Jan H. Swiegers

Yeast Modulation of Wine Flavor

Publisher Summary This chapter reviews the scientific knowledge of the role of microorganisms, especially yeast, in the development of wine flavor. Specific attention is given to the contribution of esters, higher alcohols, volatile thiols, volatile phenols, and monoterpenoids to the flavor profile. It is well known that grapes of different cultivars display characteristic aromas that are distinctive of the wines. However, it can be shown that although some volatile aroma substances arise from components of the grapes, many of these compounds are changed and a substantial portion of wine flavor substances are formed during yeast fermentation. Therefore, wine has more flavor than the grape juice it is fermented from, and the importance of yeast and other wine-related microorganisms is central to the development of wine flavor. Furthermore, different biosynthetic pathways are interactive during the formation of the aroma of alcoholic beverages, and different factors play their part in the formation of the total aroma.

Modulation of volatile sulfur compounds by wine yeast

Sulfur compounds in wine can be a ‘double-edged sword’. On the one hand, certain sulfur-containing volatile compounds such as hydrogen sulfide, imparting a rotten egg-like aroma, can have a negative impact on the perceived quality of the wine, and on the other hand, some sulfur compounds such as 3-mercaptohexanol, imparting fruitiness, can have a positive impact on wine flavor and aroma. Furthermore, these compounds can become less or more attractive or repulsive depending on their absolute and relative concentrations. This presents an interesting challenge to the winemaker to modulate the concentrations of these quality-determining compounds in wine in accordance with consumer preferences. The wine yeast Saccharomyces cerevisiae plays a central role in the production of volatile sulfur compounds. Through the sulfate reduction sequence pathway, the HS- is formed, which can lead to the formation of hydrogen sulfide and various mercaptan compounds. Therefore, limiting the formation of the HS- ion is an important target in metabolic engineering of wine yeast. The wine yeast is also responsible for the transformation of non-volatile sulfur precursors, present in the grape, to volatile, flavor-active thiol compounds. In particular, 4-mercapto-4-methylpentan-2-one, 3-mercaptohexanol, and 3-mercaptohexyl acetate are the most important volatile thiols adding fruitiness to wine. This paper briefly reviews the metabolic processes involved in the production of important volatile sulfur compounds and the latest strategies in the pursuit of developing wine yeast strains as tools to adjust wine aroma to market specifications.

Influence of wine fermentation temperature on the synthesis of yeast-derived volatile aroma compounds

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.

The effect of increased yeast alcohol acetyltransferase and esterase activity on the flavour profiles of wine and distillates

The fruity odours of wine are largely derived from the synthesis of esters and higher alcohols during yeast fermentation. The ATF1‐ and ATF2‐encoded alcohol acetyltransferases of S. cerevisiae are responsible for the synthesis of ethyl acetate and isoamyl acetate esters, while the EHT1‐encoded ethanol hexanoyl transferase is responsible for synthesizing ethyl caproate. However, esters such as these might be degraded by the IAH1‐encoded esterase. The objectives of this study were: (a) to overexpress the genes encoding ester‐synthesizing and ester‐degrading enzymes in wine yeast; (b) to prepare Colombard table wines and base wines for distillation using these modified strains; and (c) to analyse and compare the ester concentrations and aroma profiles of these wines and distillates. The overexpression of ATF1 significantly increased the concentrations of ethyl acetate, isoamyl acetate, 2‐phenylethyl acetate and ethyl caproate, while the overexpression of ATF2 affected the concentrations of ethyl acetate and isoamyl acetate to a lesser degree. The overexpression of IAH1 resulted in a significant decrease in ethyl acetate, isoamyl acetate, hexyl acetate and 2‐phenylethyl acetate. The overexpression of EHT1 resulted in a marked increase in ethyl caproate, ethyl caprylate and ethyl caprate. The flavour profile of the wines and distillates prepared using the modified strains were also significantly altered as indicated by formal sensory analysis. This study offers prospects for the development of wine yeast starter strains with optimized ester‐producing capability that could assist winemakers in their effort to consistently produce wine and distillates such as brandy to definable flavour specifications and styles. Copyright

Engineering volatile thiol release in Saccharomyces cerevisiae for improved wine aroma

Volatile thiols, such as 4‐mercapto‐4‐methylpentan‐2‐one (4MMP), 3‐mercaptohexan‐1‐ol (3MH) and 3‐mercaptohexyl acetate (3MHA), are among the most potent aroma compounds found in wine and can have a significant effect on wine quality and consumer preferences. At optimal concentrations in wine, these compounds impart flavours of passionfruit, grapefruit, gooseberry, blackcurrant, lychee, guava and box hedge. The enzymatic release of aromatic thiols from grape‐derived, non‐volatile cysteinylated precursors (Cys‐4MMP and Cys‐3MH) and the further modification thereof (conversion of 3MH into 3MHA) during fermentation, enhance the varietal characters of wines such as Sauvignon Blanc. Wine yeast strains have limited and varying capacities to produce aroma‐enhancing thiols from their non‐volatile counterparts in grape juice. Even under optimal fermentation conditions, the most efficient thiol‐releasing Saccharomyces cerevisiae wine strain known realizes less than 5% of the thiol‐related flavour potential of grape juice. The objective of this study was to develop a wine yeast able to unleash the untapped thiol aromas in grape juice during winemaking. To achieve this goal, the Escherichia coli tnaA gene, encoding a tryptophanase with strong cysteine‐β‐lyase activity, was cloned and overexpressed in a commercial wine yeast strain under the control of the regulatory sequences of the yeast phosphoglycerate kinase I gene (PGK1). This modified strain expressing carbon–sulphur lyase activity released up to 25 times more 4MMP and 3MH in model ferments than the control host strain. Wines produced with the engineered strain displayed an intense passionfruit aroma. This yeast offers the potential to enhance the varietal aromas of wines to predetermined market specifications. Copyright

Genetic Determinants of Volatile-Thiol Release by Saccharomyces cerevisiae during Wine Fermentation

ABSTRACT Volatile thiols, particularly 4-mercapto-4-methylpentan-2-one (4MMP), make an important contribution to the aroma of wine. During wine fermentation, Saccharomyces cerevisiae mediates the cleavage of a nonvolatile cysteinylated precursor in grape juice (Cys-4MMP) to release the volatile thiol 4MMP. Carbon-sulfur lyases are anticipated to be involved in this reaction. To establish the mechanism of 4MMP release and to develop strains that modulate its release, the effect of deleting genes encoding putative yeast carbon-sulfur lyases on the cleavage of Cys-4MMP was tested. The results led to the identification of four genes that influence the release of the volatile thiol 4MMP in a laboratory strain, indicating that the mechanism of release involves multiple genes. Deletion of the same genes from a homozygous derivative of the commercial wine yeast VL3 confirmed the importance of these genes in affecting 4MMP release. A strain deleted in a putative carbon-sulfur lyase gene, YAL012W, produced a second sulfur compound at significantly higher concentrations than those produced by the wild-type strain. Using mass spectrometry, this compound was identified as 2-methyltetrathiophen-3-one (MTHT), which was previously shown to contribute to wine aroma but was of unknown biosynthetic origin. The formation of MTHT in YAL012W deletion strains indicates a yeast biosynthetic origin of MTHT. The results demonstrate that the mechanism of synthesis of yeast-derived wine aroma components, even those present in small concentrations, can be investigated using genetic screens.

Carnitine-dependent metabolic activities in Saccharomyces cerevisiae : three carnitine acetyltransferases are essential in a carnitine-dependent strain

L‐Carnitine is required for the transfer of activated acyl‐groups across intracellular membranes in eukaryotic organisms. In Saccharomyces cerevisiae, peroxisomal membranes are impermeable to acetyl‐CoA, which is produced in the peroxisome when cells are grown on fatty acids as carbon source. In a reversible reaction catalysed by carnitine acetyltransferases (CATs), activated acetyl groups are transferred to carnitine to form acetylcarnitine which can be shuttled across membranes. Here we describe a mutant selection strategy that specifically selects for mutants affected in carnitine‐dependent metabolic activities. Complementation of three of these mutants resulted in the cloning of three CAT encoding genes: CAT2, coding for the carnitine acetyltransferase associated with the peroxisomes and the mitochondria; YAT1, coding for the carnitine acetyltransferase, which is presumably associated with the outer mitochondrial membrane, and YER024w (YAT2), which encodes a third, previously unidentified carnitine acetyltransferase. The data also show that (a) L‐carnitine and all three CATs are essential for growth on non‐fermentable carbon sources in a strain with a disrupted CIT2 gene; (b) Yat2p contributes significantly to total CAT activity when cells are grown on ethanol; and that (c) the carnitine‐dependent transfer of activated acetyl groups plays a more important role in cellular processes than previously realised. Copyright

Isolation of sulfite reductase variants of a commercial wine yeast with significantly reduced hydrogen sulfide production

The production of hydrogen sulfide (H(2)S) during fermentation is a common and significant problem in the global wine industry as it imparts undesirable off-flavors at low concentrations. The yeast Saccharomyces cerevisiae plays a crucial role in the production of volatile sulfur compounds in wine. In this respect, H(2)S is a necessary intermediate in the assimilation of sulfur by yeast through the sulfate reduction sequence with the key enzyme being sulfite reductase. In this study, we used a classical mutagenesis method to develop and isolate a series of strains, derived from a commercial diploid wine yeast (PDM), which showed a drastic reduction in H(2)S production in both synthetic and grape juice fermentations. Specific mutations in the MET10 and MET5 genes, which encode the catalytic alpha- and beta-subunits of the sulfite reductase enzyme, respectively, were identified in six of the isolated strains. Fermentations with these strains indicated that, in comparison with the parent strain, H(2)S production was reduced by 50-99%, depending on the strain. Further analysis of the wines made with the selected strains indicated that basic chemical parameters were similar to the parent strain except for total sulfite production, which was much higher in some of the mutant strains.

Coinoculated Fermentations Using Saccharomyces Yeasts Affect the Volatile Composition and Sensory Properties of Vitis vinifera L. cv. Sauvignon Blanc Wines

Alcoholic fermentation using Saccharomyces wine yeast is an effective means of modulating wine aroma. This study investigated the impact of coinoculating commercial yeast strains (Vin7, QA23, Vin13) on the volatile composition and sensory profile of Sauvignon Blanc wines. Small-scale replicated fermentations were conducted using single-strain and coinoculations of Vin7 with QA23 and with Vin13. The results showed that the chemical and sensory profiles of the coinoculated wines were different from both the single-strain wines and equal blends of the single-strain wines. Volatile thiol analysis indicated that the Vin7/QA23 coinoculated wines were highest in 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA), although this pattern was not observed for the Vin7/Vin13 yeast combination. The negative white vinegar aroma and high volatile acidity measured in the Vin7 single-strain wines were not present in the coinoculated wines. This study demonstrates that coinoculations can modify the aroma profile of wines, when complementary yeasts are used.

Modulation of volatile thiol and ester aromas by modified wine yeast

The volatile thiols, in particular 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA) are potent aroma shown to contribute strongly to the varietal aroma of Sauvignon Blanc wines. The thiols 4MMP and 3MH exist as non-volatile, aroma-inactive cysteine bound conjugates in the grape must and during fermentation the thiol is cleaved from the precursor. However, no cysteine conjugate for 3MHA has been identified. In this work we showed that 3MHA is formed from 3MH by the wine yeast Saccharomyces cerevisiae during fermentation. Furthermore, the alcohol acetyltransferase, Atf1p, the enzyme involved in the formation of the ester ethyl acetate, was shown to be the main enzyme responsible for the formation of 3MHA. Both a laboratory yeast and a commercial wine yeast overexpressing the ATF1 gene produced significantly more 3MHA than the wild-type. Although an atf1Δ laboratory yeast strain showed reduced 3MHA formation, it was not abolished, indicating that other enzymes are also responsible for its formation. Therefore, overexpression of the ATF1 gene in a wine yeast presents the possiblity of modulating both the thiol and ester aromas in wine.