane. The former compound is characteristic of sherry wines with biological ageing since its precursor, acetaldehyde, is a characteristic compound produced at high rate by flor yeasts (Zea, Serratosa, Mérida, & Moyano, 2015). Only one acetal, isovaleraldehyde diethylacetal, decreased in most of the wines. Total contents of ketones also increased significantly for all strains, due to acetoin. This compound is also formed by yeast from acetaldehyde (Romano & Suzzi, 1996) and some authors have observed that the production of acetoin by a flor yeast strain in synthetic medium depend on grown conditions, being favoured under biofilm formation condition (Moreno-García, García-Martínez, Millán, Mauricio, & Moreno, 2015). Yeast strains B and J produced the highest and most significant increments of acetoin compared to the other strains (Tables 2 and 3). On the other hand, the lowest significant increase was observed in the case of F strain (Table 3). 2-Nonanone, acetophenone, dihydro-3(2H)thiophenone and isovalerone presented similar trends for most strains; the two first compounds underwent augmentations for all or most strains respectively and the two last underwent decreases in most cases. These changes were generally significant with respect to base wines. The total amount of terpenes increased during biological ageing, regardless of yeast strain, these being increases significant for 14 strains, except for A strain, that produced the lowest increases (Table 2). The yeast strains that produced the highest increases were N and O (Table 4). As can be observed in Tables 2-4, six of the twelve terpenes determined increased in all cases. The terpene that underwent the highest increase was

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nerolidol, followed by farnesol, in most cases. For the terpenes that increased, the changes were significant for most strains studied except for α-terpineol and linalool oxide. Only 4 terpenes decreased in most cases, these changes being significant for α-terpinen and limonene for most of the cases. It is considered that terpenes present in wines come from grapes, especially when aromatic varieties of grapes are used as substrate (Mateo & Jiménez, 2000). However, in the biological ageing, the increases observed for most of terpenes should be due to de novo synthesis or a biotransformation of terpenes carried out by Saccharomyces cerevisiae yeast, since both processes have been reported in previous works (Carrau et al., 2005; King & Dickinson, 2000). Moreover, some authors have shown that biological ageing under yeast film increases terpene contents because these compounds are produced by flor yeast (Fagan, Kepner, & Webb, 1981). Moreover, in most cases, ethyl esters (for 13 yeasts) and aldehydes total contents (for 11 yeasts) also tended to increase significantly. We observed increases for 18 ethyl esters and decreases for 9 in most biological ageing trials. Regarding the first ones, in most cases the augmentations were significant, except for ethyl lactate. Among ethyl esters that increased in all cases, we can highlight the case of ethyl phenylacetate because their changes were significant in all cases and besides, this underwent the highest increases in most cases. Diethyl succinate was the ester that increased the most in the remaining cases, and it is a typical aroma which appears during ageing on lees (Ubeda, Kania, Del BarrioGalán, Medel, Gil, & Peña-Neira, in press). The augmentation of this compound has also been observed by other authors (Martinez de la Ossa, Caro, Bonat, Pérez, & Domecq, 1987; Cortes, Moreno, Zea, Moyano, & Medina, 1998). Biological ageing take place in a rich ethanol medium, therefore the formation of ethyl ester by chemical condensation is favoured (Ribéreau-Gayon, Glories, Maujean, & Dubourdieu, 2006).

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With respect to the loss of esters, for most yeast strains, these were significant, except for diethyl malate. Ethyl isovalerate was the ethyl ester that diminished in content in all cases, significantly so for 12 of the yeast strains studied. The increase of aldehyde total contents were due primarily to benzaldehyde, this compound increased significantly in all cases, except when the N yeast strain was used, being the compounds that increased the most in 11 trials. Moreno, Zea, Moyano, & Medida (2005) also observed an increment of this aldehyde during biological ageing of sherry Fino wine, and its production is favoured when yeast grown forming flor velum (Moreno-García et al., 2015). The total content of C13-norisoprenoids underwent a decrease due to the loss of βdamascenone, which decreased in all cases, significantly so in 14 of them. This compound have a pleasant cooked apple aroma, this loss may result in the reduction of the fruity aroma of the wine, as expected in this kind of wine. On the contrary, 1,1,6-Trimethyl-1,2dihydronaphthalene (TDN) relates to the typical “kerosene” or “petrol” aroma of some wines, which increased in all cases. This compound increases in Riesling wines, due to its chemical release from the precursor during bottle storage (Sacks, Gates, Ferry, Lavin, Kurtz, & Acree, 2012) and also in cava during the ageing which takes place in the presence of lees yeast (Riu-Aumatell, Bosch-Fusté, López-Tamames, & Buxaderas, 2006). In our case, this compound was able to increase by a similar mechanism to that which occurs in cava due to the presence of dead flor yeast or to the reductive condition of both processes. Although most strains produce an increase in most acids, particularly isobutyric and isovaleric acids that increased significantly in all trials, the total acid contents decreased for most strains. This is due to important decreases observed in octanoic and decanoic acid, except for strains D and J (Tables 2 and 3), in which cases decanoic acid was the

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one that increased the most. On the one hand, acids produce pungent and cheese odours, in large amounts, they could produce unpleasant aromas, and on the other hand, they are precursors of ethyl esters that have a fruity aroma. In the cases of octanoic (r2= 0.96) and hexanoic acid (r2= 0.83), a high correlation between ethyl ester and the corresponding acid was observed. The total content of acetic acid esters also decreased, except for B, C and D, significantly so for most strains. Contrary trends were observed in the cases of B, C and D strains, due to the significant increases of 2-phenylethanol acetate (Table 2). High losses of isoamyl acetate were also detected in all cases; this could involve an important aroma change during biological ageing since this compound contributes to fruity aroma. Although butyl acetate and cis-3-hexenyl acetate (TI) also underwent significant decreases, these were less important than the previous acetate. Therefore, our results for most strains are in agreement with other authors that reported decreases in the concentration of higher alcohol acetates during the first few months of ageing (Martinez de la Ossa et al., 1987; Useglio-Tomasset, 1983). As mentioned above, 2-phenylethanol acetate showed the contrary trend among the strains studied, on one hand, it is the compound that presented the highest and most significant increases for A, B, C and D strains (Table 2). On the other hand, this compound is one of the two acetic acid esters that decreased the most in the case of most strains. This compound provides aromatic notes of rose and honey, and has a relatively low threshold (250 μg/L) (Moreno et al., 2005). Therefore, our results reveal that the yeast strains studied would produce aromatically different wines. Finally, among 26 alcohols determined, approximately half of them tended to increase, while the other half decreased in most cases. With regard to ethanol, which is the primary alcohol in wines, significant decreases were observed in most cases, particularly in the case of strain J, which reduced the content by half during biological ageing. Martínez,

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Varcarcel, Perez & Benitez (1998) and Suarez-Lepe & Iñigo-Leal (2004) pointed out that ethanol decreases during the biological ageing of sherry because it is consumed by flor yeast as the main source of carbon and energy. On the contrary, an unexpected increase was observed in the case of the N strain (Table 4). Beside, other alcohols such as 1-octanol and 1-nonanol decreased in all cases to a significant extent. Most strains also produced a significant diminution of 1-undecanol and 1-dodecanol. Conversely, 2-phenylethanol was the alcohol that underwent the highest increments in most cases, followed by 3-methyl1-butanol. All yeast strains studied produced an augmentation of isobutanol, 1-butanol, 2-ethyl-1-butanol and 1-heptanol, these changes being significant in most cases. Similar results were observed by Moreno et al. (2005). Moreover, we observed an opposite trend with respect to 3-methyl-1-butanol, which underwent a significant augmentation in the case of most strains, whilst showing the highest decreases for strains such as G or H (Table 3). This, in conjunction with that mentioned above, corroborates the fact that each flor yeast strain has a characteristic production of volatile compounds. On the one hand, Sherry wines have a common organoleptic characteristic and, on the other hand, these wines have organoleptic characteristics typical of each winery, in order for the flor yeast to be present during biological ageing. Moreover, as we mentioned above, previous works have shown the dominance of one flor yeast strain in each winery and, even, in each wood barrel in Sherry wines (Ibeas et al., 1997b), as well as in wines from Montilla-Moriles PDO (Marin-Menguiano et al., 2017). In our work, ANOVA results showed that, in one month of biological ageing, the content of a lot of volatile compounds of wines were statistically different depending on the flor yeast strain used. This fact reveals that the peculiar aromatic characteristics of Sherry wines from different wineries may be largely due to the yeast strain present during biological ageing.

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To delve more deeply into this theory, volatile compounds were grouped according to their aromatic notes in Sherry wine, balsamic, dairy, fruity-sweet, floral-citric, greenvegetable, cheesy-pungent, spicy-toasted, waxy-fatty and chemical group and their values of relative peak area were added. The Sherry wine aroma group contained all acetals, since these compounds are characteristic of Sherry wine aged under flor velum, as mentioned above. Compounds such as high alcohols were included in the balsamic group, acetoin in the dairy group, terpenes in the floral-citric, ethyl esters and acids with high molecular weight were included in the waxy-fatty group and so on. In this way, we were able to compare the possible aroma that could be produced by each yeast strain and deduce which strain produces a volatile profile that is most typical of Sherry wine. The N strain, followed by the O strain, could apparently provide typical aromas of Sherry wine and similar values of floral-citric aroma to the other strains (Figure 1A). The wine aged with these two strains also presented predominant values of waxy-fatty and fruity-sweet aroma compared to other strains (Figure 1.B) and, on the contrary, low values of spicytoasted aroma (Figure 1.C). Therefore, we could suggest the use of N or O strains for biological ageing according to their hypothetical aromatic profile. Several PCAs were performed and Heatmaps were built with normalised data. When PCA was performed considering all volatile compounds as variables, a very low percentage of cumulative variance was explained by the first two principal components (42.3%). Samples were distributed for all the plan formed by the two first components (Figure 1S), however, 93% of the variables were located on the left side of the first component, most of them highly correlated with wine aged with N, O and C strains (Table 1S). Among them were most of alcohols, terpenes, acetic acid esters, aldehydes, volatile phenols, ketones and an important amount of ethyl esters, highlighting for their loading values (Table 1S) benzyl alcohol, 4-methyl-1-pentanol, trans-3-hexanol, 2-phenylethanol, 3-

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methyl-1-butanol, 2-methyl-1-butanol, 2,6-Dimethyl-3,7-Octadiene-2,6-diol, linalool, αterpin, benzyl acetate, 2-phenylethyl acetate, cis-3-hexenyl acetate, coumaran, 4vinylguaiacol and acetovanillone. On the left side of the first component, were also placed the yeast strains J and B, these shown a high correlation with all acetals and most of ethyl esters and acids, standing out, in these two last chemical groups, compounds such as ethyl hexanoate, ethyl butyrate, ethyl phenylacetate, ethyl dodecanoate, hexanoic and octanoic acids with high loading values. However, the best results with regard to the percentage of cumulative variance explained were obtained when we used the total values of different chemical groups as variables in PCA. Samples were distributed in similar way to the previous one for the plan formed by the two first components (Figure 2A). In this case, the first three components explained 74% of cumulative variance. Variables, except total lactones, were located on one side of the first component, with a major correlation with O, C, N, A, D, J and B strains (Figure 2B). Finally, we performed a PCA using aromatic notes as variables; the three first components explained 86.4% of cumulative variance. In this case, all variables were again located on the left side of the first component where the wines aged with N, C, O, J and B strains (Figure 3 A and B) were also located. Heatmaps performed enable to see graphically, at the same time, the correlation of the yeast strains with variables and also which yeast strains are more similar with respect to a certain volatile compounds production pattern (Figure 4) or aromatic notes (Figure 5). Heatmap for the total values of chemical groups (Figure 4) showed a high correlation between volatile phenols and the F strain, C13-norisoprenoinds with the D strain, lactones with the K strain, ketones and acid with the J strain. Strains N and C, included in the same cluster, shown a high correlation with most groups of compounds, these yeast strains had a high volatile compounds production. These yeast strains Heatmaps for aromatic notes

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variables showed a high correlation between N, C, B and J and most variables, especially in the case of the N strain (Figure 5). However, among them, the J strain was highly correlated with cheesy-pungent and waxy-fatty aroma, that produces an unpleasant aroma in wine. Therefore, the results of statistical analysis suggest that N and C strains could be the best strains to use in biological ageing to produce Sherry wines and G, H and F strains could be dispensed with for that purpose. 4. Conclusions In this work, different biological ageing trials using pure cultures of flor yeast strains have been carried out. The effect on wine volatile composition of different Saccharomyces yeast strains has been widely studied and demonstrated, however, there are few studies of the production of volatile compounds by pure cultures of flor yeast strains. Our results showed that, as expected, the yeast strains studied produced increases in acetal contents. Furthermore, these increased the ketones and terpenes contents, and decreased the acids and acetic acid esters contents. In only one month of biological ageing, the flor yeast strains produced important differences in the volatile profile of wines, suggesting that different strains of these kinds of yeasts could also produce aromatic characteristics peculiar to Sherry wine. Among the yeast strains studied, N seems to provide the most typical volatile profile to Sherry Fino wine, being, therefore, a good candidate for commercialisation. Microbiologically the visual evaluation of flor film formed for this strain was of exceptional quality. Other microbiological studies have been initiated in order to know if this yeast strain is suitable for this aim. Further studies to better understand on specific genetics trails related with the production of different aromatic profiles are needed for a complete understanding. References

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by Headspace Solid-Phase Microextraction Followed by Gas

Chromatography-Mass Spectrometry. Journal of Food Science 74, C90–C99. Figure Captions Figure 1. Spider chart of hypothetical aromatic profiles of wines submitted to biological ageing. A. Sherry wine, Balsamic, and Floral-citric aroma; B. Waxy-fatty and Fruity-

23

sweet aroma; C. Dairy, Green-vegetable, Cheesy-pungent, Spicy-toasted and Chemical aroma. Figure 2. Samples data scores (A) and variables data loading (B) plot on the plan consisting of the first two principal components (PC1 against PC2) from PCA performed using total values of each chemical group as variables. Figure 3. Samples data scores (A) and variables data loading (B) plot on the plan consisting of the first two principal components (PC1 against PC2) from PCA performed using aromatic notes as variables. Figure 4. Heatmap representation of total values of volatile compounds grouped according to chemical characteristics. Figure 5. Heatmap representation of total values of volatile compounds grouped according to aromatic notes.

24

Figure 1.A

Strain K Strain J Strain I

Strain L Strain M

Strain H

Strain N

Strain G

Strain O

Strain F

Strain A

Strain E Strain D

Strain B Strain C

Sherry wine Balsamic Floral-citric

Figure 1.B

Strain K Strain J Strain I

Strain L Strain M

Strain H

Strain N

Strain G

Strain O

Strain F Strain E Strain D

Strain A Strain B Strain C

Waxy-fatty Fruity-sweet

25

Figure 1.C

Strain K Strain J Strain I

Strain L Strain M

Strain H

Strain N

Strain G

Strain O

Strain F Strain E Strain D

Strain A Strain B Strain C

Dairy Green Cheesy-pungent Spicy-toasted Chemical

26

Figure 2.A

Str J Str B

3

2

Factor 2: 17,20%

Str H 1 Str N

Str M Str K

0

Str G

Str L

Str E Str I

Str A

Str O

-1 Str F Str C Str D

-2

-3 -6

-4

-2

0

2

4

Factor 1: 43,31%

Figure 2.B

1,0 Ketones

Factor 2 : 17,20%

0,5

Other esters Acetals Acids Terpenes Ethyl esters

Lactones

0,0

Acetic acid esters Volatile phenols Alcohols C13-norisoprenoides

-0,5

Aldehydes

-1,0 -1,0

-0,5

0,0

0,5

1,0

Factor 1 : 43,31%

27

Figure 3.A

04 Str J 03 Str B

02

Factor 2: 22,50%

Str K 01 Str A Str O

Str L

00

Str H Str E Str M

Str G

Str I -01

Str D

Str C

Str N

-02

-03

-04 -05

Str F

-04

-03

-02

-01

00

01

02

03

04

05

Factor 1: 52,76%

Figure 3. B

28

1,0 Dairy Cheesy-pungent Sherry wine 0,5

Factor 2 : 22,50%

Fruity-sweet

0,0

Waxy-fatty Chemical Floral Green-vegetable Balsamic

-0,5

Spicy-toasted

-1,0 -1,0

-0,5

0,0

0,5

1,0

Factor 1 : 52,76%

Figure 4.

29

30

Figure 5.

31

ounds

diethylacetal l-1,3-dioxolane e diethylacetal thyl propyl

l-1,3-dioxolane

oxy)-butane yl-1,3-dioxolane

Table 1. Visual appearance of flor film. Rough/smooth Thickness A

SMOOTH

++

B

ROUGH

++

C

SMOOTH

++

D

ROUGH

+++

E

ROUGH

+

F

ROUGH

+

G

ROUGH

+++

H

SMOOTH

++

I

SMOOTH

++

J

ROUGH

+++

K

SMOOTH

++

L

ROUGH

+++

M

SMOOTH

+

N

ROUGH

+++

O

ROUGH

+++

+: thin, good quality; ++: thick, very good quality; +++: very thick, exceptional quality

Table 2. Profiles of volatile compounds of base wine substrate and samples after biological ageing using different flor yeast strains. LRIb

IDc

884b 920b 942b

Base wine (1)

Strain A

A B1 B2

1.613 ± 0.020ª 0.139 ± 0.008ª nda

12.6 ± 1.0b,c 1.65 ± 0.16b,d 0.0110 ± 0.0015b,c

950b

C

0.0061 ± 0.0009ª

0.158 ± 0.021b,e

956b

C

0.0150 ± 0.0007ª

970b 987b

B3 C

0.0044 ± 0.0004ª nda

Relative peak area ± sda Strain B Strain C 12.6 ± 1.1b,c 1.47 ± 0.10b,c 0.129 ± 0.0015c

Strain D

12.0 ± 0.1b,c,d 1.910 ± 0.013d 0.0102 ± 0.0003b,c,d

10.7 ± 0.4d,e 1.714 ± 0.011b,d 0.0045 ± 0.0007e,f

0.29 ± 0.03c

0.271 ± 0.009c

0.08576 ± 0.00003d

0.239 ± 0.014b

0.32 ± 0.03c

0.220 ± 0.012b,d

0.187 ± 0.013d

0.103 ± 0.005b,d 0.069 ± 0.008b

0.205 ± 0.012c 0.044 ± 0.006c

0.107 ± 0.016d 0.0253 ± 0.0006d,h

0.072 ± 0.003e 0.0189 ± 0.0021d,e

32

z 101) ,3-dioxane 73) de diethylacetal thyl amyl acetal thyl hexyl acetal

c acid

anol anol

ntanol ntanol

opanol

nol

-1-propanol

ldehyde hylfurfural

1012 1027 1029 1044 1075 1242

C B1 B1 C

0.0047± 0.0003ª nd nda 0.0121 ± 0.0014ª 0.143 ± 0.021a 0.00107 ± 0.00013ª 1.94ª

0.163 ± 0.020b nd 0.019 ± 0.003b,e 0.043 ± 0.003b 1.79 ± 0.03b 0.011 ± 0.003b 16.9b,d,f

0.110 ± 0.015c nd 0.100 ± 0.008c 0.050 ± 0.04c 3.97 ± 0.20c 0.0254 ± 0.0017c 19.2c

0.040 ± 0.004d,f nd 0.03 ± 0.005d 0.024 ± 0.003d 2.8 ± 0.4d 0.019 ± 0.003d 17.6c,d,f

0.060 ± 0.007e nd 0.0151 ± 0.0009b 0.0113 ± 0.0003ª 2.16 ± 0.09b,e 0.0116 ± 0.0012b 15.1b,e

1442 1537 1563 1667 1741 1847 1952 1957 2072 2175 2286 2344 2352

A A A A A A B2 A A A A B4 B2

0.0343 ± 0.0022a,c,d nd nda nda nd 0.192 ± 0.018a,b,c 0.0043 ± 0.0004ª,d,e 0.00382 ± 0.00016ª,b 1.254 ± 0.008ª 0.0320 ± 0.0014ª 1.69 ± 0.21ª,e nd 0.0121 ± 0.0015ª,f 3.22ª,f

0.086 ± 0.012b nd 0.098 ± 0.014b 0.063 ± 0.009b nd 0.229 ± 0.003b 0.0130 ± 0.0016b,f 0.0055 ± 0.0022ª,b,c 0.45 ± 0.06b 0.021 ± 0.003b,c,d 0.65 ± 0.09b nd ndb 1.62b,d

0.047 ± 0.007c,g nd 0.113 ± 0.012b 0.0582 ± 0.0017b,c nd 0.22 ± 0.03b,c 0.0120 ± 0.0018b,c,f 0.0062 ± 0.0008b,c 0.61 ± 0.08c,f 0.019 ± 0.003b,c 1.25 ± 0.18c,d nd 0.0064 ± 0.0009c,d 2.34c,e

0.028 ± 0.004a,d nd 0.052 ± 0.004c 0.053 ± 0.005b,c nd 0.192 ± 0.025ª,b,c 0.0060 ± 0.0009d,e 0.0058 ± 0.0008ª,b,c 0.41 ± 0.06b 0.0171 ± 0.0025b,c 1.28 ± 0.19ª,c,d nd 0.0073 ± 0.0011d,e 2.06c,d

0.0133 ± 0.0020e nd 0.025 ± 0.003d 0.021 ± 0.003d nd 0.166 ± 0.024ª,c,d 0.00298 ± 0.00011ª 0.0063 ± 0.0009b,c 0.473 ± 0.005b,c 0.029 ± 0.004ª,d,e 1.87 ± 0.24e nd 0.0095 ± 0.0013e,g 2.61ª,c,e

944b 1022 1083 1142 1207 1226 1284 1304 1312 1325 1354 1361 1373 1380 1455 1488 1520 1558 1659 1663 1681 1723 1870 1882 1925 1971

A A A A A A B5 B2 A A A B2 B4 A A A B5 A A B5 B2 B2 B2 A A B5

26.2 ± 1.2ª 0.38 ± 0.05ª 0.20 ± 0.03ª,d 0.149 ± 0.003ª 1.31 ± 0.19ª,e 11.31 ± 0.06ª,b nda nda 0.0433 ± 0.0021ª 0.080 ± 0.003ª,b 0.66 ± 0.04ª,d,e 0.00334 ± 0.00023ª,e nda 0.039 ± 0.003ª,b,c 0.0122 ± 0.0016ª 0.031 ± 0.003ª 0.028 ± 0.003ª,d 0.101 ± 0.010ª 0.0517 ± 0.0014ª,c,e 0.129 ± 0.014ª 0.0037 ± 0.0003ª 0.0098 ± 0.0011ª 0.0142 ± 0.0003ª 0.025 ± 0.003ª,b,d 9.1 ± 1.1ª,b 0.0212 ± 0.0017ª,c 49.9ª,b,d

26.5 ± 1.3ª 0.83 ± 0.06b,f 0.365 ± 0.016b 0.184 ± 0.04ª,c 1.02 ± 0.09b,d 12.7 ± 1.5b 0.0070 ± 0.0004b 0.0174 ± 0.0024b,e 0.0384 ± 0.0024ª,b 0.092 ± 0.008b,c 0.59 ± 0.04ª,d,c 0.00312 ± 0.00006b 0.00459 ± 0.00024b,d 0.040 ± 0.003ª,b,c 0.0149 ± 0.0006ª 0.024 ± 0.003b 0.0185 ± 0.0013b,c 0.0508 ± 0.0022b 0.042 ± 0.006ª,b,d,e 0.0390 ± 0.0004b 0.002595 ± 0.000019b,d 0.0126 ± 0.0012b ndb 0.029 ± 0.003b,c,d 11.6 ± 1.3b,c,e 0.0176 ± 0.0024ª,b 54.2b

19 ± 3b 0.67 ± 0.04c 0.327 ± 0.007b,c 0.30 ± 0.03b 0.88 ± 0.12b,c 12.02 ± 1.5b 0.0138 ± 0.0014c 0.070 ± 0.003c 0.038 ± 0.003ª,b 0.103 ± 0.009c,e 0.54 ± 0.04b,c 0.00416 ± 0.00012c,d 0.00258 ± 0.00022c,f 0.0438 ± 0.0024b,c 0.024 ± 0.004b 0.028 ± 0.004ª,b 0.0243 ± 0.0017ª 0.031 ± 0.003c 0.034 ± 0.005b 0.0198 ± 0.0022c,e 0.001993 ± 0.00004c 0.00957 ± 0.00013ª 0.0112 ± 0.0016c,d 0.028 ± 0.003b,c,d 11.3 ± 1.2b,c,e 0.024 ± 0.003c 45.1ª,c,e

23 ± 3ª,c,d 0.42 ± 0.06ª,d,e 0.25 ± 0.03d,f 0.1705 ± 0.0019ª,c 1.18 ± 0.17d,e 11.57 ± 0.15ª,b 0.0009 ± 0.0003ª,d 0.0133 ± 0.0015b 0.038 ± 0.004ª,b 0.079 ± 0.009ª,b,d 0.61 ± 0.07ª,c 0.00400 ± 0.00013c,d 0.0051 ± 0.0007d,e 0.047 ± 0.006d 0.049 ± 0.007c 0.028 ± 0.004ª,b 0.025 ± 0.003ª 0.054 ± 0.008b 0.056 ± 0.008c 0.042 ± 0.006b 0.00227 ± 0.00012b,c 0.0150 ± 0.0017c 0.0119 ± 0.0016ª,d 0.031 ± 0.003c 15.4 ± 2.1d 0.0175 ± 0.0024ª,b 53.3b,d

25.4 ± 0.8ª,d 0.50 ± 0.07d,e 0.2969 ± 0.0017c,e 0.161 ± 0.007a,c 1.447 ± 0.010ª 12.3 ± 0.4b nda 0.0060 ± 0.0003b 0.0426 ± 0.0024ª 0.0660 ± 0.0008d 0.699 ± 0.004d,e 0.00384 ± 0.00016c,d,e 0.00658 ± 0.00023e 0.0483 ± 0.0009d 0.0299 ± 0.0007b,d 0.0299 ± 0.0007ª 0.023 ± 0.003ª,c 0.086 ± 0.011d 0.035 ± 0.004b 0.073 ± 0.011d 0.0037 ± 0.0005ª 0.0083 ± 0.0006ª,d 0.0072 ± 0.0008e 0.0240 ± 0.0013ª,b,d 12.4 ± 1.3c,e 0.0108 ± 0.0015d 53.8b

1040 1447 1507 1636 2489

A A A B5 A

0.00286 ± 0.00017ª 0.064 ± 0.006ª,b,d 0.0119 ± 0.0005ª 0.0140 ± 0.0019ª 0.0044 ± 0.0004ª 0.097ª

ndb 0.065 ± 0.009ª,b,d 0.063 ± 0.007b 0.0227 ± 0.0011b 0.0145 ± 0.0018b 0.164b

ndb 0.043 ± 0.006c 0.0533 ± 0.0012b,d 0.0229 ± 0.0020b 0.0058 ± 0.0007ª,c 0.125ª,e

ndb 0.079 ± 0.011d 0.116 ± 0.016c 0.0292 ± 0.0008c 0.0084 ± 0.0012e 0.232c

ndb 0.051 ± 0.006ª,c 0.117 ± 0.013c 0.01868 ± 0.00024d 0.00654 ± 0.00006ª,c,d,e 0.194d

33

acetate

acetate acid esters

butyrate

exanoate

y-4-

lthio)propionate

etate oate tridecanoato noate

dodecanoato

anoate noate 117)

canoate

873b 1035 1083 1255 1298 1717 1807

A A A A A A A

0.37 ± 0.04ª 0.0058 ± 0.0007ª 0.81 ± 0.07ª 0.002496 ± 0.000018ª 0.0040 ± 0.0005ª 0.0023 ± 0.0003ª 1.50 ± 0.14ª,e 2.69ª,b

0.1142 ± 0.0021b,c ndb 0.188 ± 0.024b 0.0050 ± 0.0007b 0.00122 ± 0.00015b,c,d 0.0035 ± 0.0003b,c 2.05 ± 0.22b,c 2.36ª,b

0.112 ± 0.016b,c ndb 0.172 ± 0.015b,c 0.0045 ± 0.0004b,c 0.00123 ± 0.00010b,c,d 0.0039 ± 0.0004c,d 2.5 ± 0.4b,c,d 2.79ª

0.085 ± 0.013c ndb 0.108 ± 0.015d,e 0.00375 ± 0.00021c,d 0.00141 ± 0.00018d,e 0.0043 ± 0.0006d 2.5 ± 0.4c,d 2.70ª,b

0.10739 ± 0.00017b,c ndb 0.138 ± 0.016b,c,d,e 0.00603 ± 0.00008e 0.00124 ± 0.00017b,c,d 0.0043 ± 0.0006d 2.6 ± 0.4d 2.87ª

948b 1001 1015 1031 1093 1125 1207 1282 1318 1329 1333 1422 1493 1523

A A A A A B5 A B3 A B2 A A B2 A

0.0094 ± 0.0013ª,b 0.273 ± 0.006ª 0.0066 ± 0.0006ª 0.0167 ± 0.0009ª 0.00143 ± 0.00020ª,e 0.00482 ± 0.00023ª 0.49 ± 0.05ª,f 0.0078 ± 0.0011ª 0.00154 ± 0.00020ª 0.0043 ± 0.0006ª,d 0.355 ± 0.019ª,b,c 0.63 ± 0.09ª,d 0.0099 ± 0.0012ª,b 0.0028 ± 0.0004ª,g

0.0085 ± 0.0012ª,b 0.27 ± 0.04ª 0.0114 ± 0.0008b 0.0073 ± 0.0006b 0.00226 ± 0.00024b,d 0.036 ± 0.004b 0.68 ± 0.10b,c 0.0028 ± 0.0003b,c,e 0.00492 ± 0.00006b 0.0070 ± 0.0009b,f 0.357 ± 0.013ª,b,c 0.49 ± 0.03b,c 0.0141 ± 0.0006b,c,e 0.0023 ± 0.0003ª,b

0.0079 ± 0.0009ª 0.38 ± 0.04b 0.0145 ± 0.0016c 0.0090 ± 0.0011c 0.0046 ± 0.0004c 0.0125 ± 0.0016c 0.746 ± 0.009c 0.0035 ± 0.0005c,e 0.01216 ± 0.00002c 0.0060 ± 0.0003b,d 0.34 ± 0.03ª,b,c 0.501 ± 0.007ª,b,c 0.0106 ± 0.0015c,e 0.0048 ± 0.0003c,e

0.0037 ± 0.0004c,d 0.136 ± 0.016c,d 0.0053 ± 0.0007ª 0.0036 ± 0.0005d,e 0.0024 ± 0.004b,d,f 0.0068 ± 0.0006ª,d 0.69 ± 0.08b,c 0.0047 ± 0.0003d 0.0145 ± 0.0021d 0.0052 ± 0.0007ª,c,d,g 0.41 ± 0.06c 0.46 ± 0.06b 0.0126 ± 0.018d 0.0042 ± 0.0006c

0.00548 ± 0.00003d 0.122 ± 0.008c,d 0.0120 ± 0.0005b,c 0.0026 ± 0.0003d 0.002 ± 0.003b,e 0.00535 ± 0.00016ª,d 0.34 ± 0.03d 0.00211 ± 0.00017b 0.00828 ± 0.00016e 0.003109 ± 0.000020e,h 0.38 ± 0.03b,c 0.59 ± 0.09ª,c,d 0.0077 ± 0.0011ª,b,c 0.0094 ± 0.0014d

1535

B2

0.0157 ± 0.0013ª,b

0.0193 ± 0.0020b,c,e

0.0196 ± 0.0015c,e

0.023 ± 0.003d

0.0170 ± 0.0010ª,b,c

1554 1606 1626 1652 1671 1681 1774 1833 1896 2045 2050 2108 2131 2145 2252 2321 2564

B2 A A A A B6 A A C A B7 B6 A B7 A A

0.0029 ± 0.0004ª 0.030 ± 0.003ª 0.061 ± 0.008ª 0.020 ± 0.003ª 1.67 ± 0.22ª nda 0.079 ± 0.008ª 0.0031 ± 0.0004ª,e 0.0143 ± 0.0014ª 0.0074 ± 0.0011ª 0.0041 ± 0.0005ª,c 0.0790 ± 0.0019ª,f nda 0.003178 ± 0.000024ª 0.0155 ± 0.0018ª,f 0.0087 ± 0.0011ª 0.00157 ± 0.00015ª 3.81ª,e

0.00205 ± 0.00006b 0.030 ± 0.003ª 0.085 ± 0.013ª,b,c 0.0126 ± 0.0011b,c,e 1.97 ± 0.18ª,b 0.00271 ± 0.00016b,d 0.36 ± 0.04b 0.0097 ± 0.0013b 0.2280 ± 0.0020b 0.0031 ± 0.0004b 0.0035 ± 0.0005ª,b 0.204 ± 0.010b 0.00445 ± 0.00017b 0.00170 ± 0.00025b 0.0077 ± 0.0011b,d,e 0.040 ± 0.004b 0.003045 ± 0.00023b,c,d 4.88b,d

0.00213 ± 0.00023b 0.030 ± 0.003ª 0.109 ± 0.011b,f 0.0122 ± 0.0008b,c 1.82 ± 0.24ª 0.00199 ± 0.00004c,e 1.36 ± 0.14c 0.029 ± 0.004c 0.122 ± 0.018c,f 0.0075 ± 0.0011ª 0.0037 ± 0.0005ª,b,c 0.117 ± 0.017c,d 0.00662 ± 0.00013c 0.0020 ± 0.0003b 0.069 ± 0.010c 0.021 ± 0.003c,f 0.0032 ± 0.0003c,d 5.79c

0.0033 ± 0.0005ª 0.039 ± 0.005b 0.092 ± 0.013b,c 0.017 ± 0.003ª,d 2.5 ± 0.3c 0.00227 ± 0.00012b,d 0.70 ± 0.05d 0.0087 ± 0.0005b,d 0.161 ± 0.022d 0.0059 ± 0.0007ª 0.0056 ± 0.0007d 0.128 ± 0.018d 0.00192 ± 0.00014d,f 0.00261 ± 0.00011ª,b 0.0114 ± 0.0008ª,b,d,e 0.026 ± 0.003d 0.003501 ± 0.000019d 5.49c,d

0.0027 ± 0.0003ª 0.027 ± 0.003ª 0.069 ± 0.003ª,c 0.019 ± 0.003ª,d 2.4 ± 0.3b,c 0.00048 ± 0.0003b,d 0.33 ± 0.04b 0.0067 ± 0.0010b,d,e,f 0.0742 ± 0.0015e,h 0.0062 ± 0.0005ª 0.00286 ± 0.00025b 0.048 ± 0.007ª,e 0.0048 ± 0.0003b 0.0036 ± 0.0005ª,c 0.0137 ± 0.0017ª,d,e 0.0093 ± 0.0004ª,e 0.00210 ± 0.00024ª,e 4.55b,e

1372 1580 1760 2207 2210 2298 2559

B2 A B2 B2 A B8 B2

0.0031 ± 0.0004ª,c 0.0030 ± 0.0004ª,f 0.0219 ± 0.0020ª,e 0.0125 ± 0.0018ª,d 0.0071 ± 0.0010ª 0.025 ± 0.004ª,c,e,g 0.00196 ± 0.00012ª 0.075ª,d,g

0.0020 ± 0.0003b,g 0.00154 ± 0.00022b 0.01761 ± 0.00015b,c,d 0.030 ± 0.004ª 0.0076 ± 0.0010b 0.056 ± 0.008b 0.0035 ± 0.0004b,c,e 0.119b

0.00313 ± 0.00018c 0.0055 ± 0.0008c,e 0.0208 ± 0.0019ª,c,e 0.043 ± 0.005b 0.0099 ± 0.0015c 0.053 ± 0.008b 0.0038 ± 0.0005b,c,d,e 0.139c

0.0024 ± 0.0003b,d 0.0050 ± 0.0007c,d 0.025 ± 0.004ª 0.015 ± 0.014ª 0.0077 ± 0.0004ª,d 0.021 ± 0.003ª,c 0.0044 ± 0.0005d 0.080d,g

0.0028 ± 0.0004ª,c,d 0.0018 ± 0.0003b 0.0153 ± 0.0006b 0.0112 ± 0.0016c 0.0028 ± 0.0004ª 0.036 ± 0.005d,g 0.00252 ± 0.00022ª,f 0.072ª,d

969b 1138 1151 1276 1322

B1 C B2 A A

0.0047 ± 0.0006ª,d,e 0.0107 ± 0.0015ª nd 0.0090 ± 0.0007ª 0.0054 ± 0.0006ª,c

0.00559 ± 0.00020b 0.0085 ± 0.0009b nd 0.228 ± 0.017b,d,e 0.0023 ± 0.0003b

0.0037 ± 0.0005c 0.0061 ± 0.0007c nd 0.385 ± 0.009c 0.0053 ± 0.0006ª,c

0.00402 ± 0.00022ª,c 0.0097 ± 0.0014ª,b nd 0.200 ± 0.003b,d 0.0054 ± 0.0006ª,c

0.0042 ± 0.0004ª,c,d 0.0088 ± 0.0006b nd 0.2336 ± 0.0007b,d,e 0.0071 ± 0.0006d,g

sters

ntanone -heptanone

pten-2-one

34

0.0066

0.0025

-thiophenone

nedione

noids l-1,2alene

1375 1492 1515 1641 1773

B2 A C A C

0.0049 ± 0.0004ª 0.0085 ± 0.0010ª,c,e 0.028 ± 0.003ª 0.0150 ± 0.0020ª 0.059 ± 0.003ª 0.145ª

0.0089 ± 0.0009b,d 0.0065 ± 0.0005ª,b 0.0262 ± 0.0018ª,b 0.035 ± 0.005b 0.034 ± 0.005b 0.355b

0.0070 ± 0.0007ª,b 0.0062 ± 0.0003b 0.023 ± 0.003ª,b,c 0.035 ± 0.004b 0.0343 ± 0.0025b 0.506c

0.0126 ± 0.0014c 0.0090 ± 0.0013c,e 0.022 ± 0.003b,c 0.0208 ± 0.0012b,c,d 0.055 ± 0.008ª,c 0.339b

0.0118 ± 0.0014c,d 0.00603 ± 0.00012b 0.0272 ± 0.0025ª 0.024 ± 0.003d 0.0330 ± 0.0004b 0.356b

1623 2039 2159

A B A

0.0476 ± 0.0021ª,b,c 0.033 ± 0.004ª 0.0095 ± 0.0007ª,c 0.090ª,b,c

0.04889 ± 0.00014ª,b,c,d 0.0439 ± 0.0021b,c 0.0142 ± 0.0020b,d 0.107b,c

0.046 ± 0.003ª,b,c 0.046 ± 0.007c,d 0.0124 ± 0.0018b,c,d 0.104ª,b,c

0.061 ± 0.009d 0.055 ± 0.005d 0.0151 ± 0.0007d 0.131d

0.03815 ± 0.00019ª 0.035 ± 0.005ª,b,e 0.0084 ± 0.0011ª 0.082ª

1727

A

0.0068 ± 0.0010ª

0.0199 ± 0.0025b

0.0131 ± 0.0019c

0.017 ± 0.003b,d

0.018 ± 0.003b

1814

A

0.080 ± 0.009ª 0.087ª

0.0402 ± 0.0026b,d,f 0.060b

0.0296 ± 0.0021b,c 0.043c,d

0.045 ± 0.006d 0.062b

0.063 ± 0.009e 0.082ª

1147 1469 1505 1543 1602 1646 1700

A B2 C A C C A

0.051 ± 0.007ª 0.0056 ± 0.0007ª,b 0.0069 ± 0.0006ª 0.173 ± 0.019ª,e 0.0227 ± 0.0014ª,c 0.00132 ± 0.00014ª 0.051 ± 0.006ª

0.076 ± 0.011b 0.0063 ± 0.0008ª,b 0.0031 ± 0.0003b 0.155 ± 0.011ª 0.0154 ± 0.0011b 0.0029 ± 0.0004b 0.052 ± 0.003ª

0.036 ± 0.005c,d 0.0070 ± 0.0010b 0.00319 ± 0.00020b 0.27 ± 0.03b,c 0.0206 ± 0.0024ª 0.0037 ± 0.0004b,d 0.055 ± 0.007ª

0.046 ± 0.003ª,d 0.0066 ± 0.0009b 0.0044 ± 0.0007c 0.29 ± 0.04c 0.0229 ± 0.0003ª,c 0.0091 ± 0.0004c 0.088 ± 0.009b

0.0152 ± 0.0022e 0.0040 ± 0.0005c 0.0051 ± 0.0006c 0.229 ± 0.012b,d 0.0139 ± 0.0009b 0.0041 ± 0.0005b,d 0.059 ± 0.006ª

1702

C

0.0218 ± 0.0022ª,b

0.0191 ± 0.0009ª,b

0.021 ± 0.003ª,b

0.0275 ± 0.0024c

0.0168 ± 0.0022ª

1768 1852 2041 2364

A A A B2

0.039 ± 0.003ª 0.051 ± 0.004ª 0.0126 ± 0.0009ª 0.02767 ± 0.00012ª 0.46ª

0.052 ± 0.005ª,b 0.0992 ± 0.0006b 0.0478 ± 0.0015ª,b 0.0844 ± 0.0017b,c 0.61ª,b

0.117 ± 0.016c 0.138 ± 0.020c 0.24 ± 0.03c,d 0.112 ± 0.016c,d 1.03e,f,g

0.093 ± 0.013d 0.146 ± 0.012c 0.28 ± 0.04d,g 0.142 ± 0.021d,e 1.16f,g

0.064 ± 0.008b,e 0.122 ± 0.018b,c 0.0889 ± 0.0012b,e 0.063 ± 0.009b 0.68b

2027 2171 2185 2203 2402 2504 2577

A A A B2 C B9 C

0.79 ± 0.08ª 0.0035 ± 0.0003ª 0.23 ± 0.03ª,c 0.0060 ± 0.0008ª 0.0124 ± 0.0009ª 0.00133 ± 0.00013ª 0.0029 ± 0.0004ª,e 1.05ª 63.58ª,b

0.743 ± 0.022ª 0.0056 ± 0.0006b,c,e 0.195 ± 0.015ª,b 0.0160 ± 0.0007b,c,e 0.0172 ± 0.0016b,c 0.00199 ± 0.00021b,c,e 0.0044 ± 0.0003b,c 0.98ª 82.39c,d

0.72 ± 0.10ª 0.0057 ± 0.0007b,c 0.20 ± 0.03ª,b 0.0161 ± 0.0024b,c,e 0.0155 ± 0.0017ª,b,c 0.0022 ± 0.0003c,e 0.0046 ± 0.0005b,c 0.96ª 78.11c,d

0.97 ± 0.14b 0.0072 ± 0.0009d 0.255 ± 0.034c 0.026 ± 0.004d 0.022 ± 0.003d 0.00265± 0.00019d 0.0059 ± 0.0005d 1.29b 84.41d

0.71 ± 0.09ª 0.00363 ± 0.00003ª,f 0.171 ± 0.012b 0.0194 ± 0.0022e 0.0150 ± 0.0013ª,b,c 0.0019 ± 0.0003b,c,e 0.0029 ± 0.0004ª 0.93ª 81.32c,d

orisoprenoids

oxide

3,7-Octadiene-

e phenols e compounds ntion index values obtained from samples. of identification: A, mass spectrum and LRI agreed with standards; B, mass spectrum agreed with mass spectral data base and LRI agreed with the literature data (T d with mass spectral data base. ected. cript letter in the same row indicates no significant statistically differences (p<0.05). ted by linear regression. rence agreed with LRI data:1: Morales at al. 2019; 2: Kim et al. 2019; 3: Carlin et al., 2016; 4: Nicolli et al., 2018; 5: http://www.chemspider.com/Default.aspx; 6: et al., 2009; 8: Ka et al., 2005; 9: Fernández de Simón et al., 2015.

Table 3. Profiles of volatile compounds of samples after biological ageing using different flor yeast strains (wine base 1). Volatile compounds

LRI

ID

b

c

Strain F

Relative peak area ± sda Strain G Strain H Strain I

Strain J

35

Acetals Acetaldehyde diethylacetal 2,4,5-Trimethyl-1,3dioxolane Propanaldehyde diethylacetal Acetaldehyde ethyl propyl acetal 2,4,5-Trimethyl-1,3dioxolane (isomer) 1-(1-Ethoxyethoxy)butane 2-Ethyl-5-methyl-1,3dioxolane

884b

A

3.69 ± 0.09f

920b

B1

0.82 ± 0.04d

942b

B2

0.0019 ± 0.0003ª,e

950b

C

0.0235 ± 0.0023ª

956b

C

0.0829 ± 0.0003e

970b

B3

0.021 ± 0.003ª

987b

C

Unknown (m/z 101)

1012

-

2,4-Dimethyl-1,3dioxane

1027

Unknown (m/z 73) Isovaleraldehyde diethylacetal Acetaldehyde ethyl amyl acetal Acetaldehyde ethyl hexyl acetal Total of acetals Acids

0.189 ± 0.007d 0.109 ± 0.016d 0.0522 ± 0.0020f 0.037 ± 0.003d,f

11.3 ± 0.6c,d,e 1.66 ± 0.14b,d 0.0073 ± 0.0010d,f,g 0.156 ± 0.019b,e 0.198 ± 0.024d 0.091 ± 0.012b,d,e 0.0279 ± 0.0020h 0.039 ± 0.006d,f

9.92 ± 0.19e

11.96 ± 0.17b,c,d 1.66 ± 0.21b,d 0.030 ± 0.004h 0.197 ± 0.022e

0.01046 ± 0.00006g 0.07015 ± 0.00003e

1.25 ± 0.08c,e 0.0055 ± 0.0008e,f,g 0.127 ± 0.013b,d 0.193 ± 0.021d 0.098 ± 0.005b,d 0.0161 ± 0.0016e,g 0.028 ± 0.003d

C

nd

nd

nd

nd

nd

1029

-

0.0040 ± 0.0004ª

1044

B1

0.0081 ± 0.0011ª,f

0.029 ± 0.003d,f 0.00673 ± 0.00020e,f

0.031 ± 0.004d,f 0.01149 ± 0.00007ª

0.0262 ± 0.0025d,e,f 0.0206 ± 0.0010d

0.063 ± 0.009g 0.0079 ± 0.0010ª,e,f

1075

B1

0.353 ± 0.012ª

2.33 ± 0.18e

2.17 ± 0.09b,e

2.0 ± 0.3b,e

4.15 ± 0.08c

1242

C

0.0039 ± 0.0006ª

0.018 ± 0.003d,e 14.6e

0.0143 ± 0.0021b,e 14.0e

0.0115 ± 0.0005b 15.5b,d,e

0.027 ± 0.003c 18.7c,f

0.030 ± 0.007ª,d nd 0.046 ± 0.007c 0.037 ± 0.005e nd

0.067 ± 0.010f nd 0.115 ± 0.016b 0.048 ± 0.006c nd

0.0099 ± 0.0014c 0.0100 ± 0.0010d

0.021 ± 0.003ª,e nd 0.036 ± 0.003c,d 0.035 ± 0.005e nd 0.150 ± 0.022ª,d 0.0111 ± 0.0016b,c 0.0073 ± 0.0010c,d

0.66 ± 0.06d,f

0.34 ± 0.05b

1.09 ± 0.16ª

0.031 ± 0.004ª,e

0.0142 ± 0.0021b 0.94 ± 0.14b,c,d nd 0.0083 ± 0.0012d,e 1.56b,d

0.053 ± 0.008g

2.92ª,e

0.035 ± 0.005c,d nd 0.045 ± 0.005c 0.0269 ± 0.0007d,e nd 0.134 ± 0.020d 0.0035 ± 0.0004ª,d 0.0035 ± 0.0005ª,b 0.162 ± 0.023e 0.0162 ± 0.0024b,c 0.94 ± 0.14b,c nd 0.0041 ± 0.0004c 1.37b

5.10g

Acetic acid

1442

A

0.051 ± 0.003g

Propanoic acid

1537

A

nd

Isobutyric acid

1563

A

0.159 ± 0.013e

Isovaleric acid

1667

A

0.051 ± 0.007c

Pentanoic acid

1741

A

nd

Hexanoic acid

1847

A

0.038 ± 0.005e

2-Ethylhexanoic acid

1952

B2

0.0067 ± 0.0010e

Heptanoic acid

1957

A

0.00320 ± 0.00004ª

Octanoic acid

2072

A

0.17 ± 0.03e

Nonanoic acid

2175

A

0.042 ± 0.006f

Decanoic acid

2286

A

2.38 ± 0.17f

9-Decanoic acid

2344

B4

nd

2352

B2

Geranic acid

10.5 ± 1.0e

Total of acids Alcohols

0.0139 ±

0.0010f

1.16 ± 0.17e 0.0089 ± 0.0013b,d,g 0.26 ± 0.03c

0.21 ± 0.03b,c

1.35 ± 0.18ª,d nd 0.0109 ± 0.0011ª,g 2.40c,e

Ethanol

944b

A

19.3 ± 2.0b,c

18 ± 3b,e

19.34 ± 0.10b,c

20.4 ± 2.1b,c

1-Propanol

1022

A

0.49 ± 0.05ª,d,e

0.41 ± 0.06ª,d

0.92 ± 0.06b

0.54 ± 0.06e

Isobutanol

1083

A

0.340 ± 0.015b

0.18 ± 0.03ª

1-Butanol

1142

A

0.150 ± 0.020ª

0.152 ± 0.012ª

0.235 ± 0.015e,f 0.195 ± 0.003c

0.258 ± 0.013e,f 0.156 ± 0.010ª

0.30 ± 0.03c 0.212 ± 0.015c 0.029 ± 0.003h 0.052 ± 0.006e,f

0.32 ± 0.05f 0.0142 ± 0.0021f 0.020 ± 0.003e

1.84 ± 0.27e nd 0.0129 ± 0.0019ª,f 3.59f 13.8 ± 2.1e 0.213 ± 0.023g 0.28 ± 0.04c,e,f 0.25 ± 0.04d

36

2-Methyl-1-butanol

1207

A

1.56 ± 0.11ª

0.73 ± 0.10c

0.95 ± 0.10b,c,d

3-Methyl-1-butanol

1226

A

11.95 ± 0.23b

9.2 ± 0.6c

9.5 ± 1.0c,d

4-Heptanol

1284

B5

nda

nda

2-Ethyl-1-butanol

1304

B2

4-Methyl-1-pentanol

1312

A

0.00229 ± 0.00004ª,d 0.0377 ± 0.0023ª,b

3-Methyl-1-pentanol

1325

A

0.075 ± 0.005ª,d

1-Hexanol

1354

A

0.72 ± 0.06e

trans-3-Hexenol

1361

B2

0.0043 ± 0.0005d

3-Ethoxy-1-propanol

1373

B4

0.0163 ± 0.0023g

cis-3-Hexen-1-ol

1380

A

0.048 ± 0.003d

1-Heptanol

1455

A

0.040 ± 0.004e,f

2-Ethyl-1-hexanol

1488

A

2-Nonanol

1520

B5

1-Octanol

1558

A

0.073 ± 0.005d

Furfuryl alcohol

1659

A

0.0438 ± 0.0004ª,b,d,e

1-Nonanol

1663

B5

0.063 ± 0.005d

cis-3-Nonen-1-ol

1681

B2

3-(Methylthio)-1propanol

1723

B2

1-Undecanol

1870

B2

Benzyl alcohol

1882

A

0.00304 ± 0.00010d 0.0086 ± 0.0010ª,d 0.00898 ± 0.00014c,e,f 0.0263 ± 0.0010b,d

0.01920 ± 0.00023e 0.033 ± 0.003b 0.075 ± 0.004ª,d 0.507 ± 0.024b 0.00330 ± 0.00004ª,e 0.00155 ± 0.00007ª,c 0.03542 ± 0.00007ª 0.03938 ± 0.000007e,f 0.0130 ± 0.0014c,d 0.0174 ± 0.0008b 0.034 ± 0.004c 0.052 ± 0.008c,e 0.0227 ± 0.0015c,e 0.001980 ± 0.000020c 0.00718 ± 0.00017d 0.0099 ± 0.0012c,d,f 0.0234 ± 0.0012ª

2-Phenylethanol

1925

A

11.3 ± 1.2ª,b,c,e

9.1 ± 0.4ª,b

8.8 ± 1.0a

1-Dodecanol

1971

B5

Total of alcohols Aldehydes Hexanal

0.0181 ± 0.0010ª,b 46.3ª,d,e

0.0147 ± 0.0021b,d 38.5c

0.0219 ± 0.0019ª,c 40.9c,e

0.01454 ± 0.00013b,e 0.033 ± 0.004b 0.078 ± 0.007ª,d 0.508 ± 0.025b 0.0033 ± 0.0003ª,e 0.0034 ± 0.0004b,c,d,f 0.038 ± 0.004ª,b 0.0379 ± 0.0017e,f 0.0157 ± 0.0014c 0.0176 ± 0.0017b 0.035 ± 0.003c 0.047 ± 0.007ª,c,d,e 0.029 ± 0.004b,e 0.00268 ± 0.00014b,d 0.009763 ± 0.000021ª 0.0109 ± 0.0016c,d 0.0247 ± 0.0005ª,b,d 13.13 ± 0.22c,d 0.0231 ± 0.0011c 46.5ª,d,e

1040

A

ndb

2-Furfuraldehyde

1447

A

0.057 ± 0.006ª,b,c

Benzaldehyde

1507

A

0.053 ± 0.007b,d

3-Methylbenzaldehyde

1636

B5

0.0243 ± 0.0013b,e

ndb 0.070 ± 0.010b,d 0.048 ± 0.007b,d 0.019 ± 0.003d

ndb 0.057 ± 0.007ª,b,c 0.041 ± 0.006d 0.0169 ± 0.0023ª,d

ndb 0.062 ± 0.009ª,b,d 0.064 ± 0.006b 0.0183 ± 0.0023d

ndb 0.051 ± 0.007ª,c 0.0578 ± 0.0009b,d 0.0273 ± 0.0003c,e

2489

A

0.0058 ± 0.0006ª,c,d

0.0079 ± 0.0012d,e

0.0078 ± 0.0012c,d,e

0.0061 ± 0.0007ª,c,d

0.0046 ± 0.0007ª

0.140b,e

0.145b,e

0.123ª,e

0.151b,e

0.141b,e

0.089 ± 0.012c ndb 0.150 ± 0.014b,c,e

0.095 ± 0.005c ndb 0.110 ± 0.010d,e

0.084 ± 0.010c ndb 0.129 ± 0.019c,d,e

0.132 ± 0.015b ndb 0.127 ± 0.019c,d,e

5Hydroxymethylfurfura l Total of aldehydes Acetic acid esters

0.01651 ± 0.00012c 0.0191 ± 0.0013b,c

Ethyl acetate

873b

A

0.117 ± 0.005b,c

Butyl acetate

1035

A

ndb

Isoamyl acetate

1083

A

0.085 ± 0.008d

0.00123 ± 0.00009d 0.0352 ± 0.0011f 0.037 ± 0.005ª,b 0.085 ± 0.007ª,b 0.51 ± 0.05b 0.0036 ± 0.00004ª,c,e 0.0104 ± 0.0015h 0.039 ± 0.003ª,b,c 0.042 ± 0.003f 0.0099 ± 0.0005d 0.0167 ± 0.0009b 0.0325 ± 0.0010c 0.039 ± 0.003b,d 0.0197 ± 0.0022c,e 0.0021 ± 0.0003c 0.0067 ± 0.0007d 0.0108 ± 0.0007c,d 0.0209 ± 0.0022ª

0.91 ± 0.11b,c 10.2 ± 0.3ª,c,d nda

1.08 ± 0.10b,d,e 11.00 ± 0.04ª,b,d 0.015330 ± 0.000022e 0.071 ± 0.007c 0.0426 ± 0.0016ª 0.1161 ± 0.0022e 0.613 ± 0.019ª,c,d 0.00415 ± 0.00009c,d 0.00300 ± 0.00005b,c,f 0.0451 ± 0.0003c 0.0500 ± 0.0015c 0.01557 ± 0.00008c 0.033 ± 0.003d 0.025 ± 0.004c 0.044 ± 0.006ª,b,c,d,e 0.0134 ± 0.0013c 0.00197 ± 0.00003c 0.00741 ± 0.00005d 0.0100 ± 0.0008c,d,f 0.026 ± 0.003b,d 11.9 ± 0.5c,e 0.0163 ± 0.0024b 39.7c,e

37

Hexyl acetate

1255

A

cis-3-Hexenyl acetate

1298

A

Benzyl acetate

1717

A

2-Phenylethyl acetate

1807

A

Total of acetic acid esters Ethyl esters

0.0035 ± 0.0005ª,c,d 0.00169 ± 0.00008e 0.0024 ± 0.0003ª,e

0.0061 ± 0.0005e 0.00134 ± 0.00007c,d,e 0.00225 ± 0.00005ª

0.00119 ± 0.00013b,c,d 0.00195 ± 0.00016ª

1.44 ± 0.05ª

1.41 ± 0.09ª

1.41 ± 0.05ª

1.65c,d

1.66c,d

1.62c

2.19b,d

1.56c

0.0040 ± 0.0006c,d 0.156 ± 0.019ª 0.0074 ± 0.0010ª 0.0038 ± 0.0006d,e 0.0029 ± 0.0004f 0.0045 ± 0.0006ª 0.492 ± 0.012ª,f 0.0037 ± 0.0005d,e 0.0064 ± 0.0005b,f 0.00457 ± 0.00024ª,d,g 0.285 ± 0.017ª 0.085 ± 0.011e 0.0135 ± 0.0020ª 0.00096 ± 0.00009f 0.0141 ± 0.0012ª 0.001025 ± 0.000013c 0.0244 ± 0.0006ª 0.0293 ± 0.0006e 0.0111 ± 0.0004b 1.53 ± 0.05ª 0.00205 ± 0.00011g 0.530 ± 0.016f 0.0041 ± 0.0006ª,e,f 0.052 ± 0.008e 0.0060 ± 0.0009ª 0.0034 ± 0.0004ª,b 0.043 ± 0.006e

0.001708 ± 0.000018e 0.204 ± 0.007b 0.0126 ± 0.0015b,c 0.0040 ± 0.0003e,f 0.0020895 ± 0.0000013b,d 0.007624 ± 0.000005d

0.0037 ± 0.0004c,d 0.160 ± 0.014c,d 0.0058 ± 0.0008ª 0.0042 ± 0.0006e,f 0.0026 ± 0.0004d,f 0.0058 ± 0.0005ª,d 0.68 ± 0.08b,c 0.00333 ± 0.00003c,e 0.0123 ± 0.0011c 0.0043 ± 0.0005ª,h 0.34 ± 0.05ª,b,c

0.0098 ± 0.0014b

Ethyl isobutyrate

948b

A

0.0090 ± 0.0011ª,b

Ethyl butyrate

1001

A

0.148 ± 0.006ª

Ethyl 2-methylbutyrate

1015

A

0.0078 ± 0.0005ª

Ethyl isovalerate

1031

A

Ethyl valerate

1093

A

Ethyl 2-butenoate

1125

B5

Ethyl hexanoate

1207

A

0.19 ± 0.03e

Ethyl (3Z)-3hexanoate

1282

B3

0.0026 ± 0.0004b,c

Ethyl heptanoate

1318

A

nda

Ethyl 2-hexenoate

1329

B2

0.0078 ± 0.0008f

Ethyl lactate

1333

A

0.41 ± 0.03c

Ethyl octanoate

1422

A

0.154 ± 0.022e

Ethyl sorbate

1493

B2

Ethyl nonanoate

1523

A

1535

B2

0.022 ± 0.003d,e

1554

B2

0.00213 ± 0.00008b

Ethyl furoate

1606

A

0.030 ± 0.003ª

Ethyl decanoate

1626

A

0.0254 ± 0.0016e

Ethyl benzoate

1652

A

Diethyl succinate

1671

A

Ethyl 9-decenoate

1681

B6

Ethyl phenylacetate

1774

A

0.40 ± 0.05b,e

Ethyl dodecanoate

1833

A

0.00164 ± 0.00020ª

Ethyl 3hydroxytridecanoate

1896

C

0.28 ± 0.04g

Ethyl tetradecanoate

2045

A

Diethyl malate

2050

B7

Ethyl 3hydroxydodecanoate

2108

B6

Ethyl 2-hydroxy-4methylpentanoate Ethyl 3(methylthio)propionate

0.0036 ± 0.0004d,e 0.00209 ± 0.00023b,d 0.01172 ± 0.00010c

0.0121 ± 0.0016d,e 0.00120 ± 0.00012b,f

0.0152 ± 0.0009c,d,e 2.5 ± 0.3c 0.0051 ± 0.0006b,d

0.00244 ± 0.00020b 0.0034 ± 0.0003ª,b 0.21 ± 0.03b

ndf

0.58 ± 0.08b,f 0.0030 ± 0.0004b,c,e 0.0107 ± 0.0009c 0.0055 ± 0.0007c,d,g 0.33 ± 0.05ª,b 0.50 ± 0.07ª,b,c 0.0096 ± 0.0014b,c,e 0.0039 ± 0.0005c,g 0.0186 ± 0.0009b,c,e ndd 0.025 ± 0.004ª 0.1211 ± 0.0024f 0.0093 ± 0.0004b 1.65 ± 0.24ª 0.0054 ± 0.0003c,d 0.302 ± 0.004b 0.0065 ± 0.0009b,d,e,f 0.112 ± 0.016c,f,h 0.0062 ± 0.0009ª 0.0039 ± 0.0005ª,c 0.108 ± 0.016c,d,f

0.0049 ± 0.0006b 0.00114 ± 0.00005b,c,d 0.00312 ± 0.00008b,e 1.97 ± 0.16b,e

0.39 ± 0.05b 0.0100 ± 0.0015ª,b,c 0.00266 ± 0.00018ª,g 0.0158 ± 0.0006ª,b,c 0.00203 ± 0.00013b 0.02614 ± 0.00025ª 0.060 ± 0.003ª 0.0116 ± 0.0014b,c 1.88 ± 0.03ª 0.0040 ± 0.0003b 0.52 ± 0.06e,f 0.0050 ± 0.0007ª,de,f 0.110 ± 0.016c,h 0.0032 ± 0.0005b 0.00443 ± 0.00017c 0.082 ± 0.012f

0.0031 ± 0.0004ª,d 0.00093 ± 0.00005b 0.00220 ± 0.00014ª 1.30 ± 0.15ª

0.31 ± 0.05c 0.022 ± 0.003d 0.0053 ± 0.0006f 0.0023 ± 0.0003b,d 0.0010 ± 0.0011c 0.763 ± 0.008c 0.00466 ± 0.00013d 0.01919 ± 0.00008g 0.00561 ± 0.00010c,g 0.379 ± 0.009b,c 0.485 ± 0.012b,c 0.0103 ± 0.0015d,e 0.0061 ± 0.0003e 0.02077 ± 0.00005d,e 0.00122 ± 0.00006c 0.0310 ± 0.0025ª 0.160 ± 0.023d 0.0162 ± 0.0023ª,d,e 1.88 ± 0.09ª 0.0101 ± 0.0009g 1.358 ± 0.024f 0.019 ± 0.003g 0.091 ± 0.012c,h 0.0099 ± 0.0004c 0.0035 ± 0.0003ª,b,c 0.094 ± 0.014c,f

38

Ethyl cinnamate

2131

A

0.001528 ± 0.000011d,f

Ethyl pentadecanoate

2145

B7

0.0030 ± 0.0004ª

Ethyl hexadecanoate

2252

A

0.0042 ± 0.0005b

Unknown (m/z 117)

2321

-

Ethyl vanillate

2564

A

Methyl octanoate

1372

B2

Methyl decanoate

1580

A

0.0062 ± 0.0009e

Methyl salicylate

1760

B2

0.0169 ± 0.0015b,d

Hexyl salicylate

2207

B2

0.0044 ± 0.0004c,e

Methyl hexadecanoate

2210

A

0.0082 ± 0.0004ª

Methyl jasmonate

2298

B8

0.023 ± 0.003ª,c,e

Methyl vanillate

2559

B2

969b

B1

1138

C

1151

B2

0.00487 ± 0.00013b,d,e 0.00927 ± 0.00013ª,b nd

Acetoin

1276

A

0.0779 ± 0.0025f

6-Methyl-5-hepten-2one

1322

A

0.0064 ± 0.0004c,d,f

2-Nonanone

1375

B2

0.020 ± 0.003e

2-Acetylfuran

1492

A

0.0124 ± 0.0013d

Dihydro-3(2H)thiophenone

1515

C

0.0206 ± 0.0009c

Acetophenone

1641

A

0.0172 ± 0.0010ª,c

1,2-Cyclopentanedione

1773

C

0.042 ± 0.006b,d

Total of ethyl esters Others esters

Total of other esters Ketones 4-Methyl-2-pentanone 2,6-Dimethyl-4heptanone 2-Heptanone

Total of ketones Lactones γ-Butyrolactone

1623

A

γ-Nonalactona

2039

B

γ-Decalactona

2159

A

1727

A

1814

A

Total of lactones C13-Norisoprenoids 1,1,6-Trimethyl-1,2dihydronaphthalene β-damascenone Total of C13Norisoprenoids

0.00113 ± 0.00015d 0.0043 ± 0.0006c,d 0.0128 ± 0.0019ª,d,e 0.025 ± 0.003d,f 0.00240 ± 0.00012e,f 4.08ª,b,e

0.00140 ± 0.00020d,f 0.001765 ± 0.000020b 0.0082 ± 0.0003b,d,e 0.0175 ± 0.0016c,g 0.00290 ± 0.00011b,c,f 4.39b,e

0.001399 ± 0.000010d,f 0.0048 ± 0.0007d 0.0211 ± 0.0009f 0.0141 ± 0.0021e,g 0.0028 ± 0.0004b,c,f 5.77c

0.0025 ± 0.0004ª,b,d 0.0040 ± 0.0006d,f 0.0148 ± 0.0014b

0.0018 ± 0.0003e,g 0.0021 ± 0.0003ª,b 0.0174 ± 0.0019b,c,d

0.0025 ± 0.0003ª,b,c,d 0.00270 ± 0.00024ª,b 0.0202 ± 0.0010c,d,e

e

0.0053 ± 0.0004d,e

0.0073 ± 0.0010ª

0.0062 ± 0.0008ª,d,e

0.0147 ± 0.0021ª,d 0.0117 ± 0.0017ª 0.0031 ± 0.0004b,c,f 0.055e

0.0245 ± 0.0036b,d 0.051 ± 0.007b,f 0.0029 ± 0.0003b,f 0.105f

0.0145 ±0.0018ª,d 0.039 ± 0.006d,f 0.0035 ± 0.0004b,c,e 0.086g

0.0099 ± 0.0014ª 0.033 ± 0.005d,e,g 0.0040 ± 0.0004d,e 0.079d,g

0.0047 ± 0.0003ª,d,e 0.00797 ± 0.00009b,e nd 0.251 ± 0.017e 0.0036 ± 0.0004e 0.0166 ± 0.0025f 0.0095 ± 0.0014e 0.0113 ± 0.0013d,e 0.0144 ± 0.0005ª 0.0389 ± 0.0006b,d 0.358b

0.0050 ± 0.0004b,e 0.0067 ± 0.0007c,e nd 0.21 ± 0.03b,d 0.0070 ± 0.0010d,f,g 0.0090 ± 0.0010b,d 0.0077 ± 0.0011ª,b,c,e 0.0155 ± 0.0019e 0.0151 ± 0.0009ª 0.0207 ± 0.0014e 0.291d

0.00369 ± 0.00009c 0.0041 ± 0.0006d nd

0.228e

0.0036 ± 0.0004c 0.0054 ± 0.0005c,d nd 0.244 ± 0.019d,e 0.0058 ± 0.0004c,f 0.00857 ± 0.00024b 0.00856 ± 0.00017ª,c,e 0.0125 ± 0.0006d,e 0.0118 ± 0.0012ª 0.058 ± 0.003ª 0.359b

0.04270 ± 0.00024ª,b 0.0427 ± 0.0004b,c,e 0.0133 ± 0.0010b,d 0.099ª,b,c

0.048 ± 0.007ª,b,c 0.037 ± 0.003ª,b,c,e 0.0114 ± 0.0017ª,c,d 0.097ª,b,c

0.040 ± 0.006ª,b 0.035 ± 0.005ª,e 0.0112 ± 0.0007ª,c,d 0.086ª,b

0.048 ± 0.005ª,b,c 0.042 ± 0.005ª,b,c,e 0.0114 ± 0.0017ª,c,d 0.102ª,b,c

0.056 ± 0.008c,d 0.040 ± 0.006ª,b,c,e 0.0136 ± 0.0020b,d 0.110c,d

0.0121 ± 0.0018c,e 0.037 ± 0.004b,c,d,f 0.049b,c,d

0.0109 ± 0.0016ª,c,e 0.026 ± 0.003c 0.037c

0.0084 ± 0.0012ª 0.027 ± 0.004c 0.036c

0.0139 ± 0.0020c,d 0.030 ± 0.003b,c,f 0.044c,d

0.0124 ± 0.0015c,e 0.042 ± 0.004d,f 0.055b,d

0.0178 ± 0.0021c,g 0.0025 ± 0.0004b,e,f 4.48b,e 0.00130±0.00009 e

0.0038 ± 0.0006c,d,e 0.064ª,e

0.00205 ± 0.00011f 0.0031 ± 0.0004ª 0.0149 ± 0.0022ª,e,f 0.0069 ± 0.0010ª 0.0027 ± 0.0004b,c,f 3.35ª ndf 0.0027 ± 0.0004ª,b 0.0167 ± 0.0003b,d 0.005917 ± 0.000003ª,d,

0.46 ± 0.05g 0.0080 ± 0.0003g 0.0119 ± 0.0011c,d 0.0069 ± 0.0010ª,b 0.0133 ± 0.0020d,e 0.0170 ± 0.0019ª,c 0.047 ± 0.007c,d 0.574f

39

Terpenes Limonene

1147

A

0.0149 ± 0.0020e

trans-Linalool oxide

1469

B9

0.00471 ± 0.00004ª,c

α-Terpinen

1505

C

0.0061 ± 0.0006ª

Linalool

1543

A

0.284 ± 0.018c

Hotrienol

1602

C

0.0202 ± 0.0012ª

α-Farnesene

1646

C

0.0119 ± 0.0014e

α-Terpineol

1700

A

0.064 ± 0.009ª

2,6-Dimethyl -3,7Octadiene-2,6-diol

1702

C

0.0191 ± 0.0022ª,b

Citronellol

1768

A

0.064 ± 0.008b,e

Geraniol

1852

A

0.141 ± 0.020c

Nerolidol

2041

A

0.22 ± 0.03c

Farnesol

2364

B2

0.1578 ± 0.0024e 1.01e,f

Total of Terpenes Volatile phenols 4-Ethylguaiacol

2027

A

Eugenol

2171

A

4-Ethylphenol

2185

A

0.20 ± 0.03ª,b

4-Vinylguaiacol

2203

B2

0.0255 ± 0.0018d

Coumaran

2402

C

Methoxyeugenol

2504

B10

Acetovanillone

2577

C

1.091 ± 0.016b 0.0047 ± 0.0003c,e,f

0.0181 ± 0.0020c,d 0.00158 ± 0.00020ª,b 0.0049 ± 0.0007c,d

0.0199 ± 0.0025e,f 0.0056 ± 0.0008ª,b 0.00290 ± 0.00014b 0.209 ± 0.010d,e 0.0164 ± 0.0022b 0.0044 ± 0.0005d 0.061± 0.003ª 0.017 ± 0.003ª 0.068 ± 0.006b,e,f 0.093 ± 0.013b 0.172 ± 0.003f 0.087 ± 0.013b,c 0.76b,c,d

0.026 ± 0.004c,f 0.0049 ± 0.0006ª,c 0.0023 ± 0.0003b 0.204 ± 0.009ª,d,e 0.0151 ± 0.0018b 0.00620 ± 0.00015f 0.051 ± 0.008ª 0.0169 ± 0.0025ª 0.0847 ± 0.0019d,f 0.181 ± 0.013d 0.111 ± 0.015e 0.154 ± 0.006e 0.86c,d,e

0.63 ± 0.04ª 0.0045 ± 0.0005ª,e,f 0.174 ± 0.014b 0.0120 ± 0.0012b 0.0147 ± 0.0016ª,b,c 0.00167 ± 0.00021ª,b 0.0039 ± 0.0005ª,b,c,e

0.66 ± 0.10ª 0.0053 ± 0.0006c,e 0.19 ± 0.03ª,b 0.0153 ± 0.0022b,c,e 0.0139 ± 0.014ª,b 0.001642 ± 0.000014ª,b 0.00367 ± 0.00017ª,b,e

0.0210 ± 0.0018e,f 0.0057 ± 0.0004ª,b 0.0029 ± 0.0003b 0.228 ± 0.013b,d 0.01446 ± 0.00022b 0.0066 ± 0.0010f 0.066 ± 0.009ª 0.0207± 0.0014ª,b 0.0679 ± 0.0003b,e,f 0.101 ± 0.012b 0.24 ± 0.03c 0.108±0.016

0.022 ± 0.003e,f 0.0060 ± 0.0008ª,b 0.0030733 ± 0.0000015b 0.268 ± 0.023b,c 0.0243 ± 0.0013c 0.0064 ± 0.0005f 0.063 ± 0.007ª 0.022 ± 0.003b 0.129 ± 0.013c 0.124 ± 0.017b,c 0.3199 ± 0.0021g

c

0.20 ± 0.03f

0.88d,e

1.19g

0.70 ± 0.07ª 0.0050 ±0.0005c,e 0.195 ± 0.014ª,b 0.0141 ± 0.0013b,c 0.0150 ± 0.0022ª,b,c 0.00173 ± 0.00022ª,b,c 0.0039 ± 0.0005b,c,e

0.76 ± 0.06ª 0.0066 ± 0.0007b,d 0.208 ± 0.021ª,b,c 0.0166 ± 0.0024c,e 0.0160 ± 0.0020ª,b,c 0.0023 ± 0.0003d,e 0.0045 ± 0.0005b,c

Total of volatile 1.35b 0.84ª 0.89ª 0.94ª 1.02ª phenols Total of volatile 63.38ª,b 61.73b 65.46ª,b 72.65ª,c 72.45ª,c compounds LRI: linear retention index values obtained from samples. ID: reliability of identification: A, mass spectrum and LRI agreed with standards; B, mass spectrum agreed with mass spectral data base and LRI agreed with the literature data (TI); C, mass spectrum agreed with mass spectral data base. nd: no peak detected. aSimilar superscript letter in the same row indicates no significant statistically differences (p<0.05). bValues estimated by linear regression. cLiterature reference agreed with LRI data:1: Morales at al. 2019; 2: Kim et al. 2019; 3: Carlin et al., 2016; 4: Nicolli et al., 2018; 5: http://www.chemspider.com/Default.aspx; 6: Ferrari et al., 2004; 7: Zhao et al., 2009; 8: Ka et al., 2005; 9: Fernández de Simón et al., 2015.

40

Table 4. Profiles of volatile compounds of base wine substrate and samples after biological ageing using different flor yeast strains. Volatile compounds Acetals Acetaldehyde diethylacetal 2,4,5-Trimethyl-1,3dioxolane Propanaldehyde diethylacetal Acetaldehyde ethyl propyl acetal 2,4,5-Trimethyl-1,3dioxolane (isomer) 1-(1-Ethoxyethoxy)butane 2-Ethyl-5-methyl-1,3dioxolane

LRI b

IDc

Base wine (2)

Strain K

18.4 ± 0.9c

24.93 ± 0.23d

1.29 ± 0.11c

0.85 ± 0.11b

1.47 ± 0.09c

0.0067 ± 0.0008c

0.246 ± 0.018c,d

0.0067 ± 0.0006c 0.202 ± 0.010c 0.21 ± 0.03b,c

0.18 ± 0.03b

0.25 ± 0.03c

0.24 ± 0.03c

0.0123 ± 0.0014b 0.022 ± 0.003b 0.0067 ± 0.0006b 0.035 ± 0.004b 0.0082 ± 0.0007b,c

0.018 ± 0.003c,e 0.039 ± 0.006c 0.0138 ± 0.0018c 0.073 ± 0.005c 0.0072 ± 0.0010b

0.0142 ± 0.0012b,c 0.0260 ± 0.0005b 0.0086 ± 0.0008b 0.057 ± 0.006d 0.0117 ± 0.0009c,d

0.0093 ± 0.0013d 0.352 ± 0.021d 0.299 ± 0.021d 0.390 ± 0.003d 0.02342 ± 0.00024d 0.050912 ± 0.000006d 0.0133 ± 0.0009c 0.100 ± 0.005e 0.0201 ± 0.0016a

1.00 ± 0.13b 0.0056 ± 0.0008b,c 0.297 ± 0.019e 0.23 ± 0.03c 0.366 ± 0.021d 0.0190 ± 0.0023e 0.044 ± 0.003c,d 0.0128 ± 0.0017c 0.108 ± 0.008e 0.0147 ± 0.0017d

3.7 ± 0.5b

4.9 ± 0.6b

4.9 ± 0.6b

7.7 ± 0.3c

8.6 ± 1.1c

0.0513 ± 0.007b 17.6b

0.066 ± 0.007b 25.1c

0.072 ± 0.010b 22.6c

0.098 ± 0.004c 35.4d

0.107 ± 0.014c 29.2e

0.022 ± 0.003ª 0.137 ± 0.013b,c 0.090 ± 0.012b 0.0091 ± 0.0013c,d

0.63 ± 0.03b,c 0.0233 ± 0.0019ª 0.131 ± 0.014b 0.077 ± 0.010b 0.0036 ± 0.0005ª

0.73 ± 0.06b,c 0.0326 ± 0.0025b 0.136 ± 0.006b 0.088 ± 0.003b 0.0109 ± 0.0012d 0.60 ± 0.05b 0.0136 ± 0.0014b 0.0293 ± 0.0019c,d 2.96 ± 0.19ª,c 0.092 ± 0.003ª,b,c 1.21 ± 0.08ª,c 0.090 ± 0.006c 0.035 ± 0.004ª,c 6.04c,d

1.40 ± 0.09a

12.7 ± 1.1b

920b

B1

0.056 ± 0.006a

942b

B2

nda

950b

C

956b

C

0.957 ± 0.025b 0.0045 ± 0.0006b 0.144 ± 0.012b 0.178 ± 0.021b

970b

B3

987b

C

nda

Unknown m/z 101

1012

-

0.00425 ± 0.00008a

2,4-Dimethyl-1,3dioxane

1027

C

nda

Unknown m/z 73

1029

-

nda

1044

B1

1075

B1

1242

C

Acetic acid

1442

A

Propanoic acid

1537

A

Isobutyric acid

1563

A

Isovaleric acid

1667

A

Pentanoic acid

1741

A

Hexanoic acid

1847

A

2-Ethylhexanoic acid

1952

B2

Heptanoic acid

1957

A

Octanoic acid

2072

Nonanoic acid

0.0190 ± 0.0024a 0.169 ± 0.020a 0.0035 ± 0.0005a 1.67a

Strain O

16.0 ± 1.5c

A

Isovaleraldehyde diethylacetal Acetaldehyde ethyl amyl acetal Acetaldehyde ethyl hexyl acetal Total of acetals Acids

Strain N

18.0 ± 2.4c

884b

0.0065 ± 0.0008a 0.00670 ± 0.00019a 0.0489 ± 0.0007a

Relative peak area ± sda Strain L Strain M

0.21 ± 0.03c

0.29 ± 0.04a

0.78 ± 0.10b

0.0183 ± 0.0025ª 0.031 ± 0.004a 0.033 ± 0.005a 0.0056 ± 0.0005ª,b

0.021 ± 0.003ª 0.1455 ± 0.0014b,c 0.0829 ± 0.0016b 0.0066 ± 0.0009b

0.63 ± 0.09b,c 0.021 ± 0.003ª 0.163 ± 0.016c 0.090 ± 0.013b 0.0071 ± 0.0003b,c

0.43 ± 0.06a

0.41 ± 0.03ª

0.51 ± 0.06ª,b

0.53 ± 0.07ª,b

0.59 ± 0.04b

0.0097 ± 0.0014a 0.0135 ± 0.0020a

0.0101 ± 0.0014ª 0.0213 ± 0.0023b

0.0135 ± 0.0013b 0.032 ± 0.004c

0.0112 ± 0.0003ª,b 0.030 ± 0.004c,d

A

3.4 ± 0.5ª,d

1.90 ± 0.23b

2.80 ± 0.23ª,c

2175

A

0.082 ± 0.012ª,b

0.078 ± 0.011ª,b

0.098 ± 0.004b,c

Decanoic acid

2286

A

1.35 ± 0.20a

0.74 ± 0.11b

0.89 ± 0.04b

9-Decanoic acid

2344

B4

Geranic acid

2352

B2

0.071 ± 0.010ª,b 0.035 ± 0.005ª,c 5.80ª,c

0.056 ± 0.008ª 0.023 ± 0.003b 4.27b

0.087 ± 0.006b,c 0.032 ± 0.003ª 5.38ª,c

0.01184 ± 0.00025ª,b 0.024 ± 0.003b,d 2.43 ± 0.18b,c 0.073 ± 0.005ª 0.96 ± 0.06b,c 0.065 ± 0.005ª 0.0292 ± 0.0011ª,b 4.97ª,b

Total of acids

0.59 ± 0.09c

3.65 ± 0.03d 0.111 ± 0.014c 1.62 ± 0.05d 0.112 ± 0.003d 0.0427 ± 0.0011c 7.03d

41

Alcohols Ethanol

944b

A

1-Propanol

1022

A

Isobutanol

1083

A

1-Butanol

1142

A

2-Methyl-1-butanol

1207

A

3-Methyl-1-butanol

1226

4-Heptanol

17.9 ± 2.2ª

27 ± 4b

23 ± 3ª,b

0.26 ± 0.03b

0.33 ± 0.04b

0.32 ± 0.04b

0.111 ± 0.009ª 0.105 ± 0.004b

0.19 ± 0.03b,c 0.183 ± 0.025c

0.158 ± 0.022b 0.129 ± 0.004b

39.3 ± 0.6c 0.425 ± 0.006c 0.210 ± 0.005c 0.195 ± 0.008c

0.92 ± 0.13ª

1.03 ± 0.10ª,b

1.28 ± 0.07b

1.15 ± 0.06ª,b

1.72 ± 0.10c

A

8.2 ± 0.6ª

8.18 ± 0.17ª

10.7 ± 1.5b,c

9.3 ± 0.8ª,b

12.7 ± 0.4c

1284

B5

nd

2-Ethyl-1-butanol

1304

B2

nda

4-Methyl-1-pentanol

1312

A

3-Methyl-1-pentanol

1325

A

0.0193 ± 0.0019ª,b 0.0251 ± 0.0020ª

nd 0.037 ± 0.003b 0.01644 ± 0.00012ª 0.033 ± 0.003b

nd 0.100 ± 0.012c 0.021 ± 0.003ª,b 0.036 ± 0.004b

nd 0.054 ± 0.006b 0.0193 ± 0.0012ª,b 0.0388 ± 0.0025b,c

nd 0.078 ± 0.008d 0.0228 ± 0.0016b 0.0452 ± 0.0007c

1-Hexanol

1354

A

0.94 ± 0.11ª

0.70 ± 0.03b

0.84 ± 0.10ª,b

0.83 ± 0.03ª,b

0.96 ± 0.04ª

0.0071 ± 0.0009ª,b,c nd 0.077 ± 0.009ª,b 0.021 ± 0.003ª 0.048 ± 0.006ª 0.0106 ± 0.0013ª 0.047 ± 0.006ª 0.057 ± 0.008ª 0.032 ± 0.004ª nd 0.0122 ± 0.0018ª 0.096 ± 0.006ª 0.0083 ± 0.0012ª 6.3 ± 0.9ª 0.162 ± 0.016ª 35.8a

0.00580 ± 0.00020ª nd 0.0707 ± 0.0011ª 0.033 ± 0.003b 0.050 ± 0.003ª 0.0109 ± 0.0010ª 0.0186 ± 0.0003b 0.057 ± 0.007ª 0.0106 ± 0.0011b nd 0.0136 ± 0.0019ª 0.0676 ± 0.0004b 0.00868 ± 0.00015ª 6.56 ± 0.23ª,b 0.033 ± 0.005b,c 35.3ª

0.0068 ± 0.0006ª,b nd 0.078 ± 0.009ª,b 0.047 ± 0.005c,d 0.0625 ± 0.0014b 0.023 ± 0.003b 0.0247 ± 0.0009b,c,d 0.087 ± 0.010b 0.0147 ± 0.0009b,c nd 0.0134 ± 0.0015ª 0.045 ± 0.006c 0.0099 ± 0.0009ª,b 8.6 ± 1.0b,c,d 0.030 ± 0.003b 50.1b

0.0079 ± 0.0006b,c,d nd 0.0820 ± 0.0011ª,b 0.0397 ± 0.0013b,c 0.061 ± 0.005b 0.0120 ± 0.0004ª 0.0219 ± 0.0012b,c 0.087 ± 0.011b 0.0142 ± 0.0017b,c nd 0.0119 ± 0.0017ª 0.071 ± 0.003b 0.0096 ± 0.0010ª,b 7.8 ± 1.2ª,b,c 0.024 ± 0.003b 43.3ª,b

0.00866 ± 0.00023c,d nd 0.090 ± 0.005b 0.0538 ± 0.0013d 0.0649 ± 0.0005b 0.0186 ± 0.0013c 0.0299 ± 0.0006d 0.1012 ± 0.0021b,c 0.0175 ± 0.0012c nd 0.0179 ± 0.0012b 0.034 ± 0.005c 0.0116 ± 0.0006b 10.2 ± 0.3d 0.034 ± 0.005b,c 66.3c

26 ± 4b 0.309 ± 0.017b 0.1815 ± 0.0020b,c 0.190 ± 0.03c 1.75 ± 0.25c 10.3 ± 1.1ª,b nd 0.071 ± 0.006d 0.0233 ± 0.0019b 0.046 ± 0.004c 0.97 ± 0.10ª 0.0095 ± 0.0011d nd 0.090 ± 0.005b 0.051 ± 0.006d 0.071 ± 0.005b 0.0168 ± 0.0015c 0.028 ± 0.003c,d 0.118 ± 0.015c 0.0146 ± 0.0020b,c nd 0.0119 ± 0.0010ª 0.163 ± 0.015d 0.0117 ± 0.0010b 9.3 ± 1.1c,d 0.050 ± 0.003c 49.5b

trans-3-Hexenol

1361

B2

3-Ethoxy-1-propanol

1373

B4

cis-3-Hexen-1-ol

1380

A

1-Heptanol

1455

A

2-Ethyl-1-hexanol

1488

A

2-Nonanol

1520

B5

1-Octanol

1558

A

Furfuryl alcohol

1659

A

1-Nonanol

1663

B5

cis-3-Nonen-1-ol 3-(Methylthio)-1propanol

1681

B2

1723

B2

1-Undecanol

1870

B2

Benzyl alcohol

1882

A

2-Phenylethanol

1925

A

1-Dodecanol

1971

B5

Hexanal

1040

A

2-Furfuraldehyde

1447

A

Benzaldehyde

1507

A

1636

B5

2489

A

Total of alcohols Aldehydes

3Methylbenzaldehyde 5Hydroxymethylfurfura l Total of aldehydes

18.6 ± 0.5ª 0.160 ± 0.017a 0.077 ± 0.011ª 0.0400 ± 0.0003ª

0.0037 ± 0.0003a 0.072 ± 0.010a 0.0093 ± 0.0010a 0.062 ± 0.009a

ndb

ndb

ndb

ndb

ndb

0.0751 ± 0.0006ª 0.0266 ± 0.0005b

0.102 ± 0.015ª,b 0.0128 ± 0.0018c

0.098 ± 0.014ª,b 0.0128 ± 0.0019c

0.124 ± 0.006b,c 0.0067 ± 0.0006a

0.134 ± 0.020c 0.04688 ± 0.00014d

ndb

ndb

ndb

ndb

ndb

0.0044 ± 0.0006a

0.0045 ± 0.0006ª

0.0050 ± 0.0007ª,c

0.0092 ± 0.0009b

0.00887 ± 0.00010b

0.0063 ± 0.0009c

0.152a

0.106b

0.120b,c

0.120b,c

0.140ª,c

0.188d

42

Acetic acid esters 0.346 ± 0.005ª 0.00151 ± 0.00010b 0.136 ± 0.015b 0.2958 ± 0.0013b 0.0060 ± 0.0009b

0.68 ± 0.10c

ndb

ndb

5.4 ± 0.8ª

1.90 ± 0.21b

9.50ª

Ethyl acetate

873b

A

0.32 ± 0.04ª

Butyl acetate

1035

A

0.004723 ± 0.000005a

Isoamyl acetate

1083

A

3.1 ± 0.4ª

Hexyl acetate

1255

A

0.63 ± 0.09ª

cis-3-Hexenyl acetate

1298

A

Benzyl acetate

1717

A

2-Phenylethyl acetate

1807

A

Total of acetic acid esters Ethyl esters

0.44 ± 0.03ª 0.0038 ± 0.0004c 0.467 ± 0.015b,c 0.051 ± 0.007b 0.0155 ± 0.0011b,c 0.00184 ± 0.00007c 2.90 ± 0.25c,d

0.44 ± 0.06ª

0.38 ± 0.05ª

0.65 ± 0.09b

0.0033 ± 0.0005c

0.003541 ± 0.000016c 0.53 ± 0.03b,c 0.052 ± 0.007b 0.0179 ± 0.0003c 0.00185 ± 0.00003c

2.51 ± 0.09b,c

0.00214 ± 0.00007b 0.1861 ± 0.0016b 0.0117 ± 0.0017b 0.0080 ± 0.0005b 0.00149 ± 0.00006c 2.50 ± 0.14b,c

2.40b

3.71b,c,d

3.09b,c

4.88d

3.88c,d

0.0133 ± 0.0020ª 0.370 ± 0.009ª 0.0127 ± 0.0007ª 0.0200 ± 0.0003ª 0.0024 ± 0.0003ª 0.0064 ± 0.0007ª

0.0065 ± 0.0010b 0.203 ± 0.017b 0.0115 ± 0.0008ª 0.0084 ± 0.0010b 0.00160 ± 0.00012b 0.0090 ± 0.0012ª,c

0.0088 ± 0.0013b

0.0093 ± 0.0013ª,b

0.020 ± 0.003b 0.0171 ± 0.0025ª 0.0033 ± 0.0005c 0.018 ± 0.003b

0.0056 ± 0.0006b 0.243 ± 0.013b 0.0136 ± 0.0009ª 0.0098 ± 0.0012b 0.00177 ± 0.00009ª,b 0.01102 ± 0.00020c

1.32 ± 0.19ª

0.96 ± 0.14b

1.45 ± 0.21ª

1.01 ± 0.15ª,b

2.1 ± 0.3c

0.0121 ± 0.0018ª 0.0053 ± 0.0008ª 0.0056 ± 0.0008ª

0.0060 ± 0.0003b 0.0125 ± 0.0006b 0.00475 ± 0.00016a

0.0104 ± 0.0014ª 0.0255 ± 0.004c 0.0108 ± 0.0016b

0.0076 ± 0.0007b 0.0139 ± 0.0020b 0.00676 ± 0.00016ª

0.0127 ± 0.0004ª 0.0279 ± 0.0012c 0.0139 ± 0.0020b

0.90 ± 0.06ª

0.82 ± 0.12ª

0.82 ± 0.10ª

0.92 ± 0.06ª

nd 0.0111 ± 0.0016ª 0.04764 ± 0.00008b

1.49 ± 0.21b,c nd 0.0043 ± 0.0006b 0.0481 ± 0.0023b

2.36 ± 0.11ª,d nd 0.0099 ± 0.0014ª 0.0518 ± 0.0016b,c

0.021 ± 0.003c 0.42 ± 0.04ª,c 0.0347 ± 0.0025c 0.0164 ± 0.0008ª 0.0035 ± 0.0003c 0.0177 ± 0.0026b 2.06 ± 0.10c 0.0111 ± 0.0011ª 0.028 ± 0.003c 0.019 ± 0.003c 0.942 ± 0.007ª 2.27 ± 0.21ª,d nd 0.0160 ± 0.0016c 0.056 ± 0.004c

0.065 ± 0.009ª 0.0035 ± 0.0005ª

0.064 ± 0.009b 0.0153 ± 0.0023b,c

3.62 ± 0.04d

Ethyl isobutyrate

948b

A

Ethyl butyrate

1001

A

Ethyl 2methylbutyrate

1015

A

Ethyl isovalerate

1031

A

Ethyl valerate

1093

A

Ethyl 2-butenoate

1125

B5

Ethyl hexanoate

1207

A

Ethyl (3Z)-3hexenoate

1282

B3

Ethyl heptanoate

1318

A

Ethyl 2-hexenoate

1329

B2

Ethyl lactate

1333

A

0.79 ± 0.11ª

Ethyl octanoate

1422

A

2.4 ± 0.4ª

Ethyl sorbate

1493

B2

Ethyl nonanoate

1523

A

1535

B2

nd 0.0095 ± 0.0013ª 0.028 ± 0.004ª

1.066 ± 0.021b nd 0.003601 ± 0.000020b 0.0460 ± 0.0013b

1554

B2

0.0051 ± 0.0008ª

0.00136 ± 0.00020b

0.00131 ± 0.00003b

0.001709 ± 0.000025b,c

0.00220 ± 0.00007c

0.00226 ± 0.00015c

Ethyl 2-furoate

1606

A

0.031 ± 0.005ª,b,c

0.0295 ± 0.0024ª,b

0.0295 ± 0.0007ª,b

0.0337 ± 0.0016b,c

Ethyl decanoate

1626

A

0.53 ± 0.07ª

0.24 ± 0.03b

0.23 ± 0.03b

0.42 ± 0.05c

Ethyl benzoate

1652

A

0.0036 ± 0.0005ª

0.0251 ± 0.0013a 0.196 ± 0.014b 0.00321 ± 0.00019a

0.0053 ± 0.0008b,c

Diethyl succinate

1671

A

1.8 ± 0.3ª

2.13 ± 0.14ª,b

2.6 ± 0.3b,c

Ethyl 9-decenoate

1681

B6

0.0176 ± 0.0016b

0.027 ± 0.004ª,c

Ethyl phenylacetate

1774

A

0.76 ± 0.07b

1.11 ± 0.10c

0.93 ± 0.07b

Ethyl dodecanoate

1833

A

0.034 ± 0.005ª 0.072 ± 0.010ª 0.0112 ± 0.0015ª

0.0041 ± 0.0003ª,b 2.59 ± 0.23b,c 0.019 ± 0.003b,c

0.057 ± 0.009b

0.049 ± 0.007b

0.029 ± 0.004c

0.00577 ± 0.00005c,d 3.16 ± 0.07c,d 0.033 ± 0.003ª 1.378 ± 0.016d 0.044 ± 0.006b,c

0.0361 ± 0.0017c 0.513 ± 0.024ª,c 0.0070 ± 0.0010d 2.96 ± 0.25d 0.035 ± 0.004a 1.18 ± 0.11c 0.115 ± 0.010d

Ethyl 2-hydroxy-4methylpentanoate Ethyl 3(methylthio)propionat e

0.43 ± 0.06ª,c

1.9 ± 0.3c,d

0.50 ± 0.03c 0.0334 ± 0.0019c 0.0192 ± 0.0024ª 0.00318 ± 0.00022c 0.02324 ± 0.00008d

43

Ethyl 3hydroxytridecanoate

1896

C

Ethyl tetradecanoate

2045

A

Diethyl malate

2050

B7

2108

B6

2131

A

Ethyl pentadecanoate

2145

B7

Ethyl hexadecanoate

2252

A

Unknown (m/z 117)

2321

-

Ethyl vanillate Total of ethyl esters Others esters

2564

A

Methyl octanoate

1372

B2

Methyl decanoate

1580

A

Methyl salicylate

1760

B2

Hexyl salicylate

2207

B2

Methyl hexadecanoate

2210

A

Methyl jasmonate

2298

B8

Methyl vanillate

2559

B2

4-Methyl-2-pentanone

969b

B1

2,6-Dimethyl-4heptanone

1138

C

2-Heptanone

1151

B2

Acetoin

1276

A

6-Methyl-5-hepten-2one

1322

A

2-Nonanone

1375

B2

2-Acetylfuran

1492

A

Dihydro-3(2H)thiophenone

1515

C

Acetophenone

1641

A

1773

C

1623

A

γ-Nonalactona 57,89

2039

B

γ-Decalactona

2159

A

Ethyl 3hydroxydodecanoate Ethyl cinnamate

Total of other esters Ketones

1,2Cyclopentanedione Total of ketones Lactones γ-Butyrolactone

Total of lactones

0.018 ± 0.003ª 0.0136 ± 0.0020ª 0.0048 ± 0.0007ª 0.070 ± 0.010ª nd 0.0077 ± 0.0008ª 0.0387 ± 0.0006ª 0.0037 ± 0.0005ª nd 7.68ª,b

0.088 ± 0.007b 0.025 ± 0.004ª,b 0.0040 ± 0.0003a 0.116 ± 0.016b nd 0.0086 ± 0.0013ª,b 0.080 ± 0.012ª 0.024 ± 0.004b nd 6.77a

0.0097 ± 0.0014ª 0.0030 ± 0.0004ª 0.0167 ± 0.0025ª 0.0128 ± 0.0019ª 0.0068 ± 0.0010ª 0.039 ± 0.006ª,c 0.0034 ± 0.0005ª 0.091ª,c

0.130 ± 0.010c 0.034 ± 0.004b 0.00461 ± 0.00010ª

nd 0.0138 ± 0.0020b,c 0.089 ± 0.013ª 0.036 ± 0.005c nd 9.33b

0.119 ± 0.016c,d 0.00515 ± 0.00014ª 0.0052 ± 0.0007ª 0.164 ± 0.007d nd 0.00171 ± 0.00012ª 0.045 ± 0.007ª 0.0275 ± 0.0020b,c nd 7.88b

0.1551 ± 0.0019e 0.062 ± 0.009c 0.00510 ± 0.00005ª 0.333 ± 0.022e nd 0.024 ± 0.004c 0.090 ± 0.013ª 0.077 ± 0.009d nd 11.9c

0.099 ± 0.008b,c 0.116 ± 0.017d 0.0048 ± 0.0007ª 0.1340 ± 0.0024b,d nd 0.064 ± 0.009d 0.32 ± 0.05b 0.0330 ± 0.0006b,c nd 11.5c

0.00303 ± 0.00012b 0.00059 ± 0.00009b,c 0.0085 ± 0.0007b 0.0068 ± 0.0010b 0.0200 ± 0.0025b 0.0255 ± 0.0022b 0.00308 ± 0.00003ª 0.068b

0.0055 ± 0.0008c,d 0.00102 ± 0.00015c,d 0.0134 ± 0.0015c,d 0.0083 ± 0.0011b 0.0208 ± 0.0010b 0.030 ± 0.004ª,b 0.0037 ± 0.0004ª 0.083ª

0.0045 ± 0.0007b,c 0.00154 ± 0.00023d 0.0110 ± 0.0003b,c 0.0150 ± 0.0022ª 0.030 ± 0.004c 0.03882 ± 0.00019ª,c 0.0036 ± 0.0005ª 0.105c,d

0.00648 ± 0.00012d 0.0023 ± 0.0003e 0.01571 ± 0.00019ª,d 0.0082 ± 0.0006b 0.031 ± 0.004c 0.048 ± 0.007c 0.00483 ± 0.00023b 0.116d

0.00595 ± 0.00018c,d

0.0044 ± 0.0006ª 0.0100 ± 0.0015ª 0.0036 ± 0.0004ª 0.041 ± 0.003ª 0.0059 ± 0.0008ª 0.0094 ± 0.0014ª 0.0082 ± 0.0012ª 0.075 ± 0.011ª 0.0065 ± 0.0009ª 0.057 ± 0.008ª 0.221ª

0.00322 ± 0.00012b 0.0098 ± 0.0004a 0.0124 ± 0.0012b 0.2958 ± 0.0013b 0.00715 ± 0.00008ª,b 0.0109 ± 0.0011ª,b 0.0110 ± 0.0012ª,b 0.0335 ± 0.0022b,c 0.018 ± 0.003b 0.0309 ± 0.0024b 0.433b

0.0034 ± 0.0003b 0.0100 ± 0.0005ª 0.0079 ± 0.0008c

0.0035 ± 0.0004b 0.0100 ± 0.0015ª 0.0123 ± 0.0009b

0.00380 ± 0.00009ª,b 0.0141 ± 0.0010b 0.0208 ± 0.0005d

0.34 ± 0.05b

0.30 ± 0.04b

0.35 ± 0.03b

0.0082 ± 0.0010b 0.0113 ± 0.0015ª,b 0.0154 ± 0.0023b 0.030 ± 0.003b 0.0161 ± 0.0004b,c 0.053 ± 0.003ª 0.496b,c

0.0093 ± 0.0008b 0.0129 ± 0.0014b 0.0154 ± 0.0020b 0.037 ± 0.003b,c 0.0106 ± 0.0004d 0.083 ± 0.006c 0.495b,c

0.0124 ± 0.0012c 0.0188 ± 0.0008c 0.0218 ± 0.0019c 0.0447 ± 0.0009c 0.01187 ± 0.00006d,e 0.079 ± 0.009c,d 0.579c

0.0033 ± 0.0003b 0.0093 ± 0.0010ª 0.0136 ± 0.0020b 0.30 ± 0.03b 0.0151 ± 0.0009d 0.0183 ± 0.0021c 0.023 ± 0.003c 0.046 ± 0.005c 0.0148 ± 0.0015c,e 0.063 ± 0.009ª,d 0.504b,c

nd 0.033 ± 0.005ª 0.0103 ± 0.0015ª 0.043ª

nd 0.023 ± 0.003b 0.078 ± 0.011b 0.101b

nd 0.0296 ± 0.0007ª,b 0.008554 ± 0.00004ª 0.038ª

nd 0.0280 ± 0.0025ª,b 0.00997 ± 0.00015ª 0.038ª

nd 0.0345 ± 0.0013ª 0.01210 ± 0.00017ª 0.047ª

nd 0.031 ± 0.004ª 0.0100 ± 0.0008ª 0.041ª

0.22 ± 0.03c

ndb 0.0137 ± 0.0003ª,c,d 0.0125 ± 0.0015ª 0.030 ± 0.005c 0.039 ± 0.005ª,c 0.0040 ± 0.0006ª,b 0.105c,d

44

C13-Norisoprenoids 1,1,6-Trimethyl-1,2dihydronaphthalene β-damascenone

1727

A

1814

A

Total of C13Norisoprenoids Terpenes Limonene

1147

A

trans-Linalool oxide

1469

B9

α-Terpinen

1505

C

Linalool

1543

A

Hotrienol

1602

C

α-Farnesene

1646

C

α-Terpineol

1700

A

2,6-Dimethyl -3,7Octadiene-2,6-diol

1702

C

Citronellol

1768

A

Geraniol

1852

A

Nerolidol

2041

A

Farnesol

2364

B2

Total of Terpenes Volatile phenols 4-Ethylguaiacol

2027

A

Eugenol

2171

A

4-Ethylphenol

2185

A

4-Vinylguaiacol

2203

B2

Coumaran

2402

C

Methoxyeugenol

2504

B10

Acetovanillone

2577

C

0.0136 ± 0.0020ª 0.077 ± 0.011ª

0.025 ± 0.004b 0.037 ± 0.004b

0.028 ± 0.004b 0.052 ± 0.007b,c

0.0162 ± 0.0024ª 0.040 ± 0.004b

0.0168 ± 0.0005ª 0.064 ± 0.003ª,c

0.0462 ± 0.0019c 0.058 ± 0.006c

0.091ª,c

0.062b

0.080c

0.056b

0.081c

0.105ª

0.0165 ± 0.0025ª,c 0.0042 ± 0.0005ª 0.0054 ± 0.0008ª 0.1297 ± 0.019ª 0.020 ± 0.003ª

0.0056 ± 0.0008b 0.0052 ± 0.0008ª,b 0.0025 ± 0.0004b 0.169 ± 0.014ª,b 0.019 ± 0.003a 0.0087 ± 0.0013b 0.038 ± 0.004ª 0.0084 ± 0.0010ª 0.049 ± 0.007b 0.082 ± 0.011ª

0.021 ± 0.003c 0.005072 ± 0.000008ª,b 0.0039 ± 0.0006c

0.0117 ± 0.0017ª,b 0.0056 ± 0.0008ª,b 0.00257 ± 0.00005b 0.208 ± 0.018b,d 0.023 ± 0.003ª,b 0.0093 ± 0.0005b 0.0471 ± 0.0011b 0.0115 ± 0.0011b,c 0.0708 ± 0.0008b 0.106 ± 0.005b 0.203 ± 0.012b 0.147 ± 0.022b 0.85b

0.0066 ± 0.0009b 0.0060 ± 0.0003b 0.00472 ± 0.00003ª,c 0.313 ± 0.005c 0.02200 ± 0.00008ª,b 0.0228 ± 0.0017c 0.0553 ± 0.0013b 0.0151 ± 0.0005d 0.122 ± 0.003d 0.157 ± 0.005c

0.041 ± 0.006d 0.0102 ± 0.0007c 0.00474 ± 0.00020ª,c 0.26 ± 0.03c,d 0.0260 ± 0.0003b 0.0134 ± 0.0020d 0.053 ± 0.004b 0.0118 ± 0.0015b,c 0.100 ± 0.014c 0.121 ± 0.018b 0.3747 ± 0.0012c,d 0.2775 ± 0.0018d 1.30c

nda 0.033 ± 0.005ª 0.0095 ± 0.0014ª,c 0.026 ± 0.004ª 0.065 ± 0.010ª 0.0183 ± 0.0003ª 0.018 ± 0.003ª 0.35ª nd 0.0024 ± 0.0004ª,b nda 0.088 ± 0.013ª 0.0154 ± 0.0023ª,d 0.00113 ± 0.00017ª,b 0.0031 ± 0.0005ª,b

0.30 ± 0.03c 0.01950 ± 0.00023ª 0.00944 ± 0.00012b 0.051 ± 0.003b 0.0125 ± 0.0006b,d 0.119 ± 0.014c,d 0.12927 ± 0.00023b

0.21 ± 0.03b

0.31 ± 0.05c

0.154 ± 0.023b 0.75b

0.194 ± 0.010b 1.18c

nd 0.00202 ± 0.00022ª 0.00068 ± 0.00010b 0.023 ± 0.003b 0.0088 ± 0.0013b 0.001055 ± 0.000012ª 0.00271 ± 0.00007ª

nd 0.00258 ± 0.00017ª,b 0.00101 ± 0.00015c 0.029 ± 0.004b,c 0.0114 ± 0.0016b,c 0.00123 ± 0.00016ª,b,c 0.00341 ± 0.00022ª,b,c

0.45 ± 0.07d 0.41 ± 0.05c 1.59d

nd 0.00253 ± 0.00025ª,b

nd 0.0036 ± 0.0003c

nda

nda

0.033 ± 0.005b,c 0.0119 ± 0.0018ª,b,c 0.00123 ± 0.00018ª,b,c 0.0031 ± 0.0005ª,b

0.0429 ± 0.0005c 0.0157 ± 0.0004d 0.001517 ± 0.000018c 0.0042 ± 0.0003c

nd 0.00291 ± 0.00009b 0.00131 ± 0.00011d 0.034 ± 0.003b,c 0.0151 ± 0.0009ª,c,d 0.00142 ± 0.00019b,c 0.0038 ± 0.0004b,c

Total of volatile 0.110ª 0.038b 0.048b,c 0.052b,c 0.068c 0.059c phenols Total of volatile 61.55a 67.87ª,b 95.76c,d 83.57b,d 128.10e 102.48c compounds LRI: linear retention index values obtained from samples. ID: reliability of identification: A, mass spectrum and LRI agreed with standards; B, mass spectrum agreed with mass spectral data base and LRI agreed with the literature data (TI); C, mass spectrum agreed with mass spectral data base. nd: no peak detected. aSimilar superscript letter in the same row indicates no significant statistically differences (p<0.05). bValues estimated by linear regression. cLiterature reference agreed with LRI data:1: Morales at al. 2019; 2: Kim et al. 2019; 3: Carlin et al., 2016; 4: Nicolli et al., 2018; 5: http://www.chemspider.com/Default.aspx; 6: Ferrari et al., 2004; 7: Zhao et al., 2009; 8: Ka et al., 2005; 9: Fernández de Simón et al., 2015.

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GRAPHICAL ABSTRACT

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HIGHLIGHTS Volatile profiles of 15 pure culture flor yeasts were studied Volatile profiles were analysed by dual sequential SBSE-HSSE-GC-MS During biological ageing increased primary acetals and also terpenes PCAs and Heatmaps were performed. Two strains could be selected to be used in biological ageing to produce Sherry wines.

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