**2.4** *Metschnikowia*

*Chemistry and Biochemistry of Winemaking, Wine Stabilization and Aging*

and a better transformation potential of 2-PE.

**2.3** *Hanseniaspora*

by *H. vineae.*

residual concentration of 2-PE was twice lower in *K. marxianus* 35 and the efficiency was found to be 73% for this strain. Additionally, the sequence variability in the genes encoding the key enzymes of the Ehrlich pathway suggests that in addition to the physiological advantages *Kluyveromyces* have probably undergone substantial evolutionary genetic alterations that result in higher enzymatic activities

Species of the genus *Hanseniaspora* are ubiquitous in the winemaking environ-

Fermentations of mixed cultures by wild yeasts, such as *H. guilliermondii*, together with *S. cerevisiae* have shown higher concentrations of acetate ester compared to fermentations with *S. cerevisiae* alone, without significantly affecting acetaldehyde, acetic acid, glycerol and higher total alcohols [43]. However, Lleixà et al. [44] reported that the use of the *H. vineae* species as an initiator is capable of granting aromatic complexity in wines, producing key aromatic compounds. However, the sensory evaluation of the wines produced by this apiculate yeast is still limited and the results have not been consistent. In this regard, Medina et al. [45] reported that fermentation using *H. vineae* produced up to 10 times higher levels of 2-phenylethyl acetate in wine, compared to conventional and spontaneous fermentations. However, the opposite was observed for the concentration of 2-PE, which was significantly lower. Similar results were reported by Viana et al. [46, 47], regarding the high production of 2-phenylethyl acetate

It should be noted that the aromatic contribution of 2-PE is controversial. Fuente-Blanco [97] reported that the contribution of 2-PE in the aroma of red wine

On the other hand, Viana et al. [16] reported that *Hanseniaspora* spp. produce high levels of ethyl acetate. In this regard, it is important to highlight that ethyl acetate at low levels, below 80 mg/L, confers aromatic complexity on the wine, giving it a "fruity" aroma. However, over 150 mg/L is responsible for the typical

The acetic acid concentration in wines is also important, becoming a defect near its flavor threshold of 0.7–1.1 g/L. Some *H. uvarum* species have been reported to

Other compounds have been associated with the metabolism of *H. vineae* such as β-damascenone, isoamyl acetate and phenylacetaldehyde, which have been identi-

Seixas et al. [99] reported the reconstruction of the metabolic network for *H. guilliermondii* UTAD222, noting that this strain of yeast contains four genes that code for β-glucosidases, as well as the genes necessary for the synthesis of acetaldehyde, ethyl esters and higher alcohols. Surprisingly, no *S. cerevisiae* acetyl transferase-like proteins, involved in the synthesis of acetate esters, were found in the ORFeome of *H. guilliermondii* UTAD222. This is contradictory because it has been described that the synthesis of these compounds is high in this species [43]. Likewise, no sequences associated with aryl alcohol dehydrogenases were found, enzymes necessary for the synthesis of higher alcohols from aldehydes, which could contribute to the lower reported capacity of this species to produce these com-

Giorello et al. [100] recently reported genome sequencing, assembly and phylogenetic analysis of two strains of *H. vineae*. When these genomes were compared with 14 genomes of *S. cerevisiae*, specific flavor gene duplications and absences

was insignificant, in addition to depending on the aromatic context.

altered sensory properties of acescence [98].

fied in ice wine fermentations [48].

produce acetic acid levels of up to more than 3.4 g/L [42].

pounds, especially in comparison with *S. cerevisiae*.

ment, and some of them have been proposed as wine yeast starters [96].

**78**

*Metschnikowia pulcherrima* is one of the non-*Saccharomyces* yeast species with the greatest capacity to express extracellular hydrolytic enzymes. In *M. pulcherrima*, the presence of enzymes with pectinase, protease, glucanase, lichenase, β-glucosidase, cellulase, xylanase, amylase, sulfite reductase, lipase and β-lyase activity [49, 101–103] has been described. Also, its high proteolytic activity makes it a candidate to be used in fermentations with *S. cerevisiae*, releasing amino acids and increasing the available nitrogen sources for the growth of *S. cerevisiae* [104, 105]. It also stands out for its glucosidase-dependent strain activity [106, 107], which increases in aerobic conditions [50], promoting the release of varietal aromas by hydrolyzing bound monoterpenes. The expression of β-D-glucosidase favors the release of free terpenes and this activity has been evaluated using the 4-methylumbelliferyl-β-D-glucoside (MUG) and p-nitrophenyl-β-D-glucoside (pNPG) substrates [108].

Terpenes are relevant in the varietal character of various white grape varieties, being the main descriptors of varieties such as Muscat, Riesling or Alvariño [51]. Their presence and relevance in certain red grape varieties are also specific. However, the composition of free terpenes in the must is scarce, with a large amount of glycosylated terpenes [52]. These can be released by enzymatic hydrolysis by glycosidase enzymes [53, 109]. Within this group, linalool, geraniol, nerol, citronellol and alpha-terpineol stand out [51, 53].

The enzymatic hydrolysis of glycosides is mainly carried out by several enzymes that act sequentially, according to two steps: first, α-L-rhamnosidase, α-L-arabinosidase or β-D-apiosidase make the cleavage from terminal sugar and rhamnose, arabinose or apiose and the corresponding β-D-glycosides are released. Subsequently, the release of terpene occurs after the action of a β-Dglucosidase [110].

Likewise, mixed fermentations between *M. pulcherrima* and *S. cerevisiae* have identified higher levels of acetate esters and β-damascenone, and lower levels of C6 alcohols in ice wines of Vidal Blanc grape variety [48]. Similarly, a higher production of higher alcohols has been reported, with a greater amount of isobutanol and phenylethanol [54].

Another aspect to highlight for *M. pulcherrima* is that it has the ability to produce biogenic amines (histamine, tyramine and putrescine); however, this phenomenon would be strain dependent [49].

To date, only the genome of one *M. pulcherrima* strain has been reported [111], and genetic studies are scarce. Reid et al. [103] identified and characterized the gene that codes for an aspartic protease of *M. pulcherrima* IWBT Y1123, called MpAPr1. The results indicated that this protein presented homology with proteases of the yeast genera. Likewise, aspartic protease activity was confirmed by heterologous expression in *S. cerevisiae* YHUM272. This gene was found in 12 other strains of *M. pulcherrima*; however, analyzes revealed that the intensity of the enzyme activity was strain dependent and was not related to the gene sequence.

#### **2.5** *Brettanomyces* **spp.**

The yeast *Brettanomyces bruxellensis* is one of the main contaminant yeasts in wines, with the ability to metabolize hydroxycinnamic acids, which are naturally present in grapes, into volatile phenols [55, 56]. It has been described that this yeast can grow in various stages of wine production, for example, after alcoholic fermentation, during malolactic fermentation, during maturation in barrels or in already bottled wine. This characteristic is due to its ability to tolerate high ethanol variables [57].

Volatile phenols represent a large family of aromatic compounds where vinyl and ethyl derivatives are involved with product deterioration [55, 58]. These volatile phenols, especially 4-ethylphenol, are responsible for odors that have been described as "animal," "medicine," "leather" and "stable," which at concentrations above their perception threshold are detrimental to the aromatic profile of wines [55, 58].

The production of these compounds by *Brettanomyces* spp. is the result of the enzymatic transformation of hydroxycinnamic acids (3-methoxy-4-hydroxycinnamic acid (ferulic acid) and 4-hydroxycinnamic acid (*p*-coumaric acid)) by the action of two specific enzymes: cinnamate decarboxylase (CD) and vinylphenol reductase (VR) [112–115]. Also, *Brettanomyces* yeast species are capable of producing 2-acetyl-3,4,5,6-tetrahydropyridine, 2-acetyl-1,2,5,6-tetrahydropyridine and 2-ethyl-3,4,5,6-tetrahydropyridine. These compounds are responsible for "mousy taint" produced by microorganisms in the presence of lysine and ethanol [59].

It has been described that the ability of these yeasts to produce volatile phenols is variable [116, 117]. Factors such as the pH of the wine, the concentration of sugar and the moment in which this yeast is inoculated influence this capacity [118]. Along with this, it has been observed that the production of 4-ethylphenol in red wines is related to population growth, a phenomenon that would be strain dependent [119].

From the genetic point of view, there is a great intraspecific diversity of strains of *B. bruxellensis* [120–123], which translates into the different phenotypes of production of reported volatile phenols. The number of chromosomes in this species can vary between 4 and 9, with chromosome sizes in the range of 1 to 6Mb, and total genome size between 20 and 30Mb [124, 125]. Also, karyotypic studies suggest speciation due to genome rearrangements. However, available genetic studies are of a limited number of strains [121, 126–130]. In this regard, a transcriptomic analysis of the strain of *B. bruxellensis* LAMAP2480 exposed to *p*-coumaric acid indicates that this acid generates a stress condition, inducing the expression of the proton pump together with the output of toxic compounds, as well as the output of nitrogen compounds, reducing intracellular concentration and triggering the expression of nitrogen metabolism genes (**Figure 3**) [121].

Additionally, sequencing and genome analysis of the strain of *B. bruxellensis* AWRI1499 reported the presence of three homologous proteins with the isoamyl acetate hydrolysis enzymes of *S. cerevisiae* that are related to isoamyl alcohol concentrations and isoamyl acetate produced in fermentation. This strain was evaluated under fermentation conditions in model wine and produced higher levels of esters [60]. In this sense, it has been described that the formation of esters between *Brettanomyces* strains is variable.

The positive aromatic contribution of these yeasts has been studied mainly in beer. *Brettanomyces* spp. are able to esterify medium and long chain fatty acids in their respective esters, influencing the sensory profile of beers. Likewise, it has been reported that *Brettanomyces* has β-glucosidase activity, which would be responsible for breaking down the cellobiose present in the barrels, explaining its survival during the wine aging stage [131]. Crauwels et al. [120] reported that *B. bruxellensis* has

**81**

approximately 0.4.

both ORFs.

**Figure 3.**

**2.6** *Schizosaccharomyces* **spp.**

*Model of early response to stress by* p*-coumaric acid.*

*Formation of Aromatic and Flavor Compounds in Wine: A Perspective of Positive and Negative…*

two genes that encode β-glucosidases. Most strains from beer, with the exception of strain ST05.12/22, have only one copy, while strains isolated from the wine have

degrade malic acid into ethanol and deacidify musts of grapes and wines.

(SO2) level affect the successful completion of MLF [133].

While *Schizosaccharomyces* genus yeasts have been associated with the production of compounds such as hydrogen sulfide (H2S) and acetaldehyde [61] that negatively impact the aromatic quality of wines, *S. pombe* stands out mainly for its ability to

L-Malic acid is a compound that is present in grape must and its concentration depends on the grape varieties and climatic conditions. When malolactic fermentation (MLF) occurs, the lactic acid bacteria transform L-malic acid into lactic acid, reducing the total acidity and thereby increasing the pH of the grape must [132]. However, factors such as ethanol concentration, pH, temperature and sulfur dioxide

An alternative to this process is the malo-ethanolic deacidification carried out by *S. pombe* [62, 134]. This yeast exhibits a high tolerance to low pH and high levels of SO2, characteristics that make it highly compatible for use during the winemaking process [135]. Benito et al. [63] reported that the conversion of malic acid to ethanol decreases the total acidity by approximately 4 g/L and increases the final pH by

Other interesting characteristics of this yeast are associated with its ability to reduce gluconic acid concentrations [64, 65]. It has also been reported that the urease activity of *Schizosaccharomyces* strains could reduce the content of ethyl carbamate and biogenic amines in wine by reducing the concentrations of urea [66, 67]. Another application that *Schizosaccharomyces* has is aging on the lees, thanks to the strong autolytic release of the polysaccharides from the cell wall [136, 137].

*DOI: http://dx.doi.org/10.5772/intechopen.92562*

*Formation of Aromatic and Flavor Compounds in Wine: A Perspective of Positive and Negative… DOI: http://dx.doi.org/10.5772/intechopen.92562*

**Figure 3.** *Model of early response to stress by* p*-coumaric acid.*

two genes that encode β-glucosidases. Most strains from beer, with the exception of strain ST05.12/22, have only one copy, while strains isolated from the wine have both ORFs.

#### **2.6** *Schizosaccharomyces* **spp.**

*Chemistry and Biochemistry of Winemaking, Wine Stabilization and Aging*

The yeast *Brettanomyces bruxellensis* is one of the main contaminant yeasts in wines, with the ability to metabolize hydroxycinnamic acids, which are naturally present in grapes, into volatile phenols [55, 56]. It has been described that this yeast can grow in various stages of wine production, for example, after alcoholic fermentation, during malolactic fermentation, during maturation in barrels or in already bottled wine. This characteristic is due to its ability to tolerate high ethanol variables [57]. Volatile phenols represent a large family of aromatic compounds where vinyl and ethyl derivatives are involved with product deterioration [55, 58]. These volatile phenols, especially 4-ethylphenol, are responsible for odors that have been described as "animal," "medicine," "leather" and "stable," which at concentrations above their perception threshold are detrimental to the aromatic profile of wines

The production of these compounds by *Brettanomyces* spp. is the result of the enzymatic transformation of hydroxycinnamic acids (3-methoxy-4-hydroxycinnamic acid (ferulic acid) and 4-hydroxycinnamic acid (*p*-coumaric acid)) by the action of two specific enzymes: cinnamate decarboxylase (CD) and vinylphenol reductase (VR) [112–115]. Also, *Brettanomyces* yeast species are capable of producing 2-acetyl-3,4,5,6-tetrahydropyridine, 2-acetyl-1,2,5,6-tetrahydropyridine and 2-ethyl-3,4,5,6-tetrahydropyridine. These compounds are responsible for "mousy taint" produced by microorganisms in the presence of lysine and ethanol [59].

It has been described that the ability of these yeasts to produce volatile phenols

From the genetic point of view, there is a great intraspecific diversity of strains

Additionally, sequencing and genome analysis of the strain of *B. bruxellensis* AWRI1499 reported the presence of three homologous proteins with the isoamyl acetate hydrolysis enzymes of *S. cerevisiae* that are related to isoamyl alcohol concentrations and isoamyl acetate produced in fermentation. This strain was evaluated under fermentation conditions in model wine and produced higher levels of esters [60]. In this sense, it has been described that the formation of esters between

The positive aromatic contribution of these yeasts has been studied mainly in beer. *Brettanomyces* spp. are able to esterify medium and long chain fatty acids in their respective esters, influencing the sensory profile of beers. Likewise, it has been reported that *Brettanomyces* has β-glucosidase activity, which would be responsible for breaking down the cellobiose present in the barrels, explaining its survival during the wine aging stage [131]. Crauwels et al. [120] reported that *B. bruxellensis* has

is variable [116, 117]. Factors such as the pH of the wine, the concentration of sugar and the moment in which this yeast is inoculated influence this capacity [118]. Along with this, it has been observed that the production of 4-ethylphenol in red wines is related to population growth, a phenomenon that would be strain

of *B. bruxellensis* [120–123], which translates into the different phenotypes of production of reported volatile phenols. The number of chromosomes in this species can vary between 4 and 9, with chromosome sizes in the range of 1 to 6Mb, and total genome size between 20 and 30Mb [124, 125]. Also, karyotypic studies suggest speciation due to genome rearrangements. However, available genetic studies are of a limited number of strains [121, 126–130]. In this regard, a transcriptomic analysis of the strain of *B. bruxellensis* LAMAP2480 exposed to *p*-coumaric acid indicates that this acid generates a stress condition, inducing the expression of the proton pump together with the output of toxic compounds, as well as the output of nitrogen compounds, reducing intracellular concentration and triggering the expression

of nitrogen metabolism genes (**Figure 3**) [121].

*Brettanomyces* strains is variable.

**2.5** *Brettanomyces* **spp.**

[55, 58].

dependent [119].

**80**

While *Schizosaccharomyces* genus yeasts have been associated with the production of compounds such as hydrogen sulfide (H2S) and acetaldehyde [61] that negatively impact the aromatic quality of wines, *S. pombe* stands out mainly for its ability to degrade malic acid into ethanol and deacidify musts of grapes and wines.

L-Malic acid is a compound that is present in grape must and its concentration depends on the grape varieties and climatic conditions. When malolactic fermentation (MLF) occurs, the lactic acid bacteria transform L-malic acid into lactic acid, reducing the total acidity and thereby increasing the pH of the grape must [132]. However, factors such as ethanol concentration, pH, temperature and sulfur dioxide (SO2) level affect the successful completion of MLF [133].

An alternative to this process is the malo-ethanolic deacidification carried out by *S. pombe* [62, 134]. This yeast exhibits a high tolerance to low pH and high levels of SO2, characteristics that make it highly compatible for use during the winemaking process [135]. Benito et al. [63] reported that the conversion of malic acid to ethanol decreases the total acidity by approximately 4 g/L and increases the final pH by approximately 0.4.

Other interesting characteristics of this yeast are associated with its ability to reduce gluconic acid concentrations [64, 65]. It has also been reported that the urease activity of *Schizosaccharomyces* strains could reduce the content of ethyl carbamate and biogenic amines in wine by reducing the concentrations of urea [66, 67]. Another application that *Schizosaccharomyces* has is aging on the lees, thanks to the strong autolytic release of the polysaccharides from the cell wall [136, 137].

The contribution from the aromatic point of view of *S. pombe* has been recently reported where it has been observed that it stands out mainly for producing fewer amounts of higher alcohols in comparison to *S. cerevisiae*, which could be attributed as a strain-dependent characteristic [62, 63, 66, 68, 69].

Benito et al. [63] reported a lower production of isobutanol, 2-methyl-butanol, 3-methyl-butanol and 2-phenyl-ethanol in white wines by *S. pombe* in comparison to *S. cerevisiae*. Similar results have been reported by Mylona et al. [66] where, fermenting red must, they observed a decrease in 2-methyl-butanol, 3-methyl-butanol and isobutanol by *S. pombe* in comparison to *S. cerevisiae*. On the other hand, Chen et al. [69] observed that *S. pombe* possesses a special ability to produce more 2, 3-butanediol, which contributes to the fruity aroma described as banana to wines.

In the case of esters, a similar phenomenon occurs, observing that *Schizosaccharomyces* shows a tendency to produce lower concentrations of esters in comparison to *S. cerevisiae.* It has been reported to produce lower concentrations of isoamyl acetate and 2-phenyl-ethyl acetate in comparison to *S. cerevisiae* [62]. Likewise, lower production of total esters was reported by Del Fresno et al. [68] in comparison to *S. cerevisiae*.

Finally, *S. pombe* fermentations have been reported to show higher levels of acetoin in comparison to *S. cerevisiae* controls. Also, they are commonly associated with high levels of acetic acid. These levels might vary from strain to strain [68–70].
