**2. Wine aromas produced by non-***Saccharomyces* **yeasts**

The formation of aromatic compounds has been extensively studied in *S. cerevisiae*. In this regard, higher alcohols are synthesized from amino acids by transamination and decarboxylation reactions (**Figure 1**). Permeases of amino acids participate in these reactions, which are encoded by the *GAP1, BAP2* and *MEP2* genes. Subsequently, the transamination reactions are carried out by enzymes encoded by the *BAT1* and *BAT2* genes, which code for branched-chain amino acid transaminases, and the *ARO8* and *ARO9* genes that code for aromatic amino acid aminotransferases, which catalyze the transfer of amines between amino acids and their respective α-keto acid. Subsequently, the decarboxylation reactions of the α-keto acid occur to form the respective aldehydes, where the *PDC1*, *PDC5*, *PDC6*, *THI3* and *ARO10* genes are responsible for coding for enzymes with decarboxylase activity and, finally, dehydrogenases act, which reduce aldehydes to alcohols, a reaction that is carried out by alcohol dehydrogenases, encoded by the *ADH1–7* and *SFA1* genes, and aryl alcohol dehydrogenases, encoded by *AAD* genes [71–73].

Other important compounds are acetate esters, and their synthesis occurs by condensation between higher alcohols and acetyl-CoA (**Figure 2**). This reaction is carried out by acetyltransferases, encoded by the *ATF1* and *ATF2* genes. The ethyl esters are produced by condensation between ethanol and acyl-CoA, a reaction mediated by acyltransferases encoded by the genes *EHT1*, *EEB1* and YMR210W, encoding for a monoacylglycerol lipase [74].

Likewise, it has been reported that *S. cerevisiae* participates in the primary release of aromas through the activity of glucosidase enzymes [76].

#### **2.1** *Torulaspora delbrueckii*

Among the non-*Saccharomyces* yeasts, *T. delbrueckii* has gained interest in the vitiviniculture industry because it modifies the aromatic properties of final wines in a very positive way, producing higher levels of fruity esters, thiols and terpenes and lower amounts of higher alcohols, thus respecting the initial character of the grape [6, 18, 19]. Also, *T. delbrueckii* typically produces low concentrations of acetic acid [12], one of the main quality parameters in wine production. It has also been reported that *T. delbrueckii* produces wines with higher levels of glycerol [5] and, consequently, with lower concentrations of ethanol [20]. This is currently a relevant feature because as a consequence of climate change, an increase in sugar concentration in the must has been observed, resulting in wines with higher alcohol content.

During alcoholic fermentation, the ethanol production is usually higher than 12% (v/v), so the associated microorganisms must have resistance mechanisms for this compound. In practice, the phenotype of ethanol resistance among wine yeasts

**75**

**Figure 2.**

*Acetate ester and ethyl ester biosynthesis (adapted from [75]).*

**Figure 1.**

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

is heterogeneous, *S. cerevisiae* being the one with the highest level of resistance and the one in charge of leading the alcoholic fermentation. However, non-*Saccharomyces* yeast species play an important role during the early stages of spontaneous alcoholic

fermentation, when the ethanol concentration is not very high [77].

*Ehrlich pathway for higher alcohol production (adapted from [71]).*

*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 1.** *Ehrlich pathway for higher alcohol production (adapted from [71]).*

is heterogeneous, *S. cerevisiae* being the one with the highest level of resistance and the one in charge of leading the alcoholic fermentation. However, non-*Saccharomyces* yeast species play an important role during the early stages of spontaneous alcoholic fermentation, when the ethanol concentration is not very high [77].

**Figure 2.** *Acetate ester and ethyl ester biosynthesis (adapted from [75]).*

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

Acetaldehyde Pyruvic acid 2, 3-butanediol Acetoin Acetic acid

*Schizosaccharomyces* spp H2S

*fermentations with* S. cerevisiae*.*

**Table 1.**

**2. Wine aromas produced by non-***Saccharomyces* **yeasts**

encoding for a monoacylglycerol lipase [74].

**2.1** *Torulaspora delbrueckii*

The formation of aromatic compounds has been extensively studied in *S. cerevisiae*. In this regard, higher alcohols are synthesized from amino acids by transamination and decarboxylation reactions (**Figure 1**). Permeases of amino acids participate in these reactions, which are encoded by the *GAP1, BAP2* and *MEP2* genes. Subsequently, the transamination reactions are carried out by enzymes encoded by the *BAT1* and *BAT2* genes, which code for branched-chain amino acid transaminases, and the *ARO8* and *ARO9* genes that code for aromatic amino acid aminotransferases, which catalyze the transfer of amines between amino acids and their respective α-keto acid. Subsequently, the decarboxylation reactions of the α-keto acid occur to form the respective aldehydes, where the *PDC1*, *PDC5*, *PDC6*, *THI3* and *ARO10* genes are responsible for coding for enzymes with decarboxylase activity and, finally, dehydrogenases act, which reduce aldehydes to alcohols, a reaction that is carried out by alcohol dehydrogenases, encoded by the *ADH1–7* and *SFA1* genes, and aryl alcohol dehydrogenases, encoded by *AAD* genes [71–73]. Other important compounds are acetate esters, and their synthesis occurs by condensation between higher alcohols and acetyl-CoA (**Figure 2**). This reaction is carried out by acetyltransferases, encoded by the *ATF1* and *ATF2* genes. The ethyl esters are produced by condensation between ethanol and acyl-CoA, a reaction mediated by acyltransferases encoded by the genes *EHT1*, *EEB1* and YMR210W,

*Metabolites produced in wine by non-*Saccharomyces *yeasts in mixed fermentations compared to* 

**Species Metabolites References Increase Decrease**

> Esters Higher alcohols Gluconic acid

[61–70]

Likewise, it has been reported that *S. cerevisiae* participates in the primary

Among the non-*Saccharomyces* yeasts, *T. delbrueckii* has gained interest in the vitiviniculture industry because it modifies the aromatic properties of final wines in a very positive way, producing higher levels of fruity esters, thiols and terpenes and lower amounts of higher alcohols, thus respecting the initial character of the grape [6, 18, 19]. Also, *T. delbrueckii* typically produces low concentrations of acetic acid [12], one of the main quality parameters in wine production. It has also been reported that *T. delbrueckii* produces wines with higher levels of glycerol [5] and, consequently, with lower concentrations of ethanol [20]. This is currently a relevant feature because as a consequence of climate change, an increase in sugar concentration in the must has been observed, resulting in wines with higher alcohol content. During alcoholic fermentation, the ethanol production is usually higher than 12% (v/v), so the associated microorganisms must have resistance mechanisms for this compound. In practice, the phenotype of ethanol resistance among wine yeasts

release of aromas through the activity of glucosidase enzymes [76].

**74**

Therefore, currently, the strategy of mixed and/or sequential fermentations is used, which combines non-*Saccharomyces* yeasts with a yeast with a higher fermentative profile such as *S. cerevisiae*, which in most of the cases is necessary to properly end the industrial process of alcoholic fermentation.

*T. delbrueckii* has been described as capable of fermenting and tolerating up to an ethanol concentration slightly higher than 9% (v/v) [21]. On the other hand, Bely et al. [12] have reported that this value is lower, reaching only 7.4% (v/v). Nevertheless, Belda et al. [5, 6], through studies of population kinetics in sequential fermentation, observed that *T. delbrueckii* suffered a significant decrease in the cellular viability when ethanol levels exceed 8% (v/v). This suggests that the ethanol resistance of *T. delbrueckii* is limited and much lower than that of *S. cerevisiae*, which complicates its use in industrial fermentations. Nevertheless, to improve the fermentation rate of the selected nonconventional yeasts, sequential cultures are used, but this is to the detriment of the diversity of aromas that could be present in the final product. Given this context, ethanol resistance is an important factor in the selection of industrial non-*Saccharomyces* strains and particularly of *T. delbrueckii*.

Non-*Saccharomyces* yeast species can produce the aromatic volatiles that are known to be important for industrial beer and wine fermentations and that are produced by *Saccharomyces* species [78]. For the case of *T. delbrueckii*, several studies have indicated how beneficial its, from the aromatic point of view, incorporation into fermentations is [79–81]. Belda et al. [6], evaluating a sequential fermentation using Verdejo variety must, observed a higher aroma quality, intensity and fruity character. Chromatographic analysis indicated that this effect was due to an increase in the levels of the main ones, mainly 4-methyl-4-sulfanyl-pentan-2-one (4-MSP), which is represented in this grape variety. Likewise, Renault et al. [22] reported that mixed inoculations of *T. delbrueckii* and *S. cerevisiae* allowed the increase of some esters specifically produced by *T. delbrueckii*, which correlated with the maximum population reached by it in mixed cultures. Among the reported compounds were ethyl propanoate, ethyl isobutanoate and ethyl dihydrocinnamate, which are considered activity markers for this yeast.

The signaling pathways involved in the formation of aroma and flavor compounds, such as the Ehrlich pathway, or the specific enzymes responsible for the synthesis of ester, are also present in nonconventional yeast. This route has been studied extensively in *S. cerevisiae* [71, 82, 83]. This pathway consists of a step of transamination of amino acids to α-keto acids, followed by decarboxylation to "fusel aldehydes." These fusel aldehydes can be reduced or oxidized in fusel alcohols or fusel acids, respectively [71, 84]. Subsequently, aromatic esters can be formed from alcohols and fusel acids [23], and these compounds are responsible for the characteristic aroma and flavor of the final fermented product [72]. In addition, these aromatic esters have a low detection threshold, which is why minimum amounts of these compounds are required for the perception of the human olfactory senses [23].

It has been reported that the concentration of assimilable nitrogen has a significant effect on the production of fermentation aromas [85, 86]. A higher concentration of higher alcohols at the end of fermentation has been observed in media with low nitrogen content [24–26]. Likewise, there is a directly proportional relationship between the concentration of nitrogen and the synthesis of the ethyl esters, in which the initial content of nitrogen is associated with an increase in the production of esters [27, 28]. In this sense, Bloem et al. [87] observed that the nitrogen composition of the medium could influence the redox balance in the yeast cells during alcoholic fermentation and that variations in this balance could change the final concentrations of certain volatile compounds. Changes in the levels of these compounds were closely related to the effects of redox status on the availability of

**77**

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

acetyl-CoA, an intermediate of central carbon metabolism and precursor of α-keto acids. Similar results were reported by Rollero et al. [88] who observed that a small change in the acetyl-CoA pool would affect the bioconversion of acetate esters from higher alcohols. These results suggest that it is possible to increase the aromatic potential of *T. delbrueckii* by modulating the availability of nitrogen in the medium, which would influence the redox balance of the cells directly affecting the final

Through next-generation sequencing, Tondini et al. [89] characterized the transcriptome of *T. delbrueckii* COFT1 observing differences in glucose fermentation pathways and the formation of aromatic and flavoring compounds, such as glycerol, esters and acetic acid with respect to *S. cerevisiae*. These differences are partly explained by the absence of paralogous genes in glycolysis and glycerol biosynthesis in *T. delbrueckii*. It has been reported that T*. delbrueckii* produces less acetic acid [29], and this phenomenon depends on increased expression of genes related to alcoholic fermentation, while acetate ester levels were influenced by the absence of esterases, *ATF1–2.* Likewise, a lower production of ethyl esters was observed in *T. delbrueckii* COFT1, which suggests a negative regulation in the fatty acid pathway

*Kluyveromyces* species do not usually intervene in spontaneous fermentation processes because they have a low fermentation capacity and slow multiplication [31]. However, they are capable of producing considerable amounts of lactic acid (1.5– 1.8 g/L) and low amounts of acetic acid. It has been reported that *Kluyveromyces* species produce aromatic compounds such as esters, monoterpenic alcohols, carboxylic acids, ketones, furans and isoamyl acetate in liquid phase fermentation. Of all these compounds, the production of 2-phenylethanol (2-PE) stands out [32], with the aroma of rose petals, which is commercially important, since it gives characteristics that positively influence wine quality, among others [90]. In particular, the influence of the carbon source [91, 92], the aeration rate [92], the composition of the medium [93] and growing conditions [94] on the production of aromas in *K.* 

*K. marxianus* produces polygalacturonases, enzymes that added in the fermentation of musts favor the release of aromatic compounds, resulting in citrus, balsamic and floral wines [33]. Other studies have demonstrated the fermentation capacity of *K. marxianus* in pure culture for the production of tequila; however, in mixed cultures with *S. cerevisiae*, the activity of *K. marxianus* is negatively affected [95]. Another group of important aromatic compounds is monoterpenoids. The common precursor of these compounds is geranyl pyrophosphate (GPP). Although plants, such as *Vitis vinifera* and *Humulus lupulus*, produce monoterpenoids [36], it has been reported that yeasts can also produce them [37], highlight-

Marcišauskas et al. [34], using the strain of *K. marxianus* iSM996, constructed the first genome-scale metabolic model for this yeast. This model contains several unique biosynthetic pathways for aromatic compounds such as 2-PE, phenethyl acetate and ethyl acetate. The *K. marxianus* iSM996 model is a solid tool to evaluate the metabolic characteristics of *K. marxianus*, allowing the integration of experi-

Ivanov et al. [35] studied the production potential of 2-PE by the strain of *K. marxianus* 35. The results revealed that the enzymatic activity of aminotransferase, pyruvate decarboxylase and alcohol dehydrogenase, key enzymes of the Ehrlich pathway, was almost twice as large compared to *S. cerevisiae*. In addition, the

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

concentrations of certain volatile compounds.

biosynthesis.

**2.2** *Kluyveromyces* **spp.**

*marxianus* has been studied.

ing *K. lactis* [38–40].

mental data and strain design based on the model.

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

acetyl-CoA, an intermediate of central carbon metabolism and precursor of α-keto acids. Similar results were reported by Rollero et al. [88] who observed that a small change in the acetyl-CoA pool would affect the bioconversion of acetate esters from higher alcohols. These results suggest that it is possible to increase the aromatic potential of *T. delbrueckii* by modulating the availability of nitrogen in the medium, which would influence the redox balance of the cells directly affecting the final concentrations of certain volatile compounds.

Through next-generation sequencing, Tondini et al. [89] characterized the transcriptome of *T. delbrueckii* COFT1 observing differences in glucose fermentation pathways and the formation of aromatic and flavoring compounds, such as glycerol, esters and acetic acid with respect to *S. cerevisiae*. These differences are partly explained by the absence of paralogous genes in glycolysis and glycerol biosynthesis in *T. delbrueckii*. It has been reported that T*. delbrueckii* produces less acetic acid [29], and this phenomenon depends on increased expression of genes related to alcoholic fermentation, while acetate ester levels were influenced by the absence of esterases, *ATF1–2.* Likewise, a lower production of ethyl esters was observed in *T. delbrueckii* COFT1, which suggests a negative regulation in the fatty acid pathway biosynthesis.

#### **2.2** *Kluyveromyces* **spp.**

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

end the industrial process of alcoholic fermentation.

considered activity markers for this yeast.

Therefore, currently, the strategy of mixed and/or sequential fermentations is used, which combines non-*Saccharomyces* yeasts with a yeast with a higher fermentative profile such as *S. cerevisiae*, which in most of the cases is necessary to properly

*T. delbrueckii* has been described as capable of fermenting and tolerating up to an ethanol concentration slightly higher than 9% (v/v) [21]. On the other hand, Bely et al. [12] have reported that this value is lower, reaching only 7.4% (v/v). Nevertheless, Belda et al. [5, 6], through studies of population kinetics in sequential fermentation, observed that *T. delbrueckii* suffered a significant decrease in the cellular viability when ethanol levels exceed 8% (v/v). This suggests that the ethanol resistance of *T. delbrueckii* is limited and much lower than that of *S. cerevisiae*, which complicates its use in industrial fermentations. Nevertheless, to improve the fermentation rate of the selected nonconventional yeasts, sequential cultures are used, but this is to the detriment of the diversity of aromas that could be present in the final product. Given this context, ethanol resistance is an important factor in the selection of industrial non-*Saccharomyces* strains and particularly of *T. delbrueckii*. Non-*Saccharomyces* yeast species can produce the aromatic volatiles that are known to be important for industrial beer and wine fermentations and that are produced by *Saccharomyces* species [78]. For the case of *T. delbrueckii*, several studies have indicated how beneficial its, from the aromatic point of view, incorporation into fermentations is [79–81]. Belda et al. [6], evaluating a sequential fermentation using Verdejo variety must, observed a higher aroma quality, intensity and fruity character. Chromatographic analysis indicated that this effect was due to an increase in the levels of the main ones, mainly 4-methyl-4-sulfanyl-pentan-2-one (4-MSP), which is represented in this grape variety. Likewise, Renault et al. [22] reported that mixed inoculations of *T. delbrueckii* and *S. cerevisiae* allowed the increase of some esters specifically produced by *T. delbrueckii*, which correlated with the maximum population reached by it in mixed cultures. Among the reported compounds were ethyl propanoate, ethyl isobutanoate and ethyl dihydrocinnamate, which are

The signaling pathways involved in the formation of aroma and flavor compounds, such as the Ehrlich pathway, or the specific enzymes responsible for the synthesis of ester, are also present in nonconventional yeast. This route has been studied extensively in *S. cerevisiae* [71, 82, 83]. This pathway consists of a step of transamination of amino acids to α-keto acids, followed by decarboxylation to "fusel aldehydes." These fusel aldehydes can be reduced or oxidized in fusel alcohols or fusel acids, respectively [71, 84]. Subsequently, aromatic esters can be formed from alcohols and fusel acids [23], and these compounds are responsible for the characteristic aroma and flavor of the final fermented product [72]. In addition, these aromatic esters have a low detection threshold, which is why minimum amounts of these compounds are required for the perception of the human olfac-

It has been reported that the concentration of assimilable nitrogen has a significant effect on the production of fermentation aromas [85, 86]. A higher concentration of higher alcohols at the end of fermentation has been observed in media with low nitrogen content [24–26]. Likewise, there is a directly proportional relationship between the concentration of nitrogen and the synthesis of the ethyl esters, in which the initial content of nitrogen is associated with an increase in the production of esters [27, 28]. In this sense, Bloem et al. [87] observed that the nitrogen composition of the medium could influence the redox balance in the yeast cells during alcoholic fermentation and that variations in this balance could change the final concentrations of certain volatile compounds. Changes in the levels of these compounds were closely related to the effects of redox status on the availability of

**76**

tory senses [23].

*Kluyveromyces* species do not usually intervene in spontaneous fermentation processes because they have a low fermentation capacity and slow multiplication [31]. However, they are capable of producing considerable amounts of lactic acid (1.5– 1.8 g/L) and low amounts of acetic acid. It has been reported that *Kluyveromyces* species produce aromatic compounds such as esters, monoterpenic alcohols, carboxylic acids, ketones, furans and isoamyl acetate in liquid phase fermentation. Of all these compounds, the production of 2-phenylethanol (2-PE) stands out [32], with the aroma of rose petals, which is commercially important, since it gives characteristics that positively influence wine quality, among others [90]. In particular, the influence of the carbon source [91, 92], the aeration rate [92], the composition of the medium [93] and growing conditions [94] on the production of aromas in *K. marxianus* has been studied.

*K. marxianus* produces polygalacturonases, enzymes that added in the fermentation of musts favor the release of aromatic compounds, resulting in citrus, balsamic and floral wines [33]. Other studies have demonstrated the fermentation capacity of *K. marxianus* in pure culture for the production of tequila; however, in mixed cultures with *S. cerevisiae*, the activity of *K. marxianus* is negatively affected [95].

Another group of important aromatic compounds is monoterpenoids. The common precursor of these compounds is geranyl pyrophosphate (GPP). Although plants, such as *Vitis vinifera* and *Humulus lupulus*, produce monoterpenoids [36], it has been reported that yeasts can also produce them [37], highlighting *K. lactis* [38–40].

Marcišauskas et al. [34], using the strain of *K. marxianus* iSM996, constructed the first genome-scale metabolic model for this yeast. This model contains several unique biosynthetic pathways for aromatic compounds such as 2-PE, phenethyl acetate and ethyl acetate. The *K. marxianus* iSM996 model is a solid tool to evaluate the metabolic characteristics of *K. marxianus*, allowing the integration of experimental data and strain design based on the model.

Ivanov et al. [35] studied the production potential of 2-PE by the strain of *K. marxianus* 35. The results revealed that the enzymatic activity of aminotransferase, pyruvate decarboxylase and alcohol dehydrogenase, key enzymes of the Ehrlich pathway, was almost twice as large compared to *S. cerevisiae*. In addition, the

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 and a better transformation potential of 2-PE.
