**6. Ternary sequential inoculations in warm areas: biotechnological approach to improve freshness**

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


#### **Table 1.**

*Potential combinations of three yeasts to improve freshness.*

*M. pulcherrima*, and finally the sugars are completely depleted by *S. cerevisiae* to obtain a dry wine. In ternary fermentations, the use of several non-*Saccharomyces* species to improve aroma and flavor must be completed with *L. thermotolerans* to decrease pH, improve the acidity, and, therefore, enhance the wine freshness. Lastly, the sugars are finished by *S. cerevisiae* or alternatively *S. pombe*. Using the latter species, it would be possible to make interesting wines in the absence of *S. cerevisiae*.
