Appl Microbiol Biotechnol (2007) 77:675–687 DOI 10.1007/s00253-007-1194-3
APPLIED MICROBIAL AND CELL PHYSIOLOGY
Influence of wine fermentation temperature on the synthesis of yeast-derived volatile aroma compounds Ana M. Molina & Jan H. Swiegers & Cristian Varela & Isak S. Pretorius & Eduardo Agosin
Received: 8 June 2007 / Revised: 17 August 2007 / Accepted: 6 September 2007 / Published online: 16 October 2007 # Springer-Verlag 2007
Abstract The yeast Saccharomyces cerevisiae synthesises a variety of volatile aroma compounds during wine fermentation. In this study, the influence of fermentation temperature on (1) the production of yeast-derived aroma compounds and (2) the expression of genes involved in aroma compounds’ metabolism (ADH1, PDC1, BAT1, BAT2, LEU2, ILV2, ATF1, ATF2, EHT1 and IAH1) was assessed, during the fermentation of a defined must at 15 and 28°C. Higher concentrations of compounds related to fresh and fruity aromas were found at 15°C, while higher concentrations of flowery related aroma compounds were found at 28°C. The formation rates of volatile aroma compounds varied according to growth stage. In addition, linear correlations between the increases in concentration of higher alcohol and their corresponding acetates were obtained. Genes presented different expression profiles at both temperatures, except ILV2, and those involved in common pathways were co-expressed (ADH1, PDC1 and BAT2; and ATF1, EHT1 and IAH1). These results demonstrate that the fermentation temperature plays an important role in the wine final aroma profile, and is therefore an important control parameter to fine-tune wine quality during winemaking. Keywords Fermentation temperature . Wine aroma . Wine fermentation . Wine yeast A. M. Molina : E. Agosin (*) Departamento de Ingeniería Química y Bioprocesos, Facultad de Ingeniería, Pontificia Universidad Católica de Chile, Casilla 306 Correo 22, Santiago, Chile e-mail:
[email protected] J. H. Swiegers : C. Varela : I. S. Pretorius The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, Adelaide, SA 5064, Australia
Introduction As far as consumers are concerned, the aroma and flavour of wine are among the main characteristics that determine its quality and value (Swiegers et al. 2005). The aroma of wines is a unique mixture of volatile compounds originated from grapes (varietal aromas), secondary products formed during the wine fermentation (fermentative aromas) and ageing (post-fermentative aromas; Lambrechts and Pretorius 2000; Swiegers and Pretorius 2005). The aroma complexity dramatically increases during alcoholic fermentation as a result of the synthesis of important volatile compounds by the wine yeast Saccharomyces cerevisiae and the release of some varietal aroma precursors (Swiegers et al. 2005). The nature and amount of the synthesised volatile compounds depend on multiple factors, such as the nitrogen content of the must, the temperature of fermentation and the yeast strain (Lambrechts and Pretorius 2000; Swiegers et al. 2006). The volatile compounds synthesised by wine yeasts include higher alcohols (fusel, marzipan and floral aromas), medium- and long-chain volatile acids (fatty, cheesy and sweaty aromas), acetate esters and ethyl esters (fruity and floral aromas) and aldehydes (buttery, fruity and nutty aromas), among others (Delfini et al. 2001; Lambrechts and Pretorius 2000; Stashenko et al. 1992). Higher alcohols can be synthesised either from intermediates of sugar metabolism, through anabolic reactions, or from branched-chain amino acids, through a multi-step catabolic reaction, the Ehrlich pathway (Boulton et al. 1996; Dickinson et al. 1997, 2003; Eden et al. 2001). The volatile fatty acids also contribute to the aroma of wines. Fatty acids are essential constituents of the plasma membrane and precursors of more complex molecules, such as phospholipids. They are synthesised through the repetitive condensation of acetylCoA, catalysed by the fatty acid synthetase complex
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(Lambrechts and Pretorius 2000). Finally, ester compounds are produced by condensation of an alcohol and a coenzyme-A-activated acid (acyl-CoA). Hence, in S. cerevisiae, acetate esters result from the combination of acetyl-CoA with an alcohol, by the action of the alcohol acetyl transferases Atf1p and Atf2p (Lambrechts and Pretorius 2000). Correspondingly, ethyl esters are generated from acyl-CoA and ethanol by the action of Eht1p, Eeb1p and probably other unknown enzymes (Mason and Dufour 2000; Saerens et al. 2006). The capacity of yeast to synthesise these compounds varies between different strains (Lambrechts and Pretorius 2000; Wondra and Boveric 2001). Although their exact contribution is not completely clear, grape variety and the temperature of fermentation are additional variables that affect the final concentration of yeast-derived aroma compounds in wine (Aragon et al. 1998; Gómez-Míguez et al. 2007; Guth 1997; Torija et al. 2003). In general, white wines are produced at lower fermentation temperatures than red wines to preserve the fresh and fruity characters desirable in young white wines. In fact, it has been shown that higher concentrations of esters, responsible for some fruity characters in wine, are obtained at lower temperatures of fermentation. This is due to increased stability of volatile compounds, reduced evaporative loss, and the differential metabolism of the yeast due to altered fatty acid biosynthesis to modify the composition of the cell membrane (Boulton et al. 1996; Killian and Ough 1979; Torija et al. 2003; Walker 1998). Although the fermentation temperature significantly affects the yeast growth rate and its central metabolism (Beltran et al. 2006), the impact of fermentation temperature on the yeast biosynthetic pathways of aroma-enhancing compounds is still not fully understood. The aim of this study was to quantify the differences on the synthesis of the yeast-derived volatile compounds resulting exclusively from the difference in fermentation temperature conditions between white (15°C) and red (28°C) wines. Volatile compounds were quantified at different stages of the fermentation and compared to the expression of ten genes involved in aroma biosynthetic pathways. The wine yeast S. cerevisiae EC1118 was selected for this study because it exhibits a good ability to ferment over a wide temperature range. In this paper, we provide renewed evidence of the contribution of temperature to the synthesis of volatile compounds by the wine yeast S. cerevisiae.
Appl Microbiol Biotechnol (2007) 77:675–687
cultures were grown in 3 ml of YPD medium (1% yeast extract, 2% peptone and 2% glucose) at 28°C under aerobic conditions. Modified MS300 medium, simulating standard grape juice, was used in bioreactor fermentations (Varela et al. 2004). The initial sugar concentration was 240 g/l (120 g/l of glucose and 120 g/l of fructose), and the yeast assimilable nitrogen concentration (YAN), 300 mg/l (ammonium and amino acids). Anaerobic factors were added to the medium: 15 mg/l ergosterol, 5 mg/l sodium oleate and 0.5 ml/l Tween 80, dissolved in 5 ml of ethanol. The inoculum was grown in shake flasks containing the modified MS300 medium with half the amount of sugar (60 g/l of glucose and fructose) at 25°C under aerobic conditions (no anaerobic factors were included). Fermentation conditions and sampling Fermentations were run in triplicate in 1-l Bioflo 110 bioreactors (New Brunswick Scientific, Edison, NJ, USA) with 800 ml of artificial must. Before inoculation, nitrogen was sparged to eliminate the oxygen from the medium. Temperature was maintained at either 15 or 28°C, pH at 3.5 and agitation at 120 rpm. The bioreactors were inoculated with 106 cells/ml. Samples of 15 ml of culture medium were taken at different stages of the fermentation from each bioreactor to determine cell number, optical density measured at 600 nm (OD600), dry cell weight (DCW) and for the chemical analysis of the supernatant (residual sugar, organic acids and volatile fermentation products). DCW was estimated by filtering 10 ml of medium through a Supor®-200-membrane filter (0.2-μm pore diameter, Pall Life Sciences, Melbourne, Australia), washing twice with distilled water and drying to a constant weight at 85°C. Supernatant samples were stored at −20°C and analysed later to determine concentration of glucose, fructose, organic acids, volatile fermentation products, ammonia and free amino acid nitrogen (FAN). For RNA isolation, the volume containing 2×107 cells was collected and centrifuged for 3 min at 3,000 rpm. The pellet was immediately resuspended in 600 μl of Buffer RLT (RNeasy Mini Kit, QIAGEN, Doncastor, Australia), transferred to a screw-capped tube containing 800 μl of acid-washed RNase-free glass beads. Cells were broken down in a bead beater and then centrifuged at 13,000 rpm for 3 min. The supernatant was frozen at −80°C until use. Analytical techniques
Materials and methods Yeast strain and growth conditions The commercial wine yeast S. cerevisiae Prise de Mousse EC1118 strain (Lalvin, Zug, Switzerland) was used. Initial
The concentrations of glucose, fructose, ethanol, glycerol, malic acid, citric acid and acetic acid were measured by high-performance liquid chromatography using a Bio-Rad HPX-87H column. Ammonia concentration was measured enzymatically using the Glutamate Dehydrogenase Enzymatic
Appl Microbiol Biotechnol (2007) 77:675–687
Bioanalysis UV-method (Roche, Mannheim, Germany). The FAN concentration was determined by using the o-phtaldehyde/ N-acetyl-L-cysteine spectrophotometric assay (NOPA) using a Roche Cobas FARA spectrophotometric auto-analyser (Roche, Basel, Switzerland). The YAN was obtained by adding the FAN value and 82.25% of the ammonia concentration. HS-SPME-GC-MS using SIDA Volatile compounds synthesised during fermentation were quantified 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 elsewhere (Siebert et al. 2005). Thirty-one volatile compounds were analysed, including ethyl- and acetate-esters, acids and alcohols. Small amounts of the following volatile compounds were detected in the must before inoculation: 2methyl propanol (1.38 mg/l), ethyl dodecanoate (0.04 mg/l), acetic acid (19.6 mg/l), hexanoic acid (0.16 mg/l), octanoic acid (0.33 mg/l), decanoic acid (0.25 mg/l) and ethanol (4 g/l). They were probably generated from the anaerobic factors fraction used in the preparation of artificial must (Tween 80 and oleic acid dissolved in ethanol). Possible correlations between the concentration of ethyland acetate-esters and their alcohol and acid precursors were analysed by multiple-variable analysis (P
