**3. Results**

The genetic identification of the 33 non-*Saccharomyces* strains from vineyards and cellars of D.O. "Vinos de Madrid" allowed them to be classified into 13 species belonging to 10 different genera. The initial strain selection was designed to include species frequently isolated from the winemaking environment. Moreover, our strategy for ethanol reduction was the use of one non-*Saccharomyces* strain that exhibited a low ethanol yield but consumed enough sugars to affect the ethanol concentration (Section I) and be compatible with *S. cerevisiae* in order to ensure the completion of fermentation (Section II).

#### *3.1. Section I: Pure Culture of Non-Saccharomyces Strains*

In this section of the work, we studied fermentative kinetics of non-*Saccharomyces* strains and the control strain (*S. cerevisiae* CLI 889) in pure cultures. The fermentative profiles during the first 96 h permitted the division of the strains into three different groups: A, B, and C (Figure 1). Group A was represented by four non-*Saccharomyces* species: *Wickerhamomyces anomalus* (two strains), *Candida stellata* (one strain), *Lachancea thermotolerans* (one strain), and *Hanseniaspora guilliermondii* (two strains), which showed similar CO2 released to *S. cerevisiae* CLI 889 control strain. Group B included all *Torulaspora delbrueckii* and *Hanseniaspora valbyensis* strains studied in this work together with the other three *L. thermotolerans* strains. These strains showed less fermentative capacity than the control, with a CO2 loss between 2.2−1.2 g against above 3 g liberated by *S. cerevisiae* CLI 889. Nine different non-*Saccharomyces* species were represented within group C. This latter group had 19 of 33 non-*Saccharomyces* strains studied, wherein their fermentation kinetics presented the lowest CO2 liberation observed during the first 96 h.

**Figure 1.** Release of CO2 in aerobic conditions. (**A**) Six non-*Saccharomyces* strains showed similar CO2 released to Sc CLI 889 control strain (white circles). (**B**) Eight non-*Saccharomyces* strains showed less CO2 released than the control (white circles). (**C**) Nineteen non-*Saccharomyces* strains showed values below 1 g of CO2 liberated from the control (white circles). Wa, *W. anomalus*; Cs, *C. stellata*; Lt, *L. thermotolerans*; Hg, *H. guilliermondii*; Sc, *S. cerevisiae*; Td, *T. delbrueckii*; Hv, *H. valbyensis*; Mg, *M. guilliermondii*; Hu, *H. uvarum*; Mp, *M. pulcherrima*; Pc, *P. carsonii*; Hv, *H. vineae*; Zb, *Z. bailii*; Pm, *P. membranifaciens*.

#### *3.2. Section II: Sequential Culture of Non-Saccharomyces*/*S. cerevisiae Strains*

After 96 h, *S. cerevisiae* CLI 889 was sequentially inoculated into all fermentations in Section I. A total of 8 days were needed by yeas<sup>t</sup> strains to complete the fermentation process (Sections I and II).

Yeast isolates with fermentation behavior showing in group A (Section I) did not exhibit an increase on the CO2 release, producing similar amounts of ethanol at the end of fermentation—all of these wines were about 13% (*v*/*v*).

When sequential fermentations finished, some strains combinations produced wines with ethanol concentration similar to the control (13%, *v*/*v*), and thus they were discarded as low-ethanol cultures. These sequential combinations that were not selected included the strains CLI 679, CLI 1218, 31-1C, CLI 457, CLI 72, CLI 219, CLI 461, CLI 463, CLI 1221, and CLI 903; all of them were classified into group C (Section I). Another group of non-*Saccharomyces*/*S. cerevisiae* fermentations, including CLI 918, CLI 194, CLI 1219, AMB FF4 10A, CLI 190, and LS1 FF2 3A strains from group B (Section I), and CLI 225, CLI 622, CLI 417, 6-5A, and CLI 3 strains from group C (Section I), increased by between 7% and 10% in terms of ethanol concentration, but high amounts of residual sugars were not fermented, and thus these combinations were not selected either; most of them belonged to group B (Section I) in which CO2 liberated was lower than the control with values between 1.18 and 2.19 g. Finally, four non-*Saccharomyces*/*S. cerevisiae* sequential inoculations produced wines with decreased ethanol proportions compared with the control, and the residual sugars values were suitable for dry wines (<5 g/<sup>L</sup> residual sugar) (Table 2).

**Table 2.** Oenological parameters and cell dry weight for the best non-*Saccharomyces*/*S. cerevisiae* sequential combinations to reduce ethanol concentration in wines.


Data are means ± standard deviation (*n* = 2). Data with different letters (a,b,c) within each row are significantly different (Tukey test; *p* < 0.05). (S), sequential culture; (P), pure culture.

#### *3.3. Yeast Strain Sequential Combinations Selected as Low-Ethanol Producers*

In order to reduce the ethanol content in wines, the selected non-*Saccharomyces* yeas<sup>t</sup> strains were *W. anomalus* 21A-5C, *Metschnikowia pulcherrima* CLI 68, *Meyerozyma guilliermondii* CLI 1217, and *M. pulcherrima* CLI 460 used in sequential combination with *S. cerevisiae* CLI 889. These co-cultures produced wines with between 0.8% (*v*/*v*) and 1.3% (*v*/*v*) lower ethanol concentrations in Malvar wines, showing significant di fferences from the control (Table 2).

Sequential cultures inoculated with 21A-5C, CLI 68, and CLI 460 produced more glycerol than the control, highlighting *M. pulcherrima* CLI 460 strain with values significantly higher than the control (Table 2). There were no significantly di fferences in malic and lactic acid content, and fermentations with sequential combinations generated more acetic acid than the amount produced by the *S. cerevisiae* control (Table 2). Regarding dry weight, all sequential fermentations presented greater values compared with the control; in particular, sequential culture of *M. guilliermondii* CLI 1217 was 1.6-fold higher, showing significant di fferences (Table 2).

To find the aromatic composition of these wines, we studied 32 volatile compounds classified in alcohols, esters, acids, and aldehydes/ketones (Table 3). Sequential inoculation produced Malvar wines with greater total concentration of higher alcohols. The amounts of isoamyl alcohol (harsh, bitter) and β-phenylethyl alcohol (flowery, roses) were significantly higher in wines produced in sequential culture, increasing the total concentration of alcohols. The ethyl isovalerate and isoamyl acetate ester concentration responsible for fruity and sweet aromas were significantly di fferent in wines generated from sequential inoculations. Regarding volatile acids, isobutyric acid and hexanoic acid were the main compounds responsible for the total concentration of volatile acids in all wines. The sequential culture *W. anomalus* 21A-5C/*S. cerevisiae* CLI 889 produced the highest concentration of the ketone acetoin. Finally, sequential cultures with *M. pulcherrima* strains (CLI 68 and CLI 460) and the control showed higher amounts of γ-butyrolactone, related to sweet aroma in wines.


**Table 3.** Major volatile compounds (mg/L) of wines produced in the Section II (sequential culture of non-*Saccharomyces* strains + *S. cerevisiae* CLI 889 and a control, *S. cerevisiae* CLI 889 pure culture).

Data are means ± standard deviation (*n* = 2). Data with different letters (a,b,c,d,e) within each row are significantly different (Tukey test; *p* < 0.05). n.q., not quantifiable. (S), sequential culture; (P), pure culture.

A PCA analysis was performed to cluster wines from sequential combinations and the control according to their oenological and aromatic composition. In the score plot for the first two principal components, PC1 and PC2 explain 75.9% of the total variance (Figure 2). PC1 was mainly determined by ethyl hexanoate (0.986), ethyl octanoate (0.950), decanoic acid (0.944), 2-phenylethyl acetate (0.916), total esters (0.916), 1-propanol (0.916), γ-butyrolactone (0.903), and alcohol degree (0.831); this component allowed us to differentiate the control *S. cerevisiae* fermentation from those fermentations conducted by sequential non-*Saccharomyces*/*S. cerevisiae* combinations. The principal constituents for PC2 were total volatile acids (0.903), butyric acid (0.840), hexanoic acid (0.810), glycerol (0.780), fructose (0.773), and isobutanol (0.740). PC2 differentiated the sequential fermentations among them, showing clearly two groups. One group formed by sequential inoculations with *W. anomalus* 21A-5C/*S. cerevisiae* CLI 889 and *M. guilliermondii* CLI 1217/*S. cerevisiae* CLI 889, mostly related to dry weight, isoamyl alcohol, acetoin, total aldehydes/ketones, isoamyl acetate, ethyl isovalerate, and β-phenylethyl alcohol in the loadings plot (Figure 2B). Another group contained the sequential cultures with *M. pulcherrima* strains (CLI 68 and CLI 460), mainly classified by total acids, butyric acid, ethyl lactate, hexanoic acid, glycerol, fructose, and isobutanol.

**Figure 2.** Principal component analysis (PCA) score plot (**A**) and loadings plot (**B**) using main fermentation parameters and 32 volatile compounds.
