**2.3** *Hanseniaspora*

Species of the genus *Hanseniaspora* are ubiquitous in the winemaking environment, and some of them have been proposed as wine yeast starters [96].

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 by *H. vineae.*

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

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 altered sensory properties of acescence [98].

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

Other compounds have been associated with the metabolism of *H. vineae* such as β-damascenone, isoamyl acetate and phenylacetaldehyde, which have been identified in ice wine fermentations [48].

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

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

**79**

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

were identified in the *H. vineae* genome. In this regard, the increase observed in the formation of 2-phenylethyl acetate and phenylpropanoids, such as 2-phenylethyl and benzyl alcohol, could be explained by duplications of *ARO8, ARO9* and *ARO10* genes. Similarly, the high level of acetate esters produced by *H. vineae* compared to that of *S. cerevisiae* is related to the identification of six proteins with domains of alcohol acetyltransferase (AATase). The opposite occurs with the reduced production of higher branched chain alcohols, fatty acids and ethyl esters, which responds to the absence of branched chain amino acid transaminases (*BAT2*) and acyl

*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

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

**2.4** *Metschnikowia*

glucosidase [110].

phenylethanol [54].

would be strain dependent [49].

coenzyme A (acyl-CoA)/ethanol O-acyltransferases (*EEB1*).

(MUG) and p-nitrophenyl-β-D-glucoside (pNPG) substrates [108].

citronellol and alpha-terpineol stand out [51, 53].

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,

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-D-

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

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

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.

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

were identified in the *H. vineae* genome. In this regard, the increase observed in the formation of 2-phenylethyl acetate and phenylpropanoids, such as 2-phenylethyl and benzyl alcohol, could be explained by duplications of *ARO8, ARO9* and *ARO10* genes. Similarly, the high level of acetate esters produced by *H. vineae* compared to that of *S. cerevisiae* is related to the identification of six proteins with domains of alcohol acetyltransferase (AATase). The opposite occurs with the reduced production of higher branched chain alcohols, fatty acids and ethyl esters, which responds to the absence of branched chain amino acid transaminases (*BAT2*) and acyl coenzyme A (acyl-CoA)/ethanol O-acyltransferases (*EEB1*).
