**4. Discussion**

A combination of quality, health, and economic reasons will force wine producers to find e fficient strategies that enable the production of wines with lower ethanol content without detriment on sensory properties. In this work, the strategy employed for this purpose was the use of sequential combinations between non-*Saccharomyces* and *S. cerevisiae* yeas<sup>t</sup> strains. It is well documented that non-*Saccharomyces* strains are often unable to consume all sugar present in a grape must [13,46]. Hence, sequential culture application would allow the completion of fermentation using one *S. cerevisiae* strain in a second instance [40,47–49]. The successful trials will be carried out by non-*Saccharomyces* strains with a low ethanol yield or those that are able to aerobically metabolize sugars without the simultaneous production of ethanol, prior to *S. cerevisiae* inoculation [2,50]. Regarding aeration regimen, some authors have suggested the use of aerobic yeasts in order to oxide sugars at early stages of winemaking and therefore decrease ethanol production [30,51,52]. After *S. cerevisiae* inoculation, researchers have favored anaerobic conditions to increase the ethanol yield of *Saccharomyces* strain and to avoid excessive oxidation of wine. The fermentation procedure programmed in this work was found to have positive results with other authors [50,51,53].

Several studies have evaluated the action of non-*Saccharomyces*/*S. cerevisiae* combinations in the reduction of ethanol content in wines [19–22,25–29,50,54]. In some cases, the lower ethanol yields resulted from high residual sugar at the end of fermentation [20,21,50]. By contrast, other research works have reported wines with a significant reduction in ethanol yield (0.6−1.7%, *v*/*v*) when using non-*Saccharomyces* and *S. cerevisiae* strains in mixed or sequential cultures. Contreras et al. [22] found that sequential inoculation of a selected *M. pulcherrima* strain (AWRI1149) with *S. cerevisiae* wine strain was the best combination for reducing the ethanol content in Chardonnay (0.9%, *v*/*v* lower than control) and Shiraz (1.6% *v*/*v* lower than control) wines. In the same way, Varela et al. [26] obtained Merlot wines fermented with *M. pulcherrima* with 1.0% less ethanol than *S. cerevisiae*-fermented wines at pilot scale. Further studies also showed ethanol reduction using immobilized selected strains of non-*Saccharomyces* yeasts followed by inoculation of free *S. cerevisiae* cells [25,47]. The sequential cultures of *M. pulcherrima* and *Starmerella bombicola* immobilized cells and *S. cerevisiae* free cells were the best for ethanol reduction with values 1.4% and 1.6% v/v, respectively [25]. In addition, ethanol lowering has been recorded in wines obtained by di fferent *Saccharomyces* species. Using sterile Shiraz must, sequential inoculation of *M. pulcherrima* (AWRI1149) and *S. uvarum* (AWRI2846) with *S. cerevisiae* produced wines with 0.9% *v*/*v* less ethanol than *S. cerevisiae* alone [55]. Puškaš et al. [29] also observed that sequential cultures with *M. pulcherrima, S. bayanus*, and *S. cerevisiae* generated wines with 0.9% *v*/*v* lower ethanol than control. In the present work, the application of sequential cultures of native non-*Saccharomyces* strains (*W. anomalus* 21A-5C, *M. guilliermondii* CLI 1217, and *M. pulcherrima* CLI 68 and CLI 460) and *S. cerevisiae* CLI 889 generated a reduction of alcohol content between 0.8%−1.3% *v*/*v* in Malvar wines, where *M. pulcherrima* CLI 68/*S. cerevisiae* CLI 889 sequential inoculation produced the highest decrease in alcohol degree. On the other hand, *W. anomalus* has been described as low fermentative species in pure culture compared to *S. cerevisiae* [28,31]. This statement is consistent with our results where *W. anomalus* 21A-5C presented 121 g/<sup>L</sup> of residual sugars after the first 96 h (Table S1). In sequential culture with *S. cerevisiae*, previous works denoted that the presence of *W. anomalus* does not a ffect final alcohol contents [28]. Instead, the strain studied in this work (*W. anomalus* 21A-5C) produced wines with 0.9% *v*/*v* less ethanol than control, in agreemen<sup>t</sup> with Contreras et al. [19] who studied another strain of the *W. anomalus* species. Finally, the use of *M. guilliermondii* as a low-ethanol producer has not been well documented. Some research works have studied *M. guilliermondii* as a candidate for reducing ethanol content in wines, but none have considered its use for that purpose [19,56]. In contrast, *M. guilliermondii* CLI 1217 in sequential culture was the second-best option to decrease the ethanol concentration in Malvar wines (1.2% less ethanol than control).

Beyond ethanol, the growth of the four selected non-*Saccharomyces* affected glycerol and acetic acid concentrations in Malvar wines. Several studies have reported that the production of glycerol by yeasts leads to an increase in acetic acid concentration [57,58]. Wines produced with *M. pulcherrima* strains CLI 68 and CLI 460 contained the greatest glycerol content (8.32 and 9.30 g/L, respectively) compared with other wines studied. The connection between *M. pulcherrima* and an increased glycerol production has been explained by the overexpression of the glycerol-3-phosphate dehydrogenase 1 (*GDP1*) gene in *S. cerevisiae* (associated with the conversion of dihydroxyacetone phosphate in glycerol-3-phosphate, an intermediate for glycerol formation). This gene is overinduced when *S. cerevisiae* coexists with *M. pulcherrima* in must fermentations [59]. Moreover, glycerol formation has been demonstrated as the best strategy, followed by yeasts for producing wines with lower ethanol content [60]. This compound is present in semi-sweet and dry wines ranging from 5 to 14 g/L, although glycerol imparts sweetness at a threshold of about 5.2 g/<sup>L</sup> in dry white wines [61]. Unlike glycerol, acetic acid imparts an objectionable character to wine at elevated concentrations. This volatile acid becomes undesirable at concentrations over 0.7–1.1 g/L, depending on the style of wine; its optimal concentration is 0.2–0.7 g/<sup>L</sup> [61]. One reason for elevated acetic acid levels is usually related to aeration, which could lead to elevated oxygen levels during fermentation [23,30,52]. However, more acetic acid was produced in Malvar wines during the anaerobic period than during aerobic fermentation in the current work, in agreemen<sup>t</sup> with results observed by Röcker et al. [24]. All sequential fermentation between four selected non-*Saccharomyces*/*S. cerevisiae* in this article produced wines with elevated volatile acidity (>0.7 g/<sup>L</sup> of acetic acid), significantly increased after *S. cerevisiae* inoculation (Table S1, see values of acetic acid caused by non-*Saccharomyces* fermentations). This noticeable increase could be caused by a lack of nutrition sources available for *S. cerevisiae* in the second part of fermentations [62,63]. Low YAN values (below 200 mg N/L, such as the Malvar must we studied) can also lead to elevated acetic acid concentration [64].

For selection of low-ethanol producing wine yeast, its impact on aroma profile is of grea<sup>t</sup> importance. Sequential cultures in this work had an important influence on higher alcohol proportions compared with the control. High levels of these volatile compounds (>300 mg/L) can have a detrimental e ffect on wine aroma, while concentrations below 300 mg/<sup>L</sup> can contribute positively to aroma complexity [65,66]. All wines produced using sequential inoculations presented values of higher alcohols below 300 mg/L.

It is worth noting that isoamyl alcohol (harsh, bitter) and β-phenylethyl alcohol (flowery, roses) are increased by sequential culture with *W. anomalus* 21A-5C and *M. guilliermondii* CLI 1217 strains. The ethyl isovalerate and isoamyl acetate esters, which impart fruity (banana) and sweet aromas, were also higher in these sequential cultures. In relation with *W. anomalus* species, these results agree with other publications [28,67–70]. Rojas et al. [67] indicated that one *W. anomalus* (*P. anomala*) strain produced the highest isoamyl acetate concentration in 48 h cultures in aerobiosis conditions; moreover, the increment in acetates was also observed in sequential cultures with *W. anomalus* and *S. cerevisiae* [28,70]. In addition to increasing alcohols, as well as ethyl and acetate esters [69], Airen white wines elaborated with *W. anomalus*/*S. cerevisiae* sequential cultures were judged to be better than *S. cerevisiae* monoculture due to their higher scores for descriptors as fruity and floral, and having an intense sweet smell and longer-lasting aftertaste [68]. Nevertheless, *M. guilliermondii* has been considered as a spoilage yeas<sup>t</sup> in winemaking that is able to produce large amounts of volatile phenols [71], identified with horse, stable, leather, or medicinal notes [72]; in contrast, the *M. guilliermondii* CLI 1217 strain used in sequential culture in the present work has contributed to rising amounts of fusel alcohols and some esters related to fruity and floral character in Malvar white wines.

Apart from high levels of isoamyl alcohol and β-phenylethyl alcohol previously documented by authors [22,24,73–75], sequential cultures with *M. pulcherrima* strains (CLI 68 and CLI 460) also showed an elevated proportion of isobutanol (bitter, fusel, alcohol) compared to the wine fermented solely with *S. cerevisiae*. This high isobutanol content is in good agreemen<sup>t</sup> with the experimental data reported previously [24,28,76]. While some reports [22,73,77] have stated that wines inoculated with *M. pulcherrima*/*S. cerevisiae* contain higher concentration of esters, other studies [16,54,74,78,79] have noted that wines fermented with these yeas<sup>t</sup> species in combination have lower concentrations, as in the case of this work. Moreover, it is worth mentioning that sequential culture with *M. pulcherrima* native strains presented higher concentration of esters with fruity aroma (ethyl isovalerate, isoamyl acetate

and ethyl-3-hydroxybutyrate) than the control. On the other hand, Malvar wines elaborated with *M. pulcherrima* CLI 68 and CLI 460 strains are mostly related to volatile fatty acids. These compounds are generally associated with negative aromas in wine [80], although hexanoic, octanoic, and decanoic fatty acids impart mild and pleasant notes to wine at concentrations between 4 to 10 mg/L; however, their impact can be negative on wine at levels above 20 mg/<sup>L</sup> [81]. Thus, these fatty acids might have a positive effect on the aroma of *M. pulcherrima*/*S. cerevisiae* Malvar wines since their levels are below 20 mg/L.

Relative to wine fermented with the control, *S. cerevisiae* CLI 889, we found a higher total concentration of esters in Malvar wines using *S. cerevisiae* monoculture. This *S. cerevisiae* strain produced wines with a fruity and floral character due to the greater concentration of ethyl isobutyrate (pineapple), ethyl hexanoate (pineapple, apple), and 2-phenylethyl acetate (flowery, lilac) esters, being the perfect candidate to ferment Malvar musts, and improving the typicity of the wines produced in the area "Vinos de Madrid".
