Comparison of inorganic and organic nitrogen ...

Food Chemistry 127 (2011) 1072–1083

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Comparison of inorganic and organic nitrogen supplementation of grape juice – Effect on volatile composition and aroma profile of a Chardonnay wine fermented with Saccharomyces cerevisiae yeast Diego Torrea a,b, Cristian Varela a, Maurizio Ugliano a, Carmen Ancin-Azpilicueta b, I. Leigh Francis a, Paul A. Henschke a,⇑ a b

Australian Wine Research Institute, P.O. Box 197, Glen Osmond (Adelaide), SA 5064, Australia Universidad Pubica de Navarra, Department of Applied Chemistry, Campus Arrosadia S-N, E-31006 Pamplona, Spain

a r t i c l e

i n f o

Article history: Received 2 September 2010 Received in revised form 10 December 2010 Accepted 21 January 2011 Available online 28 January 2011 Keywords: Fermentation Wine Chardonnay Yeast Saccharomyces cerevisiae Aroma Flavour Nitrogen

a b s t r a c t Inorganic nitrogen salts, and to a growing extent organic nitrogen preparations, are widely used to ameliorate a nitrogen deficiency in wine fermentation, but the impact of nitrogen supplementation on perceived wine sensory profile is essentially unknown. Supplementation of a low nitrogen Chardonnay grape juice with either ammonium nitrogen or combined amino acid and ammonium nitrogen showed that the type of nitrogen and concentration in the range 160–480 mg N/l had a substantial impact on the formation of yeast volatile compounds and perceived wine aroma. Addition of amino acid and ammonium nitrogen increased both acetate and medium chain fatty acid esters to a greater extent and decreased higher alcohols to a lesser extent than ammonium nitrogen alone whereas ammonium nitrogen substantially increased ethyl acetate and acetic acid. Low nitrogen wines were rated relatively low in floral/fruity aroma descriptors, while moderate nitrogen wines showed a good balance between desirable and less desirable attributes, whereas high nitrogen produced either an acetic/solvent character or highest ratings for floral/fruity attributes, depending on nitrogen type. These results show that amount and type of nitrogen supplement can substantially modulate Chardonnay wine volatiles composition and perceived aroma. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction The yeast assimilable nitrogen (YAN) content of grape must is a critical nutrient that has been identified as being suboptimal in many viticultural regions surveyed world-wide (see for example: Butzke, 1998; Hagen, Keller, & Edwards, 2008; Henschke & Jiranek, 1993; Nicolini, Larcher, & Versini, 2004). A concentration exceeding 140 mg N/l is generally considered the threshold concentration for low risk completion of fermentation for low solids, low temperature, anaerobic musts of moderate sugar content (reviewed by Bell & Henschke, 2005). Higher concentrations of nitrogen are generally required for the reliable fermentation of musts containing a combination of risk factors including higher sugar concentrations (reviewed by Colombié, Malherbe, & Sablayrolles, 2005; Cramer, Vlassides, & Block, 2002; Henschke & Jiranek, 1993; Jiranek, Langridge, & Henschke, 1995a).

⇑ Corresponding author. Tel.: +61 8 8303 6600; fax: +61 8 8303 6601. E-mail addresses: (P.A. Henschke).

[email protected],

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0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.01.092

The main sources of assimilable nitrogen present in grape juice include ammonium, a-amino acids, and, to a lesser extent, small peptides. During the early stages of fermentation, these compounds are rapidly accumulated by yeast, thus fulfilling the biosynthetic requirements for amino acids needed for protein synthesis and growth, and any surplus is then stored in the cell vacuole (Henschke & Jiranek, 1993; Salmon, 1996). Insufficient assimilable nitrogen results in lower biomass yields and consequently slower fermentation rates (Salmon, 1989; Varela, Pizarro, & Agosin, 2004), which increase the risk of slow or stuck fermentations (reviewed by Alexandre & Charpentier, 1998; Bisson, 1999; Henschke, 1997). Supplementation of musts with inorganic nitrogen in the form of ammonium salts, such as diammonium phosphate (DAP), can decrease such risks and may also affect the formation of undesirable volatile sulphur compounds (Bell & Henschke, 2005; Jiranek, Langridge, & Henschke, 1995b; Vos & Gray, 1979). As a result, DAP addition to grape must, in many cases without knowledge of initial YAN content, is common practice in the wine industry. Besides its effect on yeast growth and fermentation kinetics, YAN can regulate yeast metabolism at several levels including

D. Torrea et al. / Food Chemistry 127 (2011) 1072–1083

the formation of yeast volatile and non-volatile metabolites, which contribute to wine flavour (Albers, Larsson, Liden, Niklasson, & Gustafsson, 1996; Bell & Henschke, 2005; Garde-Cerdan & Ancin-Azpilicueta, 2008). Of the non-volatile compounds, glycerol, malic acid, a-ketoglutaric acid and succinic acid are affected by nitrogen source and concentration (Albers et al., 1996; Camarasa, Grivet, & Dequin, 2003; Vilanova et al., 2007). Many of the volatile compounds synthesised by yeast also change in response to nitrogen source and/or nitrogen concentration (review by Bell & Henschke, 2005). The most important compounds include acetate and ethyl esters, higher alcohols, medium chain fatty acids (MCFA) and branched-chain acids. Esters can impart fruity and floral aromas, higher alcohols are mostly associated with solvent or fusel odours, and MCFA and branched-chain acids have soapy, cheesy, sweaty or rancid odours (Francis & Newton, 2005; Lambrechts & Pretorius, 2000). Higher alcohols are generated from a-ketoacids which are derived mainly from sugars, and by anabolic reactions through a multi-step reaction from branched-chain amino acids (Dickinson et al., 1997; Eden, Van Nedervelde, Drukker, Benvenisty, & Debourg, 2001). Biosynthesis of higher alcohols from branchedchain amino acids occurs by the Ehrlich pathway which involves transamination followed by decarboxylation and reduction steps. MCFAs are formed during the early stages of lipid biosynthesis by the repetitive condensation of acetyl-CoA to the growing fatty acid chain and prematurely released from the fatty acid synthase (FAS) complex (Marchesini & Poirier, 2003). Similar to higher alcohols, branch-chain acids are also derived from branched-chain amino acids and are formed by the Ehrlich pathway, in this case the last step is an oxidation reaction (Dickinson et al., 1997). Esters are produced by condensation of an alcohol and a coenzyme-A activated acid (acyl-CoA), catalysed by an acyltransferase (Sumby, Grbin, & Jiranek, 2010). Specifically, acetate esters result from the combination of acetyl-CoA with an alcohol by the action of the ATF1 or ATF2 encoded alcohol acetyl transferases (Verstrepen, Van Laere et al., 2003). Ethyl esters are biochemically generated from acyl-CoA and ethanol, by the action of acyltransferases encoded by EEB1 and EHT1 (Saerens et al., 2008). The effect of must nitrogen on the formation of yeast derived volatile compounds is complex and depends on the type and concentration of nitrogen present and class of volatile compound. Higher alcohols show an inverse relationship to initial nitrogen concentration, except at very low nitrogen concentration (Äyräpää, 1971; Carrau et al., 2008; Guitart, Orte, Ferreira, Pena, & Cacho, 1999). Nitrogen supplementation has a complex effect on the formation of acetate and ethyl esters but in most studies, ethyl acetate concentration increases with increasing concentrations of nitrogen (Bell & Henschke, 2005; Guitart et al., 1999; Hernandez-Orte, Bely, Cacho, & Ferreira, 2006; Hernandez-Orte, Ibarz, Cacho, & Ferreira, 2005; Miller, Wolff, Bisson, & Ebeler, 2007). Volatile acids also show complex relationships with nitrogen due to their different synthetic origins (Bely, Rinaldi, & Dubourdieu, 2003; Hernandez-Orte, Bely et al., 2006; Vilanova et al., 2007). The link between nitrogen supplementation of low nitrogen musts and formation of yeast volatile compounds has been explored in several studies. However, most of these investigations have been carried out using model grape juice (Carrau et al., 2008; Hernandez-Orte, Ibarz et al., 2006; Vilanova et al., 2007), or in the case of studies using real grape juice, they were carried out with grape juice already having a relatively high YAN content (Hernandez-Orte, Ibarz, Cacho, & Ferreira, 2006; Hernandez-Orte, Ibarz et al., 2005; Miller et al., 2007) or using high solids, red must (Ugliano, Siebert, Mercurio, Capone, & Henschke, 2008; Ugliano et al., 2009). Moreover, a clear correlation between the chemical changes induced by nitrogen supplementation and the consequent sensory modifications, if any, has never been clearly established. In

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particular, the specific effects of the two major components of grape YAN, namely ammonium nitrogen and amino acid nitrogen, on wine aroma composition and sensory properties have not been fully determined. In order to establish a practical understanding of the effect of nitrogen supplementation on white wine aroma, a low nitrogen Chardonnay juice (160 mg N/l) was supplemented with two nitrogen sources [inorganic (ammonium chloride) and organic (combined amino acids and ammonium nitrogen)] and the resulting wines evaluated chemically and by quantitative descriptive sensory analysis. 2. Materials and methods 2.1. Grape juice and growth conditions A low nitrogen Chardonnay juice, prepared commercially from grapes mechanically harvested from Langhorne Creek (South Australia) in 2003, was used for wine fermentations. The grapes used were disease-free and 100 ppm potassium metabisulfite was added post-harvest; the filter clarified (0.45 lm, Millipore) cold-settled juice contained 13 and 35 mg/l free and total SO2, respectively. The juice contained a sugar concentration of 200 g/l and a low content in yeast assimilable nitrogen (YAN), 160 mg N/l, with a pH of 3.3. After adjusting the sugar concentration to 225 g/l by adding glucose and fructose, the must was divided into five aliquots. One aliquot was not supplemented and was used as a control. Two aliquots were supplemented with NH4Cl to increase the YAN concentration up to 320 and 480 mg N/l, respectively. Ammonium chloride was used in preference to the phosphate salt since chloride is believed to have less physiological impact. The last two aliquots of juice were supplemented with a mix of amino acids and NH4Cl to reach YAN concentrations of 320 and 480 mg N/l. In both cases the relative proportions of nitrogen assimilable compounds found in the original must were maintained. The concentration of nitrogen compounds in the grape must was the following (expressed as mg/l): ammonium 52, alanine 74.4, arginine 98.5, asparagine 14.9, aspartate 24.9, cysteine 1.4, c-aminobutyrate 69.7, glutamine 111.9, glutamate 75.3, glycine 4.7, histidine 19.6, isoleucine 11, leucine 11.2, lysine 5.2, methionine 3.7, ornithine 1.1, phenylalanine 20.1, serine 50.8, threonine 48.6, tryptophan 10.9, tyrosine 18.7, valine 18.6, proline 764.8, homoproline 10.4. YAN was calculated by adding the nitrogen present in ammonium to the nitrogen present in individual amino acids excepting proline. Proline is not used as a nitrogen source under anaerobic conditions (Ingledew, Magnus, & Sosulski, 1987). Once the five must fractions were prepared they were saturated with oxygen by aeration (0.22 lm, Millipore) with a stainless stain diffuser and filter sterilised (0.45 lm, Millipore) immediately before inoculation. 2.2. Fermentation conditions Saccharomyces cerevisiae AWRI 796, obtained as a freeze-dried culture from The Australian Wine Research Institute culture collection, was used throughout this study. A yeast starter culture was made in 20 ml of MYPG medium (malt extract, 3 g/l; yeast extract, 3 g/l; peptone 5 g/l; glucose, 10 g/l) and incubated at 27 °C with shaking (180 rpm). The starter culture was then used to inoculate two Erlenmeyer flasks containing 500 ml of diluted (1:1) Chardonnay must. Both flasks were incubated for 2 days at 18 °C with shaking (180 rpm) and then used to inoculate the fermentations. Fermentations were carried out in triplicate in 3 l glass fermentation flasks equipped with fermentation locks. Fermentations were inoculated at a cell density of 1 106 cells/ml and fermented at 18 °C with constant stirring and their progress monitored daily

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by measuring the refractive index of culture supernatants. At the end of fermentation the wines were cold settled and racked. Wine samples for amino acids, organic acids and basic wine composition were centrifuged for 5 min at 15,000g and the cell-free supernatants were kept at 4 °C for analysis. Samples for analysis of volatile fermentation products were centrifuged as described before, poured into glass ampoules under nitrogen gas and kept at 20 °C. Wines used for sensory analysis were adjusted to 20 mg/l of free SO2, filtered under nitrogen environment and kept in brown bottles closed with crown seals at 18 °C. Chemical and sensory analyses were carried out within 3 months after bottling. 2.3. Analytical methods Wine parameters such as density, specific gravity, pH, volatile acidity, alcohol and titratable acidity were determined by using a Foss WineScan FT 120 as described by the manufacturer (Foss, Hillerød, Denmark). Free and total SO2 were measured by the aspiration method (Rankine & Pocock, 1970). Brown pigments were estimated by absorbance at 420 nm (Skouroumounis, Kwiatkowski, Sefton, Gawel, & Waters, 2003). Yeast growth was followed spectrophotometrically by absorbance at 600 nm. Viable cells were determined by plating and counting of colonies on MYPG agar. Organic acids were measured by high-performance liquid chromatography (HPLC) using a Bio-Rad HPX-87H column as described previously (Nissen, Schulze, Nielsen, & Villadsen, 1997). Glucose, fructose, glycerol and ammonium were quantified by using the R-Biopharm (Roche, Mannheim, Germany) enzymatic kits (10 139106 035, 10 148270 035, 1 1112732 035) as described by the manufacturer. Amino acids were quantified by HPLC using the FMOC (9-fluorenylmethylchloroformate) pre column derivatisation method as described elsewhere (Melucci, Xie, Reschiglian, & Torsi, 1999). 2.4. Analysis of volatile compounds Volatile fermentation products synthesised during fermentation were analysed using headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME/ GCMS), with polydeuterated internal standards for stable isotope dilution analysis (SIDA) as described previously (Siebert et al., 2005). Twenty-six compounds, including ethyl esters and acetate esters, volatile fatty acids and higher alcohols, were quantified. Acetaldehyde was determined by using HS-SPME/GCMS/SIDA with d4-acetaldehyde as an internal standard. Briefly, a Supelco carbowax/divinylbenzene (CW/DVB, orange) 65 lm fibre was exposed to the headspace of the sample vials for 5 min at room temperature and desorbed in the GC inlet for 4 min. A HP 6890 gas chromatograph equipped with Gerstel MPS2 multipurpose sampler and coupled to a HP 5973N mass selective detector was used for gas chromatography–mass spectrometry. The instrument was fitted with a 30 m 0.25 mm Phenomenex fused silica capillary column ZB-Wax, 0.25 lm film thickness. Oven temperature was maintained at 40 °C while the SPME inlet liner was held at 200 °C. Helium (BOC Ultra High Purity) was used as carrier gas at a flow rate of 2.0 ml/min. Instrument control and data analysis were carried out with the HPG1701CA ChemStation software. Analyses were carried out in duplicate. 2.5. Quantitative descriptive sensory analysis A preliminary informal sensory assessment and inspection of the composition of wines showed that differences among replicates were small, and accordingly only fermentation duplicates were subjected to sensory analysis. In the preliminary assessment, it was noted that the wines were not as fresh and fruity as similar

wines usually made under research conditions, and that there was a degree of oxidative flavour evident across the set. Notwithstanding this observation, it was considered that there were sufficient sensory differences among the wines in other attributes to allow study. A panel of 12 judges (eight females, four males) with previous experience in wine descriptive analysis studies took part in this study. During three training discussion sessions where four samples were presented per session, the panellists generated, by consensus, a list of attributes considered necessary to differentiate and describe the samples, with 16 aroma attributes (Table 1) agreed upon. For sessions 1 and 2 aroma and also flavour descriptors were generated by the panel, however it was agreed that aroma attributes in the remaining sessions were of greater importance in discriminating the samples, and in addition some of the wines were strongly acidic in taste, masking other sensations. Following these sessions four practice rating sessions were then held under the same conditions as the subsequent formal sessions, except that each assessor was presented with the same sample presentation order. Assessor performance was evaluated prior to the formal sessions. Wines were assessed for aroma only in six formal sessions under colour masking sodium lights in isolated tasting booths. Reference standards were available to the panellists in the booths during each session. Wines from a fermentation duplicate of each nitrogen treatment were presented in random order across the judges in coded, covered ISO tasting glasses (25 ml) in triplicate. The judges rated each of the attributes on an unstructured 10 cm line scale, with anchors of ‘low’ and ‘high’ placed at 1 and 9 cm, respectively. FIZZ software (Version 2.0, Biosystemes, Couternon, France) was used for the collection of the data. 2.6. Statistical analysis Statistical differences among treatments for volatile and nonvolatile compounds measured in fermentation samples were determined using analysis of variance (ANOVA) using the Enterprise Guide 3 System Software (SAS Institute, Cary, NC, USA). The descriptive analysis data for each attribute was analysed using a nested analysis of variance (ANOVA) based on a balanced incomplete block design, testing for the effects of treatment and fermentation replicate nested within treatment, using a mixed model treating judges as a random effect (Genstat 5.4.1, Lawes Agricultural Trust, Rothamsted Experimental Station, UK). Fisher’s Least Significant Difference (LSD) means comparison test (P = 0.05) was performed. Using Unscrambler 9.5 (CAMO ASA, Oslo, Norway) partial least squares (PLS) regression was employed to identify specific volatile fermentation products related to specific sensory attributes (Aznar, Lopez, Cacho, & Ferreira, 2003; Ferreira, Fernandez, Pena, Escudero, & Cacho, 1995). PLS1 regression was employed for each of the fermentation duplicates included in the sensory study, with the y-data being the scores for each of the sensory attributes and the x-data set comprising the volatile compositional data. In order to decrease over-fitting of the PLS models full cross validation was carried out (Martens & Dardenne, 1998). 3. Results In order to determine the impact of nitrogen type and concentration on wine aroma, a low yeast assimilable nitrogen (YAN) Chardonnay juice (160 mg N/l) was supplemented with ammonium nitrogen or a mixture of amino acids and ammonium nitrogen to yield juices with a moderate (320 mg N/l) and high (480 mg N/l) concentration of nitrogen. Grape juices were fermented with a yeast widely utilised for Chardonnay (S. cerevisiae

D. Torrea et al. / Food Chemistry 127 (2011) 1072–1083 Table 1 Aroma attributes used for quantitative descriptive sensory analysis and composition of the reference standards. Attribute

Reference standard compositiona

Banana Fruity ester Artificial grape Musk Floral Tropical Stewed fruit Citrus Bruised apple Honey Stale beer Nail polish remover Acetic Cheese Sweat

500 ll Isoamyl acetate (1 g/l in ethanol) 200 ll Mixed esters stock solutionb 100 ll o-Aminoacetophenone (10 mg/l in ethanol)

Wet cardboard

1 musk Flavoured hard candy (LifesaverÒ) 800 ll 2-Phenylethanol (200 ml/l in ethanol) 7 ml Canned pineapple juice 10 ml Canned pear juice and apple sauce 10 ml Orange marmalade 100 ll Acetaldehyde (50% w/v in ethanol) 1 ml Honey 50 ml Beer left overnight in a glass 200 ll Undiluted ethyl acetate 200 ll Undiluted glacial acetic acid 10 ll 3-Methyl butyric acid (10 g/l in ethanol) 300 ll of a solution of hexanoic acid (6.7 g/l) and 3-methyl butyric acid (3.3 g/l) in ethanol Piece of wet cardboard (no wine)

a

Unless otherwise stated, solutions were added to 100 ml of neutral white wine. Ester solution contains: isobutyl acetate 5 g/l, ethyl butyrate 0.9 g/l, ethyl hexanoate 2 g/l and ethyl octanoate 2 g/l in redistilled ethanol. b

AWRI 796) under simulated commercial winemaking conditions and the young wines subjected to chemical and quantitative descriptive sensory analysis within three months from bottling. 3.1. Yeast growth and fermentation rate Yeast biomass yield increased and fermentation time decreased in response to higher initial juice nitrogen concentrations irrespective of the type of nitrogen used for supplementation (Fig. 1). Fermentation time was reduced from 13 days for the control juice (160 mg N/l) to 7 days for moderate nitrogen concentrations (320 mg N/l) and to 5 days for high nitrogen concentrations (480 mg N/l). The type of the nitrogen added, i.e. ammonium or a mixture of amino acid and ammonium nitrogen, only showed an effect at moderate nitrogen concentrations where the nitrogen mixture increased the fermentation rate (Fig. 1A). The maximum yeast number was significantly higher for the fermentations with increased nitrogen concentrations, while yeast viability, on the other hand, was higher for the control ferment (Table 2). Sugar was completely consumed in all five fermentation treatments. Ammonium nitrogen was also entirely consumed in all treatments (data not shown), while residual YAN was marginally higher only for high initial nitrogen concentrations (Table 2). The residual concentrations of most of the amino acids were not significantly different among treatments. However, statistically significant differences were observed for some amino acids (data not shown). After fermentation, the concentrations of alanine and aspartate were higher in the wines supplemented with ammonium nitrogen; glutamine, leucine and threonine were higher in the wines with high initial nitrogen; histidine was higher in the wine supplemented with a mixture of amino acid and ammonium nitrogen at high nitrogen concentrations. Lysine was consumed at a greater extent in the control ferment, whereas tyrosine was consumed in larger amounts in the wines supplemented with nitrogen. 3.2. Changes in non-volatile compounds in response to nitrogen supplementation Nitrogen supplementation had a significant effect on many nonvolatile compounds (Table 2). Titratable acidity was decreased

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substantially by nitrogen addition, with musts supplemented with a mixture of amino acid and ammonium nitrogen exhibiting the lowest values. There were lower concentrations of malic, succinic and tartaric acids in the musts supplemented with nitrogen. By contrast, wine pH was decreased following the addition of ammonium but not the nitrogen mixture, with the 480 mg/l treatment reducing the pH to a very low value. Brown pigments and total SO2 were also found to decrease in response to nitrogen supplementation. Ethanol, which ranged from 13.0% to 13.3% v/v, exhibited a small increase only in musts supplemented with ammonium. There was no effect of nitrogen addition on glycerol concentration. 3.3. Formation of volatile compounds in response to nitrogen addition Nitrogen supplementation affected the final concentrations of all the 28 fermentation-derived volatile compounds (nine ethyl esters, six acetate esters, seven volatile acids, five higher alcohols and acetaldehyde) measured in this study (Table 3). The branched-chain ethyl esters ethyl 2-methylpropanoate and ethyl 2-methylbutanoate strongly decreased in concentration with nitrogen addition, whereas ethyl 3-methylbutanoate decreased at a lower rate as nitrogen increased. The type of nitrogen had no important differential effect on these esters except for ethyl 2methylbutanoate which was less affected by moderate amino nitrogen concentrations. In general, the ethyl esters of the medium-chain fatty acids increased with nitrogen supplementation although ethyl propanoate was the only ester to show a decrease and ethyl decanoate only decreased at high nitrogen. Ethyl octanoate and ethyl decanoate increased by up to 3–5-fold with nitrogen supplementation. The effects of the type of nitrogen supplement depended on the level of supplementation and in some cases on the ester chain length. At moderate nitrogen supplementation nitrogen type had no important differential effect on ester production except for ethyl butanoate whereas at high nitrogen supplementation amino nitrogen tended to favour the longer chain ethyl esters, especially ethyl octanoate, ethyl decanoate and ethyl dodecanoate. All acetate esters showed a marked increase with nitrogen supplementation, in fact 2-methylpropyl acetate concentration increased by up to 10-fold compared to the control wine. For most of the acetate esters the biggest increase resulted with moderate nitrogen supplementation whereas 2-methylpropyl acetate and ethyl acetate showed a more linear increase according to the concentration of nitrogen in the juice. Phenylethyl acetate showed a non-linear response to nitrogen with higher concentrations at moderate nitrogen. Nitrogen type showed the greatest effects at high nitrogen supplementation with the amyl acetates responding most strongly to 480 mg/l amino-nitrogen. Amino nitrogen also favoured production of phenylethyl acetate when compared to ammonium nitrogen. Ethyl acetate responded strongly to both the type of the nitrogen added and concentration, being higher for juice supplemented with ammonium nitrogen and showing the highest concentration at 480 mg N/l. As a result of higher concentrations of both ethyl esters and acetate esters in musts supplemented with nitrogen, the concentration of total esters (excluding ethyl acetate) increased in response to nitrogen addition (Fig. 2A). Total acetate esters showed increased concentrations in musts supplemented with a mixture of amino acid and ammonium nitrogen, with the highest concentration being obtained at 480 mg N/l. The concentration of the branched-chain acids 2-methylpropanoic acid, 2-methylbutanoic acid and 3-methylbutanoic acid decreased in response to nitrogen addition. These last two compounds had lowest concentrations at higher nitrogen. The medium-chain fatty acids (MCFA) hexanoic acid, octanoic acid

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more moderate effect (Fig. 2C). Only the control wine and the wine supplemented with amino nitrogen sources of 320 mg N/l exceeded a concentration of 300 mg/l higher alcohols, the limit considered by some authors as detrimental for wine quality (Rapp & Mandery, 1986; Wagener & Wagener, 1968). 2-Methylpropanol and hexanol exhibited lower concentrations in juices supplemented with high concentrations of nitrogen (480 mg N/l), whereas 2-methylbutanol, 3-methylbutanol and 2-phenylethanol were present at lower concentrations in juices with added ammonium nitrogen when compared to amino nitrogen. Acetaldehyde was low in all wines, and increased only slightly in juices supplemented with amino nitrogen sources.

3.4. Quantitative descriptive sensory analysis

Fig. 1. Sugar consumption (A) and yeast growth (B) for control fermentation (circles) and fermentations supplemented with ammonium nitrogen (NH4) (closed symbols) and with a mixture of amino acid and ammonium nitrogen (AAC) (open symbols). YAN 320 mg N/l (squares), 480 mg N/l (triangles).

and decanoic acid, showed higher concentrations in juices supplemented with nitrogen with little significant impact of nitrogen type. Acetic acid concentration also increased in response to nitrogen addition and was higher in musts supplemented with ammonium nitrogen. The concentration of total acids (excluding acetic acid) showed an increase in response to nitrogen addition, with highest values at moderate nitrogen concentrations (320 mg N/l). This profile was driven by the MCFA which showed a similar pattern (Fig. 2B). All higher alcohols analysed showed lower concentrations in juices supplemented with nitrogen, with amino nitrogen giving a

The sensory panel selected 16 terms to describe the aroma profile of the wines (Table 1). Statistically significant differences were found among the treatments for the majority of the 16 attributes, with only ‘honey’ and ‘stewed fruit’ not being rated as significantly different. There was no significant difference between fermentation duplicates for any of the treatments for any attribute. Fig. 3 shows the mean aroma attribute intensity scores for the five treatments, averaged across fermentation replicates. All wines were rated relatively highly in the attribute ‘bruised apple’, and based on previous experience with ratings for this attribute using the sensory methodology used in our laboratory, all wines would be considered to have elevated levels of this character. There was a close and statistically significant negative correlation (r < 0.92) between scores for the fruity attributes ‘banana’, ‘fruit ester’, ‘musk’, ‘floral’ and ‘tropical’ with ‘bruised apple’ aroma. There was no significant correlation between ‘bruised apple’ aroma and ‘acetic’ or ‘nail polish remover’. The control wine was characterised by a low rating for most fruity and floral attributes, with the exception of ‘artificial grape’, and was rated high for attributes that would be considered less desirable, especially ‘bruised apple’, ‘stale beer’, ‘cheese’, ‘sweat’ and ‘wet cardboard’. Compared to the control wine, wines made from juices with moderate nitrogen concentrations (320 mg N/l) were rated higher in intensity for most of the fruity and floral attributes and lower for the other attributes. The type of nitrogen added showed a negligible effect for the 320 mg/l addition, with the wine supplemented with a mixture of amino acid and ammonium nitrogen at this concentration being rated not significantly different to the ammonium only treatment for any attribute, although there was a trend for the wine supplemented with a mixture of amino

Table 2 Basic wine composition and yeast cell data during fermentation for control wine and wines supplemented with ammonium nitrogen (NH4) and with a mixture of amino acid and ammonium nitrogen (AAC). Parameter

Yeast number (106 cells/ml)* Yeast viability (%) Residual sugar (g/l) Residual YAN (mg N/l) pH Titratable acidity (g/l)** Malic acid (g/l) Succinic acid (g/l) Brown pigments (A420 nm) Total SO2 (mg/l) Ethanol (% v/v) Glycerol (g/l)

Condition Control (160)

NH4 (320)

AAC (320)

NH4 (480)

AAC (480)

128 ± 6a 90 ± 3a 0.1 ± 0.0a 5.2 ± 0.2a 3.2 ± 0.0a 10.1 ± 0.2a 3.3 ± 0.2a 1.4 ± 0.1a 0.09 ± 0.00a 63 ± 3a 13.0 ± 0.2a 9.4 ± 0.3a

198 ± 2b 80 ± 1b 0.0 ± 0.0a 6.3 ± 0.6a 3.0 ± 0.0b 8.1 ± 0.3b 2.3 ± 0.1b 0.7 ± 0.2c 0.07 ± 0.01c 28 ± 4b 13.3 ± 0.1b 8.6 ± 0.4a

196 ± 8b 83 ± 8b 0.0 ± 0.0a 5.7 ± 0.8a 3.2 ± 0.0a 7.9 ± 0.3bc 2.4 ± 0.1b 1.1 ± 0.1b 0.08 ± 0.00b 26 ± 3b 13.0 ± 0.1a 9.0 ± 0.7a

213 ± 12bc 82 ± 6b 0.0 ± 0.0a 7.6 ± 1.9b 2.7 ± 0.0c 8.2 ± 0.1b 1.9 ± 0.1c 0.6 ± 0.1c 0.07 ± 0.00c 32 ± 1b 13.2 ± 0.1ab 9.3 ± 0.6a

225 ± 12c 75 ± 8b 0.0 ± 0.0a 7.5 ± 0.7b 3.2 ± 0.0a 7.7 ± 0.2c 2.2 ± 0.0b 1.0 ± 0.1b 0.07 ± 0.00c 32 ± 0b 13.0 ± 0.0a 9.2 ± 0.7a

Values with different superscripts are significantly different as shown by analysis of variance. YAN levels (mg N/l) are indicated in brackets. * Maximum yeast number during fermentation. ** Expressed as tartaric acid.

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Table 3 Concentration of volatile fermentation products for control wine and wines supplemented with ammonium nitrogen (NH4) and with a mixture of amino acid and ammonium nitrogen (AAC). Compound

Ethyl 2-methylpropanoate [lg/l] Ethyl 2-methylbutanoate [lg/l] Ethyl 3-methylbutanoate [lg/l] Ethyl propanoate [lg/l] Ethyl butanoate [lg/l] Ethyl hexanoate [lg/l] Ethyl octanoate [lg/l] Ethyl decanoate [lg/l] Ethyl dodecanoate [lg/l] 2-Methylpropyl acetate [lg/l] 2-Methylbutyl acetate [lg/l] Hexyl acetate [lg/l] 3-Methylbutyl acetate [mg/l] Phenylethyl acetate [mg/l] Ethyl acetate [mg/l] 2-Methylpropanoic acid [mg/l] 2-Methylbutanoic acid [mg/l] 3-Methylbutanoic acid [mg/l] Hexanoic acid [mg/l] Octanoic acid [mg/l] Decanoic acid [mg/l] Acetic acid [mg/l] 2-Methylpropanol [mg/l] 2-Methylbutanol [mg/l] 3-Methylbutanol [mg/l] 2-Phenylethanol [mg/l] Hexanol [mg/l] Acetaldehyde [mg/l]

Condition Control (160)

NH4 (320)

AAC (320)

NH4 (480)

AAC (480)

8.0 ± 1.3a 3.6 ± 0.1a 6.3 ± 0.5a 546 ± 134a 179 ± 19a 531 ± 27a 336 ± 15a 201 ± 17a 206 ± 8a 14 ± 4a 145 ± 12a 92 ± 10a 2.5 ± 0.3a 0.7 ± 0.0a 36 ± 3a 2.5 ± 0.1a 1.7 ± 0.0a 1.8 ± 0.1a 1.2 ± 0.1a 1.4 ± 0.0a 0.8 ± 0.1a 13 ± 2a 29 ± 0a 104 ± 3a 321 ± 17a 137 ± 2a 1.3 ± 0.1a 0.4 ± 0.2a

0.0 ± 0.0b 0.7 ± 0.2c 2.3 ± 0.5b 221 ± 45b 499 ± 35b 824 ± 43b 886 ± 59b 939 ± 160b 312 ± 82ab 113 ± 11b 379 ± 50bc 300 ± 11c 6.9 ± 0.7b 1.0 ± 0.1b 62 ± 2c 1.2 ± 0.1c 0.3 ± 0.0c 0.5 ± 0.0c 3.0 ± 0.2c 4.2 ± 0.6b 2.6 ± 0.6c 53 ± 12c 24 ± 2bc 38 ± 4c 183 ± 10c 50 ± 5c 0.9 ± 0.0b 0.5 ± 0.2a

0.0 ± 0.0b 1.3 ± 0.3b 2.4 ± 0.5b 291 ± 46b 335 ± 42c 779 ± 76bc 884 ± 125b 1095 ± 160b 355 ± 34b 128 ± 19b 440 ± 37c 264 ± 19b 7.4 ± 0.6b 2.0 ± 0.2d 57 ± 2b 1.6 ± 0.2b 0.5 ± 0.1b 0.8 ± 0.1b 2.6 ± 0.2bc 4.2 ± 0.6b 2.8 ± 0.6c 34 ± 5b 25 ± 3b 50 ± 8b 210 ± 19b 95 ± 11b 0.9 ± 0.0b 1.6 ± 0.7b

0.5 ± 0.1b 0.5 ± 0.2c 2.0 ± 0.4c 314 ± 39b 357 ± 60c 664 ± 91c 716 ± 80c 399 ± 73c 504 ± 94c 190 ± 15c 344 ± 37b 321 ± 18bd 7.4 ± 0.4b 0.7 ± 0.1a 102 ± 2e 1.3 ± 0.1bc 0.1 ± 0.0d 0.3 ± 0.0d 2.3 ± 0.4b 3.5 ± 0.6b 1.4 ± 0.2ab 210 ± 13e 22 ± 1bc 22 ± 2d 132 ± 13d 28 ± 5d 0.7 ± 0.0c 0.6 ± 0.2a

0.0 ± 0.0b 0.6 ± 0.1c 1.5 ± 0.3c 277 ± 20b 398 ± 71c 735 ± 76bc 1028 ± 40d 707 ± 28d 852 ± 97d 196 ± 5c 671 ± 27d 335 ± 17d 9.8 ± 0.1c 1.5 ± 0.1c 91 ± 1d 1.3 ± 0.1c 0.2 ± 0.0c 0.46 ± 0.0cd 2.7 ± 0.3bc 4.2 ± 0.5b 1.8 ± 0.3b 78 ± 11d 22 ± 2c 37 ± 6c 175 ± 6c 51 ± 4c 0.7 ± 0.0c 2.3 ± 0.2c

Values with different superscripts are significantly different as shown by analysis of variance. YAN levels (mg N/l) are indicated in brackets. Bold values exceed respective sensory detection thresholds Amerine and Roessler (1976), Etievant (1991), Ferreira, Lopez, and Cacho (2000), Guth (1997), Meilgaard (1975), Salo (1970), Shinohara and Watanabe (1976), Siebert et al. (2005), Simpson (1979).

acid and ammonium nitrogen to have slightly higher ratings for ‘citrus’, ‘nail polish remover’, ‘acetic’ and ‘sweat’, and somewhat lower values for ‘floral’. By contrast, the high nitrogen concentration treatments (480 mg N/l) showed very different sensory profiles depending on the type of nitrogen added. Addition of ammonium nitrogen only led to wines with the highest ratings for the attributes ‘nail polish remover’ and ‘acetic’, while values for the fruit-related attributes were rated very low. The addition of amino acid and ammonium nitrogen at 480 mg N/l resulted in wines exhibiting significantly more intense ‘banana’, ‘fruit ester’, ‘musk’, ‘floral’ and ‘tropical’ aromas compared to the other treatments.

3.5. Correlation between volatile fermentation products and sensory descriptors The use of PLS models revealed specific correlations between volatile fermentation products and sensory attributes (Table 4). Calibration equations for each sensory attribute showed a coefficient of determination (R2) higher than 0.94, however only those calibrations explaining more than 85% of the variation were included in Table 4. Compounds positively associated with fruit and floral attributes and negatively associated with less desirable aroma attributes (‘bruised apple’, ‘stale beer’, ‘nail polish remover’, ‘acetic’ and ‘cheese’) included ethyl octanoate, ethyl dodecanoate, 2-methylpropyl acetate, 2-methylbutyl acetate, 3-methylbutyl acetate, phenylethyl acetate, 2-methylbutanol, 3-methylbutanol and 2-phenylethanol. Conversely, compounds correlating positively with less desirable aroma attributes and negatively associated with fruit and floral attributes included ethyl 2-methyl propanoate, ethyl acetate, octanoic acid and acetic acid.

4. Discussion Over recent decades considerable research has focused on the impact of nitrogen supplementation on yeast biomass and fermentation kinetics (see for example: Blateyron & Sablayrolles, 2001; Colombié et al., 2005; Cramer et al., 2002; Henschke & Jiranek, 1993) and more recently on the formation of flavour active compounds (see for example: Carrau et al., 2008; Guitart et al., 1999; Hernandez-Orte et al., 2005; Hernandez-Orte, Ibarz et al., 2006; Miller et al., 2007; Ugliano et al., 2008; Vilanova et al., 2007; Wang, Bohlscheid, & Edwards, 2003). However, little is known about the effect of nitrogen addition on the sensory profile of wine, apart from its role in influencing the development of hydrogen sulphide and other volatile sulphur compounds (Hernandez-Orte et al., 2005; Hernandez-Orte, Ibarz et al., 2006; Ugliano et al., 2009). In order to establish a practical understanding of the impact of nitrogen addition on wine aroma, a low nitrogen Chardonnay juice was supplemented with nitrogen in order to achieve a range of nitrogen concentrations that typically exist in different wine regions, and the resulting wines evaluated chemically and by quantitative descriptive sensory analysis. Two types of nitrogen supplements were studied, inorganic nitrogen in the form of ammonium chloride and organic nitrogen in the form of a mixture of amino acids and ammonium, which simulates the nitrogen composition of the Chardonnay juice used in this work. Data from the present study shows that the concentration of nitrogen and not the nitrogen source added is driving the increase in fermentation rate and biomass yield observed previously as a result of nitrogen supplementation (Bely, Sablayrolles, & Barre, 1990; Blateyron & Sablayrolles, 2001; Cramer et al., 2002; Salmon, 1989; Vilanova et al., 2007).

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Fig. 2. Total volatile fermentation products. (A) Total esters (excluding ethyl acetate), ethyl esters and acetate esters. (B) Total acids (excluding acetic acid), branched-chain acids and medium-chain fatty acids. (C) Total higher alcohols. Values not connected by same letter are significantly different as shown by analysis of variance.

Residual concentrations of most amino acids were not significantly different among treatments including the wines made from juices supplemented with a mixture of amino acid and ammonium

nitrogen. This observation suggests that it is the total concentration of nitrogen in the medium and not the concentration of individual amino acids that dictates the uptake of most amino acids. Indeed, similar findings have been reported when mixtures of amino acids were added to Merlot juice (Hernandez-Orte, Ibarz et al., 2006). Ammonium ion uptake is associated with the excretion of proton ions into the medium, thereby decreasing extracellular pH whereas amino acid uptake leads to a lower excretion of protons (Pena, Pardo, & Ramirez, 1987; Torija et al., 2003). This differential excretion explains the lower pH values observed for wines supplemented with only ammonium. Furthermore, the chloride anion is poorly accumulated by yeast and also has poor pH buffering capacity. The extent of pH change is related to the initial concentration of ammonium, types of organic acids and buffer capacity of the medium (Delfini & Parvex, 1989; Torija et al., 2003). Among the non-volatile products analysed, malic and succinic acids showed decreased concentration in wines supplemented with nitrogen (Bell & Henschke, 2005). The effect of nitrogen supplementation on succinic acid production is still unclear. Some articles have reported an increased succinate concentration at higher concentrations of assimilable nitrogen (Camarasa et al., 2003; Varela et al., 2004) while others, as observed in this work, have shown a decreased succinate concentration in response to nitrogen addition (Beltran, Esteve-Zarzoso, Rozes, Mas, & Guillamon, 2005). Nevertheless, the addition of some amino acids, such as glutamate, asparagine, proline, glutamine, threonine and c-aminobutyric acid that contribute to the citric acid pathway, has been shown to stimulate succinate production (Albers et al., 1996; Bach, Sauvage, Dequin, & Camarasa, 2009; Camarasa et al., 2003; Roustan & Sablayrolles, 2002). This may explain the higher concentrations of succinate in wines when juices were supplemented with a mixture of amino acid and ammonium rather than ammonium nitrogen alone. Although nitrogen addition has been shown to affect glycerol concentration when using AWRI 796 in chemically defined medium (Vilanova et al., 2007) and in Shiraz must (Ugliano et al., 2008), we did not observe any significant changes in glycerol in response to nitrogen supplementation in the present Chardonnay

Fig. 3. Mean aroma attribute intensity scores (n = 12 judges three presentation replicates two fermentation replicates) and Fisher’s least significant differences values (LSD, P = 0.05) for each of the wine treatments. A, control and ammonium nitrogen (NH4) supplementation. B, control and nitrogen supplementation with a mixture of amino acid and ammonium nitrogen (AAC).

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D. Torrea et al. / Food Chemistry 127 (2011) 1072–1083 Table 4 Regression coefficients of volatile fermentation products from PLS regression models for each individual sensory descriptor. Compound Ethyl 2-methylpropanoate Ethyl 3-methylbutanoate Ethyl propanoate Ethyl butanoate Ethyl octanoate Ethyl dodecanoate 2-Methylpropyl acetate 2-Methylbutyl acetate 3-Methylbutyl acetate Phenylethyl acetate Ethyl acetate Octanoic acid Acetic acid 2-Methylpropanol 2-Methylbutanol 3-Methylbutanol 2-Phenylethanol

Banana 0.19 0.03 0.04 0.07 0.16 0.14 0.00 0.23 0.16 0.06 0.03 0.03 0.15 0.02 0.10 0.13 0.01

Fruity ester 0.18 0.05 0.02 0.03 0.17 0.15 0.02 0.23 0.16 0.08 0.02 0.01 0.13 0.01 0.09 0.11 0.02

Artificial grape 0.18 0.05 0.02 0.03 0.17 0.15 0.02 0.23 0.16 0.08 0.02 0.01 0.13 0.01 0.09 0.11 0.02

Musk

Floral

0.19 0.01 0.06 0.08 0.15 0.12 0.02 0.21 0.14 0.05 0.05 0.04 0.17 0.04 0.11 0.14 0.01

0.19 0.01 0.06 0.08 0.15 0.12 0.02 0.21 0.14 0.05 0.05 0.04 0.17 0.04 0.11 0.14 0.01

Tropical 0.18 0.04 0.03 0.07 0.17 0.16 0.03 0.25 0.18 0.04 0.00 0.04 0.13 0.01 0.08 0.10 0.02

Citrus 0.02 0.12 0.19 0.15 0.12 0.12 0.14 0.14 0.15 0.20 0.09 0.19 0.05 0.18 0.01 0.02 0.12

Bruised apple 0.12 0.08 0.01 0.03 0.15 0.14 0.06 0.19 0.16 0.13 0.00 0.06 0.10 0.04 0.09 0.08 0.07

Stale beer 0.20 0.02 0.06 0.10 0.16 0.15 0.01 0.25 0.17 0.01 0.01 0.07 0.15 0.04 0.09 0.12 0.03

Nail polish remover 0.20 0.04 0.16 0.14 0.09 0.08 0.13 0.15 0.02 0.04 0.13 0.14 0.22 0.10 0.12 0.18 0.03

Acetic 0.20 0.04 0.16 0.14 0.09 0.08 0.13 0.15 0.02 0.04 0.13 0.14 0.22 0.10 0.12 0.18 0.03

Cheese 0.19 0.03 0.04 0.07 0.16 0.14 0.00 0.23 0.16 0.06 0.03 0.03 0.15 0.02 0.10 0.13 0.01

Regression coefficients obtained from the optimum number of principal components. Bold values indicate most relevant parameters that correlate with a particular sensory descriptor. Signs indicate positive or negative correlation. Only significant parameters and descriptors are shown as described in the text.

juice. Some other reports have shown that nitrogen addition has no effect on glycerol formation and that differences in glycerol concentration are related to the wine yeast used (Remize, Sablayrolles, & Dequin, 2000). Since glycerol formation is largely the result of stress response, particularly osmoregulation, and redox balance (Hohmann, 1997; Remize, Cambon, Barnavon, & Dequin, 2003), it is plausible that the physiological stresses related to particular musts could be responsible for these differing observations on the effect of nitrogen addition on glycerol formation. Nitrogen had a considerable impact on the concentration of all volatile fermentation products measured. The increase in acetic acid concentration in response to nitrogen supplementation by AWRI 796 is consistent with previous observations made with chemically defined media (Vilanova et al., 2007) whereas a small reduction was observed in fermentation of a high solids Shiraz must (Ugliano et al., 2008). With the Chardonnay juice, the addition of ammonium nitrogen resulted in larger increases in acetic acid than combined amino acid and ammonium nitrogen. The variable responses to added nitrogen appears to be influenced by several factors including the type of nitrogen source used, initial YAN content of the medium, yeast strain, pH, organic acids present and buffer capacity of the medium (Albers et al., 1996; Beltran et al., 2005; Delfini & Parvex, 1989; Hernandez-Orte, Ibarz et al., 2006; Torija et al., 2003). Higher alcohols were inversely related to initial nitrogen with the type of nitrogen source quantitatively affecting production. For equivalent YAN, the concentration of higher alcohols was larger in wines supplemented with a mixture of amino acid and ammonium nitrogen when compared to ammonium nitrogen alone. This pattern has been observed before and corresponds to changes in the relative activity of the biosynthetic pathways and the amino acid catabolic reactions responsible for the formation of the aketoacids (Äyräpää, 1968). Although derived directly from the corresponding higher alcohol through condensation with acetyl-CoA, the production of acetate esters was not related to higher alcohol production but more closely related to acetate. The increase in the formation of acetate esters in response to nitrogen supplementation is the result of higher expression of the genes encoding for alcohol acyl transferase enzymes (Verstrepen, Derdelinckx et al., 2003; Yoshimoto, Fukushige, Yonezawa, & Sone, 2002). The type of nitrogen source generally had little effect on production of most of the acetates, although amino acid plus ammonium nitrogen most strongly

influenced 2- and 3-methylbutyl acetate production. Presumably, the type of nitrogen source influences expression of the ester synthetic/hydrolytic genes. Consistent with previous findings, nitrogen addition resulted in a reduction in the concentration of branched-chain acids (Ugliano et al., 2008; Vilanova et al., 2007). A similar trend was observed for the corresponding ethyl esters but no consistent effect of nitrogen type was found. Although previously considered of minor importance for wine flavour, some ethyl esters of branched-chain acids can be important contributors to wine aroma due to their particularly low odour threshold (between 1 and 18 lg/l) (Francis & Newton, 2005). The decrease in the formation of the higher alcohols, branchchain acids and also ethyl esters of branched-chain acids in response to nitrogen addition is explained by a repression of amino acid biosynthesis, causing a reduced carbon flow through the Ehrlich pathway (Fig. 4). Recent findings have shown that the expression of the genes involved in amino acid biosynthesis (LEU1, LEU2, BAT1 and BAT2) is initially enhanced when nitrogen is increased from very low levels but is then repressed for further nitrogen additions (Yoshimoto et al., 2002). Furthermore, nitrogen supplementation has also been shown to upregulate the genes encoding for the branched-chain amino acid transporters (BAP2 and BAP3) (Marks, van der Merwe, & van Vuuren, 2003). Even though the available concentration of higher alcohols is lower, the upregulation of the alcohol acyl transferases (ATF1p and ATF2p) causes an increase in the formation of the corresponding acetate esters. In the light of present results showing that the type of nitrogen source can differentially affect synthesis of some higher alcohols, acids and esters, further studies on gene expression patterns seem warranted so that strategies to better control wine flavour profile can be devised. The increase in the concentration of MCFA ethyl esters as a consequence of nitrogen addition is consistent with previous reports (Saerens et al., 2008). Since the synthesis of MCFA ethyl esters is a direct result of substrate availability (Saerens et al., 2006, 2008) the increase in both MCFA and MCFA ethyl esters could be related to the relative increase in fatty acid synthesis due to nitrogen supplementation. However, with total YAN concentration being the same, addition of nitrogen in the form of a combination of amino acid and ammonium nitrogen resulted in MCFA ethyl esters concentrations that were generally higher than those obtained with ammonium alone. This result contrasts with others

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(Hernandez-Orte et al., 2005; Hernandez-Orte, Ibarz et al., 2006; Miller et al., 2007) who found little consistent pattern, indeed Miller et al. (2007) suggested that in must with high YAN addition of amino acids can reduce ester concentrations due to feedback inhibition suppressing amino acid uptake. At high YAN concentration, ammonium depressed accumulation of the longer chain esters (C8–C12) relative to combined amino acid and ammonium nitrogen. A trend was observed showing the depression at high nitrogen supplementation levels of MCFA (C6–C10) synthesis by ammonium relative to combined amino acid and ammonium nitrogen. The preferential effect of nitrogen source needs further investigation as a means for elevating the desirable fruity MCFA ethyl esters. Our results suggests that, in addition to the previously reported positive correlation between total nitrogen and esters formation (Ugliano et al., 2008; Vilanova et al., 2007), the composition of the nitrogen pool, in particular with regard to the ammonium/amino acids ratio, can influence ester production. Although direct amino acid addition is not allowed by current wine regulations, the ratio between ammonium and amino acids can depend on grape variety, vintage, and viticultural practices (Bell & Henschke, 2005). Some inactive dry yeast preparations are enriched in amino acid and peptides and might provide a means to modifying the aroma composition of wine (Pozo-Bayon, Andujar-Ortiz, & MorenoArribas, 2009). The yeast metabolic implications of these variables need further investigation. A comparison between the concentrations of fermentation volatile compounds and sensory panel ratings of aroma attributes in the present study indicated a correspondence between several of them. The low nitrogen control wine, which was generally rated low in the fruity and floral descriptors, had the lowest concentration of total esters and long chain fatty acids. By contrast, this wine had the highest concentration of higher alcohols and branched-chain acids, which are generally characterised by unpleasant amylic and cheese aromas, respectively. Overall, this type of aroma composition might have accounted for, at least in part, the fact that these wines were mainly characterised by less preferred aroma descriptors, such as cheese and sweat, stale beer and bruised apple. Although wines from the low nitrogen juice had the highest score for the two latter descriptors, the concentration of the typical staling compound acetaldehyde was the lowest in these wines. Bruised apple and stale aromas can also depend on the presence of other aldehydes, particularly (E)-2-alkenals (Culleré, Cacho, & Ferreirra, 2007), which were not measured here. Alternatively, it is possible that, by increasing fruity aromas, esters can reduce the perception of bruised apple characters. Wines with moderate concentrations of nitrogen had moderate to high concentrations of esters, moderate concentrations of higher alcohols and moderate to high MCFA concentrations, which correlates with the moderate to high ratings of fruity and floral attributes exhibited by these wines. Wines with high nitrogen concentration showed high concentrations of esters and the lowest concentration of acids and higher alcohols. However, when ammonium was used as nitrogen source at the higher nitrogen addition level, the resulting wine was scored low in the fruity and floral attributes and was dominated by the acetic and nail polish remover attributes, which are strongly associated with high concentrations of ethyl acetate and acetic acid. Although the results presented here were only obtained from a single Chardonnay juice fermented with one strain of S. cerevisiae, they suggest that the initial concentration of nitrogen in the juice not only has a considerable effect on the concentration of volatile fermentation aroma compounds but also impacts highly on the sensory or perceived profile of the wine. In addition, the form of available nitrogen can also affect sensory characteristics of the wines, with musts higher in amino acids resulting in wines with increased fruity aromas. This might be due to the

increased concentration of acetates and MCFA ethyl esters of these wines. From the sensory analysis it is clear that, in the case of ammonium nitrogen addition, a moderate nitrogen concentration in the juice gave rise to wines that had lower levels of sensory attributes that most wine producers would consider undesirable. These data suggest that there may be an optimum nitrogen concentration, or range, to give the most preferred wine aroma profile, at least in terms of a clean, fruity profile. However, grape variety, initial YAN concentration and yeast strain play an important role in determining the outcomes of nitrogen supplementation (Saenz-Navajas et al., 2010; Ugliano, Travis, Francis, & Henschke, 2010) and therefore a generalisation on the range of nitrogen concentrations that is optimal to wine aroma is still not possible at this stage. Interestingly, wines obtained from the addition of a mixture of amino acid and ammonium nitrogen at high concentration did not show a solvent character (nail polish remover), but produced wine with the highest rating of pleasant fruity aromas. This observation suggests that juices with naturally high amino acid content might produce wines with more intense fruity aroma profiles than low YAN juices. Further work is needed to show if the optimum nitrogen range changes depending on the type of nitrogen source used for different yeast strain and juice combinations. PLS models have been extensively used to correlate chemical compositional data to sensory data (Escudero et al., 2004; Ferreira, Fernandez, & Cacho, 1996; Ferreira, Ortin, Escudero, Lopez, & Cacho, 2002). In this work, most of the volatile fermentation products identified as relevant in explaining a particular sensory attribute have been associated to a similar sensory descriptor. For example, ethyl octanoate, ethyl dodecanoate, 2-methylpropyl acetate, 2-methylbutyl acetate, 3-methylbutyl acetate, phenylethyl acetate and 2-phenylethanol have been associated with the following sensory descriptors: fruity, pear, banana, lollies, roses and floral. Similarly, ethyl acetate and acetic acid associated with the descriptors nail polish remover and vinegar, respectively, correlated with the attributes nail polish remover and acetic. Interestingly, 2-methylbutanol and 3-methylbutanol, which have been described as having undesirable sensory descriptors, correlated with fruity and floral attributes, whereas ethyl 2-methyl propanoate associated with fruity and berry-like descriptors correlated with less desirable sensory attributes. These associations could either be the result of a masking effect by other compounds (measured or unmeasured) or indicate that we need to revisit the aroma descriptors associated to these compounds. In summary, supplementation of a low YAN Chardonnay juice with either ammonium nitrogen or a mixture of amino acid and ammonium nitrogen not only affected yeast growth and fermentation kinetics, non-volatile and volatile compounds composition but also perceived wine aroma. The type of nitrogen source resulted in quantitative differences for most of the yeast aroma and flavour compounds studied. In general, acetate esters, MCFA ethyl esters and their corresponding acids increased whereas branched chain ethyl esters and their corresponding acids and alcohols decreased. Amino acid and ammonium nitrogen compared with ammonium nitrogen alone favoured production of ethyl and acetate esters whereas ammonium nitrogen favoured acetic acid and its ethyl acetate. Ammonium nitrogen led to greater reduction in higher alcohols. Sensory evaluation revealed that 14 of the 16 aroma descriptors were affected by nitrogen supplementation quantity and type. Wines made with low assimilable nitrogen and those with high ammonium nitrogen presented clear sensory deficiencies. The former wines, which were rated high for less preferred sensory attributes, were also high in higher alcohols and branch-chain acids. The latter wines were rated high for volatile acidity attributes reflecting high concentrations of ethyl acetate and acetic acid.


Fig. 4. Concentrations of the main volatile fermentation products associated with the Ehrlich pathway. aKG, a-ketoglutarate; Glu, glutamic acid.

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Wines made with moderate nitrogen presented a more desirable sensory profile, while wines made with high amino acid and ammonium nitrogen exhibited the highest intensity of most of the desirable sensory attributes, which correlated with the highest concentrations of acetate and medium chain fatty acid esters. Wines rated high for the positive fruity and floral sensory attributes were high in esters and medium chain fatty acids and low in higher alcohols. Further work is required to confirm the generality of these effects with different combinations of yeast and grape varieties. Acknowledgements This work was financially supported by a grant from the Spanish Government. Research at The Australian Wine Research Institute is supported by Australia’s grapegrowers and winemakers through their investment agency the Grape and Wine Research and Development Corporation, with matching funds from the Australian Government. The AWRI is part of the Wine Innovation Cluster. We thank Briony Liebich and Kate Lattey for the conduct of the sensory study. Assistance with analysis of fermentation volatiles compounds by Tracey Siebert is greatly appreciated. References Albers, E., Larsson, C., Liden, G., Niklasson, C., & Gustafsson, L. (1996). Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation. Applied and Environmental Microbiology, 62(9), 3187–3195. Alexandre, H., & Charpentier, C. (1998). Biochemical aspects of stuck and sluggish fermentation in grape must. Journal of Industrial Microbiology & Biotechnology, 20(1), 20–27. Amerine, M. A., & Roessler, E. B. (1976). Composition of wines. In M. A. Amerine & E. B. Roessler (Eds.), Wines – their sensory evaluation (pp. 72–77). New York: W H Freeman. Äyräpää, T. (1968). Formation of higher alcohols by various yeasts. Journal of the Institute of Brewing, 74, 169–178. Äyräpää, T. (1971). Biosynthetic formation of higher alcohols by yeast. Dependence on the nitrogen nutrient level of the medium. Journal of the Institute of Brewing, 77, 266–275. Aznar, M., Lopez, R., Cacho, J., & Ferreira, V. (2003). Prediction of aged red wine aroma properties from aroma chemical composition. Partial least squares regression models. Journal of Agricultural and Food Chemistry, 51(9), 2700–2707. Bach, B., Sauvage, F., Dequin, S., & Camarasa, C. (2009). Role of c-aminobutyric acid as a source of nitrogen and succinate in wine. American Journal of Enology and Viticulture, 60(4), 508–516. Bell, S. J., & Henschke, P. A. (2005). Implications of nitrogen nutrition for grapes, fermentation and wine. Australian Journal of Grape and Wine Research, 11(3), 242–295. Beltran, G., Esteve-Zarzoso, B., Rozes, N., Mas, A., & Guillamon, J. M. (2005). Influence of the timing of nitrogen additions during synthetic grape must fermentations on fermentation kinetics and nitrogen consumption. Journal of Agricultural and Food Chemistry, 53(4), 996–1002. Bely, M., Rinaldi, A., & Dubourdieu, D. (2003). Influence of assimilable nitrogen on volatile acidity production by Saccharomyces cerevisiae during high sugar fermentation. Journal of Bioscience and Bioengineering, 96, 507–512. Bely, M., Sablayrolles, J., & Barre, P. (1990). Description of alcoholic fermentation kinetics – Its variability and significance. American Journal of Enology and Viticulture, 41(4), 319–324. Bisson, L. (1999). Stuck and sluggish fermentations. American Journal of Enology and Viticulture, 50(1), 107–119. Blateyron, L., & Sablayrolles, J. (2001). Stuck and slow fermentations in enology: Statistical study of causes and effectiveness of combined additions of oxygen and diammonium phosphate. Journal of Bioscience and Bioengineering, 91(2), 184–189. Butzke, C. (1998). Survey of yeast assimilable nitrogen status in musts from California, Oregon, and Washington. American Journal of Enology and Viticulture, 49, 220–224. Camarasa, C., Grivet, J. P., & Dequin, S. (2003). Investigation by 13C-NMR and tricarboxylic acid (TCA) deletion mutant analysis of pathways for succinate formation in Saccharomyces cerevisiae during anaerobic fermentation. Microbiology-SGM, 149, 2669–2678. Carrau, F. M., Medina, K., Farina, L., Boido, E., Henschke, P. A., & Dellacassa, E. (2008). Production of fermentation aroma compounds by Saccharomyces cerevisiae wine yeasts: Effects of yeast assimilable nitrogen on two model strains. FEMS Yeast Research, 8(7), 1196–1207. Colombié, S., Malherbe, S., & Sablayrolles, J. M. (2005). Modeling alcoholic fermentation in enological conditions: Feasibility and interest. American Journal of Enology and Viticulture, 56, 238–245.

Cramer, A., Vlassides, S., & Block, D. (2002). Kinetic model for nitrogen-limited wine fermentations. Biotechnology and Bioengineering, 77(1), 49–60. Culleré, L., Cacho, J., & Ferreirra, V. (2007). An assessment of the role played by some oxidation-related aldehydes in wine aroma. Journal of Agricultural and Food Chemistry, 55, 876–881. Delfini, C., & Parvex, C. (1989). Study on pH and total acidity modifications during alcoholic fermentation. Importance of the ammoniacal salt added. Rivista di Viticoltura e di Enologia, 42(2), 43–56. Dickinson, J. R., Lanterman, M. M., Danner, D. J., Pearson, B. M., Sanz, P., Harrison, S. J., et al. (1997). A 13C nuclear magnetic resonance investigation of the metabolism of leucine to isoamyl alcohol in Saccharomyces cerevisiae. Journal of Biological Chemistry, 272(43), 26871–26878. Eden, A., Van Nedervelde, L., Drukker, M., Benvenisty, N., & 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(3), 296–300. Escudero, A., Gogorza, B., Melus, M. A., Ortin, N., Cacho, J., & Ferreira, V. (2004). Characterization of the aroma of a wine from Maccabeo. Key role played by compounds with low odor activity values. Journal of Agricultural and Food Chemistry, 52(11), 3516–3524. Etievant, P. X. (1991). Wine. In H. Maarse (Ed.), Volatile compounds of food and beverages (pp. 483–546). New York: Marcel Dekker, Inc. Ferreira, V., Fernandez, P., & Cacho, J. F. (1996). A study of factors affecting wine volatile composition and its application in discriminant analysis. Food Science and Technology-Lebensmittel-Wissenschaft & Technologie, 29(3), 251–259. Ferreira, V., Fernandez, P., Pena, C., Escudero, A., & Cacho, J. F. (1995). Investigation on the role played by fermentation esters in the aroma of young spanish wines by multivariate-analysis. Journal of the Science of Food and Agriculture, 67(3), 381–392. Ferreira, A. C. S., Lopez, R., & 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. Ferreira, V., Ortin, N., Escudero, A., Lopez, R., & Cacho, J. (2002). Chemical characterization of the aroma of Grenache rose wines: Aroma extract dilution analysis, quantitative determination, and sensory reconstitution studies. Journal of Agricultural and Food Chemistry, 50(14), 4048–4054. Francis, I. L., & Newton, J. L. (2005). Determining wine aroma from compositional data. Australian Journal of Grape and Wine Research, 11(2), 114–126. Garde-Cerdan, T., & Ancin-Azpilicueta, C. (2008). Effect of the addition of different quantities of amino acids to nitrogen-deficient must on the formation of esters, alcohols, and acids during wine alcoholic fermentation. LWT-Food Science and Technology, 41(3), 501–510. Guitart, A., Orte, P. H., Ferreira, V., Pena, C., & Cacho, J. (1999). Some observations about the correlation between the amino acid content of musts and wines of the chardonnay variety and their fermentation aromas. American Journal of Enology and Viticulture, 50(3), 253–258. Guth, H. (1997). Quantitation and sensory studies of character impact odorants of different white wine varieties. Journal of Agricultural and Food Chemistry, 45(8), 3027–3032. Hagen, K. M., Keller, M., & Edwards, C. G. (2008). Survey of biotin, pantothenic acid, and assimilable nitrogen in winegrapes from the Pacific Northwest. American Journal of Enology and Viticulture, 59(4), 432–436. Henschke, P. A. (1997). Wine yeast. In F. Zimmermann & K. Entian (Eds.), Yeast sugar metabolism: biochemistry, genetics, biotechnology, and applications (pp. 527–560). Lancaster, PA: Technomic. Henschke, P. A., & Jiranek, V. (1993). Metabolism of nitrogen compounds. In G. Fleet (Ed.), Wine microbiology and biotechnology (pp. 77–164). Harwood Academic Publishers. Hernandez-Orte, P., Bely, M., Cacho, J., & 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(2), 150–160. Hernandez-Orte, P., Ibarz, M. J., Cacho, J., & Ferreira, V. (2005). Effect of the addition of ammonium and amino acids to musts of Airen variety on aromatic composition and sensory properties of the obtained wine. Food Chemistry, 89(2), 163–174. Hernandez-Orte, P., Ibarz, M. J., Cacho, J., & Ferreira, V. (2006). Addition of amino acids to grape juice of the Merlot variety: Effect on amino acid uptake and aroma generation during alcoholic fermentation. Food Chemistry, 98(2), 300–310. Hohmann, S. (1997). Shaping up: the response of yeast to osmotic stress. In S. Homann & W. Mager (Eds.), Yeast stress response (pp. 101–145). Austin: R.G. Landes. Ingledew, W., Magnus, C., & Sosulski, F. (1987). Influence of oxygen on proline utilization during the wine fermentation. American Journal of Enology and Viticulture, 38, 246–248. Jiranek, V., Langridge, P., & Henschke, P. A. (1995a). Amino acid and ammonium utilization by Saccharomyces cerevisiae wine yeasts from a chemically-defined medium. American Journal of Enology and Viticulture, 46(1), 75–83. Jiranek, V., Langridge, P., & Henschke, P. A. (1995b). Regulation of hydrogen sulfide liberation in wine producing Saccharomyces cerevisiae strains by assimilable nitrogen. Applied and Environmental Microbiology, 61(2), 461–467. Lambrechts, M. G., & Pretorius, I. S. (2000). Yeast and its importance to wine aroma. South African Journal of Enology and Viticulture, 21, 97–129. Marchesini, S., & Poirier, Y. (2003). Futile cycling of intermediates of fatty acid biosynthesis toward peroxisomal beta-oxidation in Saccharomyces cerevisiae. Journal of Biological Chemistry, 278(35), 32596–32601.

D. Torrea et al. / Food Chemistry 127 (2011) 1072–1083 Marks, V., van der Merwe, G., & van Vuuren, H. (2003). Transcriptional profiling of wine yeast in fermenting grape juice. Regulatory effect of diammonium phosphate. FEMS Yeast Research, 3(3), 269–287. Martens, H., & Dardenne, P. (1998). Validation and verification of regression in small data sets. Chemometrics and Intelligent Laboratory Systems, 44, 99–106. Meilgaard, M. (1975). Flavour chemistry of Beer, part II: Flavour and threshold of 239 aroma volatiles. Technical quarterly – Master Brewers Association of the Americas, 12, 151–168. Melucci, D., Xie, M., Reschiglian, P., & Torsi, G. (1999). FMOC-Cl as derivatizing agent for the analysis of amino acids and dipeptides by the absolute analysis method. Chromatographia, 49(5–6), 317–320. Miller, A. C., Wolff, S. R., Bisson, L. F., & Ebeler, S. E. (2007). Yeast strain and nitrogen supplementation: Dynamics of volatile ester production in Chardonnay juice fermentations. American Journal of Enology and Viticulture, 58(4), 470–483. Nicolini, G., Larcher, R., & Versini, G. (2004). Status of yeast assimilable nitrogen in Italian grape musts and effect of variety, ripening and vintage. Vitis, 43, 89–96. Nissen, T., Schulze, U., Nielsen, J., & Villadsen, J. (1997). Flux distributions in anaerobic, glucose-limited continuous cultures of Saccharomyces cerevisiae. Microbiology, 143, 203–218. Pena, A., Pardo, J. P., & Ramirez, J. (1987). Early metabolic effects and mechanism of ammonium transport in yeast. Archives of Biochemistry and Biophysics, 253(2), 431–438. Pozo-Bayon, M., Andujar-Ortiz, I., & Moreno-Arribas, M. (2009). Scientific evidences beyond the application of inactive dry yeast preparations in winemaking. Food Research International, 42, 754–761. Rankine, B., & Pocock, K. (1970). Alkalimetric determination of sulphur dioxide in wine. Australian Wine, Brewing & Spirit Review, 88, 40–44. Rapp, A., & Mandery, H. (1986). Wine Aroma. Experientia, 42(8), 873–884. Remize, F., Cambon, B., Barnavon, L., & Dequin, S. (2003). Glycerol formation during wine fermentation is mainly linked to Gpd1p and is only partially controlled by the HOG pathway. Yeast, 20, 1243–1253. Remize, F., Sablayrolles, J., & Dequin, S. (2000). Re-assessment of the influence of yeast strain and environmental factors on glycerol production in wine. Journal of Applied Microbiology, 88(3), 371–378. Roustan, J. L., & Sablayrolles, J. M. (2002). Impact of the addition of electron acceptors on the by-products of alcoholic fermentation. Enzyme and Microbial Technology, 31(1–2), 142–152. Saenz-Navajas, M. P., Campo, E., Cullere, L., Fernandez-Zurbano, P., Valentin, D., & Ferreira, V. (2010). Effects of the nonvolatile matrix on the aroma perception of wine. Journal of Agricultural and Food Chemistry, 58(9), 5574–5585. Saerens, S. M. G., Delvaux, F., Verstrepen, K. J., Van Dijck, P., Thevelein, J. M., & Delvaux, F. R. (2008). Parameters affecting ethyl ester production by Saccharomyces cerevisiae during fermentation. Applied and Environmental Microbiology, 74(2), 454–461. Saerens, S. M. G., Verstrepen, K. J., Van Laere, S. D. M., Voet, A. R. D., Van Dijck, P., Delvaux, F. R., et al. (2006). The Saccharomyces cerevisiae EHT1 and EEB1 genes encode novel enzymes with medium-chain fatty acid ethyl ester synthesis and hydrolysis capacity. Journal of Biological Chemistry, 281(7), 4446–4456. Salmon, J. (1989). Effect of sugar transport inactivation in Saccharomyces cerevisiae on sluggish and stuck enological fermentations. Applied Environmental Microbiology, 55(4), 953–958. Salmon, J. (1996). Sluggish and stuck fermentations: Some actual trends on their physiological basis. Viticultural and Ecological Sciences, 51, 137–140. Salo, P. (1970). Determining the odor thresholds for some compounds in alcoholic beverages. Journal of Food Science, 35, 95–99. Shinohara, T., & Watanabe, M. (1976). Gas chromatographic analysis of higher alcohols and ethyl acetate in table wines. Agricultural and Biological Chemistry, 40, 2475–2477.

1083

Siebert, T., Smyth, H. E., Capone, D. L., Neuwöhner, C., Herderich, M. J., Sefton, M. A., et al. (2005). Stable isotope dilution analysis of wine fermentation products by HS-SPME-GC–MS. Analytical and Bioanalytical Chemistry, 381, 937–947. Simpson, R. F. (1979). Some important aroma compounds of white wine. Food Technology in Australia, 31, 516–522. Skouroumounis, G. K., Kwiatkowski, M., Sefton, M. A., Gawel, R., & Waters, E. J. (2003). In situ measurement of white wine absorbance in clear and in coloured bottles using a modified laboratory spectrophotometer. Australian Journal of Grape and Wine Research, 9(2), 138–147. Sumby, K. M., Grbin, P. R., & Jiranek, V. (2010). Microbial modulation of aromatic esters in wine: Current knowledge and future prospects. Food Chemistry, 121(1), 1–16. Torija, M. J., Beltran, G., Novo, M., Poblet, M., Rozes, N., Mas, A., et al. (2003). Effect of organic acids and nitrogen source on alcoholic fermentation: Study of their buffering capacity. Journal of Agricultural and Food Chemistry, 51(4), 916–922. Ugliano, M., Fedrizzi, B., Siebert, T., Travis, B., Magno, F., Versini, G., et al. (2009). Effect of nitrogen supplementation and Saccharomyces species on hydrogen sulfide and other volatile sulfur compounds in Shiraz fermentation and wine. Journal of Agricultural and Food Chemistry, 57(11), 4948–4955. Ugliano, M., Siebert, T., Mercurio, M., Capone, D., & Henschke, P. A. (2008). Volatile and color composition of young and model-aged Shiraz wines as affected by diammonium phosphate supplementation before alcoholic fermentation. Journal of Agricultural and Food Chemistry, 56(19), 9175–9182. Ugliano, M., Travis, B., Francis, I. L., & Henschke, P. (2010). Volatile composition and sensory properties of Shiraz wines as affected by nitrogen supplementation and yeast species: Rationalizing nitrogen modulation of wine aroma. Journal of Agricultural and Food Chemistry, 58(23), 12417–12425. Varela, C., Pizarro, F., & Agosin, E. (2004). Biomass content governs fermentation rate in nitrogen-deficient wine musts. Applied and Environmental Microbiology, 70(6), 3392–3400. Verstrepen, K. J., Derdelinckx, G., Dufour, J. P., Winderickx, J., Pretorius, I. S., Thevelein, J. M., et al. (2003). The Saccharomyces cerevisiae alcohol acetyl transferase gene ATF1 is a target of the cAMP/PKA and FGM nutrient-signalling pathways. FEMS Yeast Research, 4(3), 285–296. Verstrepen, K. J., Van Laere, S. D. M., Vanderhaegen, B. M. P., Derdelinckx, G., Dufour, J. P., Pretorius, I. S., et al. (2003). Expression levels of the yeast alcohol acetyltransferase genes ATF1, Lg-ATF1, and ATF2 control the formation of a broad range of volatile esters. Applied and Environmental Microbiology, 69(9), 5228–5237. Vilanova, M., Ugliano, M., Varela, C., Siebert, T., Pretorius, I. S., & Henschke, P. A. (2007). Assimilable nitrogen utilisation and production of volatile and nonvolatile compounds in chemically defined medium by Saccharomyces cerevisiae wine yeasts. Applied Microbiology and Biotechnology, 77(1), 145–157. Vos, P. J. A., & Gray, R. S. (1979). Origin and control of hydrogen sulfide during fermentation of grape must. American Journal of Enology and Viticulture, 30(3), 187–197. Wagener, W., & Wagener, G. (1968). The influence of esters and fusel alcohol content upon the quality of dry white wines. South African Journal of Science, 11, 469–476. Wang, X., Bohlscheid, J., & Edwards, C. (2003). Fermentative activity and production of volatile compounds by Saccharomyces grown in synthetic grape juice media deficient in assimilable nitrogen and/or pantothenic acid. Journal of Applied Microbiology, 94(3), 349–359. Yoshimoto, H., Fukushige, T., Yonezawa, T., & Sone, H. (2002). Genetic and physiological analysis of branched-chain alcohols and isoamyl acetate production in Saccharomyces cerevisiae. Applied Microbiology and Biotechnology, 59(4–5), 501–508.