**5. Yeast selection to improve acidity, aromatic profile, or color**

The selection of yeast strains to obtain non-*Saccharomyces* able to improve the wine freshness in terms of acidity, aromatic profile, or color starts with the isolation of a yeast collection from a vine environment, mainly grapes, and also leaves, wood, or soil. After that, the yeast can be initially classified by using both selective and differential agar media. Later, the pre-identified yeasts can be confirmed by PCR amplification of the ribosomal region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene using as primers the ITS1 and ITS4 [51], the subsequent sequencing and the comparison of the sequence in a genomic database that facilitates the proper identification of genus and species [45]. Microfermentations in triplicate can be performed in order to select specific yeast strains with improved properties, e.g., a *L. thermotolerans* strain with suitable production of lactic acid, during spontaneous fresh must fermentation. Later, the production of lactic acid and whatever other metabolites with repercussion in wine sensory quality can be evaluated by instrumental analysis (**Figure 3**).

**157**

2–4 log CFU/mL.

**Figure 4.**

*a warm region.*

*Strategies to Improve the Freshness in Wines from Warm Areas*

fermentation [52–54]. These properties can be targeted to improve color by the formation of stable pigments as vitisins [55, 56], vinylphenolic pyranoanthocyanins [57], and polymeric pigments [50, 58], the enhancement of aroma by the production of esters or enzymatic activities able to release varietal aroma [59, 60], or the improvement of the mouthfeel and flavor by the production/release of polyalco-

*Phylogenetic tree of the wild non-*Saccharomyces *yeast species that were found in the grapes of a vineyard from* 

The isolation of wild yeasts and the subsequent sequencing and comparison of the rDNA can help to elucidate the yeast microbioma from a vineyard (**Figure 4**). Normally, when the wild yeast populations are evaluated at the grape maturity stage, several mold species are frequently found together with apiculate yeasts such as those which belong to the genus *Kloeckera* or *Hanseniaspora*, making difficult to isolate and identify *S. cerevisiae* strains. Apiculate yeast can reach populations of

**6. Ternary sequential inoculations in warm areas: biotechnological** 

The use of sequential fermentations with non-*Saccharomyces* species has been used to improve wine acidity, aromatic and flavor complexity, and freshness. As reviewed in Section 3, non-*Saccharomyces* yeasts such as *H. vineae*, *T. delbrueckii*, *W. anomalus*, *M. pulcherrima*, *K. apiculata*, *S. bombicola*, and *C. stellata* improve aroma by either the increased production of acetate esters or the development of enzymatic activities that enhance the varietal aroma. Some of them can also increase sweetness and body by the production of polyalcohols such as glycerol or 2,3-butanediol. Moreover, it is currently possible to control pH in fermentation by the formation of suitable amounts of lactic acid with *L. thermotolerans*. The use of sequential combinations of two yeasts is already used at industrial level, but the combination of three yeast species (**Table 1**), namely, ternary inoculations, is less explored as a biotechnology to improve freshness in warm areas. In this case, it is more similar to what happens in a spontaneous fermentation according to the principle of succession: the fermentation is started by an apiculate yeast, followed by a medium fermentative power yeast like *T. delbrueckii*, *L. thermotolerans*, or

hols, polysaccharides [61, 62], acids [39, 45], etc.

**approach to improve freshness**

*DOI: http://dx.doi.org/10.5772/intechopen.86893*

Yeast selection can be focused on the identification of strains with specific properties of technological, fermentative, or sensory repercussion during wine

#### *Strategies to Improve the Freshness in Wines from Warm Areas DOI: http://dx.doi.org/10.5772/intechopen.86893*

#### **Figure 4.**

*Advances in Grape and Wine Biotechnology*

fermentations [39].

hyperchromic effect. Indirectly, as pH affects the levels of both molecular and free

From a sensory perspective, the biological acidification with *L. thermotolerans* produces a good and perceptible sourness, thus increasing wine freshness [39]. Usually, no unpleasant nuances of dairy foods are found, even when higher levels of ethyl lactate are produced, but the levels of acetoin and diacetyl in the sequential fermentations with *S. cerevisiae* are quite controlled and similar to single *S. cerevisiae*

The selection of yeast strains to obtain non-*Saccharomyces* able to improve the wine freshness in terms of acidity, aromatic profile, or color starts with the isolation of a yeast collection from a vine environment, mainly grapes, and also leaves, wood, or soil. After that, the yeast can be initially classified by using both selective and differential agar media. Later, the pre-identified yeasts can be confirmed by PCR amplification of the ribosomal region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene using as primers the ITS1 and ITS4 [51], the subsequent sequencing and the comparison of the sequence in a genomic database that facilitates the proper identification of genus and species [45]. Microfermentations in triplicate can be performed in order to select specific yeast strains with improved properties, e.g., a *L. thermotolerans* strain with suitable production of lactic acid, during spontaneous fresh must fermentation. Later, the production of lactic acid and whatever other metabolites with repercussion in wine

sulfur dioxide, it may also promote a protective effect on color.

**5. Yeast selection to improve acidity, aromatic profile, or color**

sensory quality can be evaluated by instrumental analysis (**Figure 3**).

*Isolation of wild yeast and selection protocol under a metabolic approach.*

Yeast selection can be focused on the identification of strains with specific properties of technological, fermentative, or sensory repercussion during wine

**156**

**Figure 3.**

*Phylogenetic tree of the wild non-*Saccharomyces *yeast species that were found in the grapes of a vineyard from a warm region.*

fermentation [52–54]. These properties can be targeted to improve color by the formation of stable pigments as vitisins [55, 56], vinylphenolic pyranoanthocyanins [57], and polymeric pigments [50, 58], the enhancement of aroma by the production of esters or enzymatic activities able to release varietal aroma [59, 60], or the improvement of the mouthfeel and flavor by the production/release of polyalcohols, polysaccharides [61, 62], acids [39, 45], etc.

The isolation of wild yeasts and the subsequent sequencing and comparison of the rDNA can help to elucidate the yeast microbioma from a vineyard (**Figure 4**). Normally, when the wild yeast populations are evaluated at the grape maturity stage, several mold species are frequently found together with apiculate yeasts such as those which belong to the genus *Kloeckera* or *Hanseniaspora*, making difficult to isolate and identify *S. cerevisiae* strains. Apiculate yeast can reach populations of 2–4 log CFU/mL.
