**3. Glycerol**

Glycerol is the most abundant yeas<sup>t</sup> metabolism by-product after ethanol and CO2. It is produced from dihydrodroxyacetone phosphate, which is first reduced to glycerol-3-phosphate via glycerol-3-phosphate dehydrogenase (GPDH), and then converted into glycerol by a specific phosphatase. This is a non-volatile 3-hydroxy alcohol, which is a polyol also known as a sugar alcohol. Glycerol is a viscous liquid at room temperature and appears to contribute to mouthfeel and viscosity at, or above 28 g/<sup>L</sup> and sweetness in the range of 5–12 g/<sup>L</sup> [42,61–63]. However, Nieuwoudt, et al. [64] did not find any link between wine quality (expressed as the number of medals received at a wine competition) and concentrations of glycerol in wine, and Goold et al. [42] concluded in their review that glycerol had only a minimal influence on the viscosity of wine.

The synthesis of glycerol and acetic acid, in addition to ethanol, are both linked to redox balance [22,42]. The significance of glycerol synthesis to redox balance has been suggested to be due to the inability of mutants (unable to synthesise glycerol) to grow in anaerobic conditions [22,65]. During the stationary phase of yeas<sup>t</sup> during fermentation, glycerol synthesis has been found to be associated with redox balance by removing excess reducing power [22,66].

Many factors can influence the production of glycerol, which is in general more abundant in wines fermented with non-*Saccharomyces* than those fermented with *S. cerevisiae* [67–69], and in red wine (approximately 10.5 g/L) compared to white wines (approximately 7 g/L) [64]. Glycerol is generally more abundant in red wines in part because red juice typically ferments at higher temperatures (20–25 ◦C) than white wines (<20 ◦C). Yet, fermenting temperatures positively influence the production of glycerol by yeasts, and non-*Saccharomyces* and *S*. non-*cerevisiae* are not an exception. For example, increasing the fermentation temperature from 16 to 20 ◦C increased the glycerol content from 1.69 to 3.04 g/<sup>L</sup> in co-fermentations of *Candida stellata* and *S. cerevisiae* [52]. A significant increase in the glycerol content of a grape juice was also observed after increasing the temperature from 12 to 25 ◦C, with fermentations carried out by pure *Saccharomyces paradoxus* reporting an increment of approximately 2.5 g/L, for example [12]. The sugar level of grapes at harvest (and therefore in the juice) also influences the production of glycerol, because this compound is accumulated by yeas<sup>t</sup> to combat dehydration by balancing the intracellular osmolarity with that of the medium [1]. This effect has become even more evident in recent times due to hotter seasonal temperatures compressing the ripening windows of different grape varieties in warm climates. This has meant that winemakers delay the harvest date because of wineries operating at full capacity, thereby causing a part of the crop to overripe in the vineyard [70]. Hranilovic, et al. [71] reported that the glycerol content of a Shiraz wine from early harvest grapes (approximately 265 g/<sup>L</sup> TSS) was much higher than those from the late harvest (approximately 325 g/<sup>L</sup> TSS), with a *M. pulcherrima* strain (followed by a *S. cerevisiae* inoculum), producing early and late harvest wines with 10.51 and 12.59 g/<sup>L</sup> glycerol, respectively. Juice with high sugar concentration also leads to an excess in acetic acid, which can be explained by yeasts trying to maintain redox balances by using surplus NAD(P)+ accumulated during the synthesis of fermentation metabolites [62,72–74].

The growth of non-*Saccharomyces* yeas<sup>t</sup> species such as *Lachancea thermotolerans*, *T. delbrueckii*, and *M. pulcherrima* strictly depends upon oxygen availability [57,75,76]. When the effect of oxygen availability on glycerol production by non-*Saccharomyces* was evaluated, oxygenation at three dissolved oxygen levels of 0.08, 0.41, and 1.71 mg/<sup>L</sup> resulted in glycerol reduction [76]. This was evident for a *T. delbrueckii* strain in co-fermentation with *S. cerevisiae*, with the glycerol content decreasing from 6.79 g/<sup>L</sup> in the *T. delbrueckii* anaerobic treatment up to 1.09 g/<sup>L</sup> in the co-inoculation treatment with 1.71 mg/<sup>L</sup> of dissolved oxygen. Different results were reported by Morales et al. [57], who observed increased glycerol yields for *M. pulcherrima*/*S. cerevisiae* mixed cultures under controlled oxygenation conditions (sparged with pure air, nitrogen, or mixtures of both) during the first 48 h of fermentation, and anaerobic conditions thereafter.

The non-*Saccharomyces* yeasts, which have the capacity to redirect the sugar consumption for the production of alternative compounds, rather than ethanol, have been studied in wines

with reduced ethanol content. These alternative compounds could be glycerol and pyruvic acid produced via glycerol–pyruvic metabolisms. Alternatively, before sugar is utilised during alcoholic fermentation, sugars can be consumed via respiratory metabolism [40], which is the case with various non-*Saccharomyces* yeasts with lower Crabtree e ffect.

Other factors can have an impact on the formation of glycerol by non-*Saccharomyces*. These include the concentration of nitrogen and sulfites [8]. Limited nitrogen concentrations in the must (in the form of amino acids and ammonium) can lead to a significant increase of glycerol production. By contrast, higher levels of sulfur dioxide lead to higher levels of glycerol [30]. Increased glycerol production has been found to be linked to increased acetic acid, which is easily detected due to its vinegar smell [3,18,77].

Upon a thorough literature review, we identified five non-*Saccharomyces* yeasts that have been widely studied due to their di fferent oenological traits; *Schizosaccharomyces pombe, Metschnikowia pulcherrima, Lachancea thermotolerans, Candida stellata,* and *Torulaspora delbrueckii.* These five non-*Saccharomyces* yeasts have di fferent pathways that result in ethanol reduction and glycerol production while influencing di fferent parameters of the chemical composition of wines. The following sections of this review present current knowledge regarding the e ffect of alternative yeasts that influence ethanol and glycerol concentrations while highlighting gaps in our knowledge that require further research.

## **4. Schizosaccharomyces Pombe**

*Schizosaccharomyces pombe* is a widely studied yeas<sup>t</sup> due to its particular ability to moderate wine acidity via malic acid degradation [78]. Among its other promising traits is the ability to enhance the color of red wines and reduce Ochratoxin A, biogenic amines, and ethyl carbamate [79–81]. The most detrimental metabolites produced by spoilage yeasts in pure culture or spoiled juices have been found to decease in mixed fermentations carried out at the laboratory scale [2,82]. The main characteristics of *S. pombe* and its application in winemaking were reviewed recently [83,84]. *S. pombe* is commercially available as an alternative method to de-acidity wine [19].

Unlike some non-*Saccharomyces* yeas<sup>t</sup> species, *S. pombe* is capable of fermenting wines up to comparable concentrations of *Saccharomyces,* in the range of 10–15% *v*/*v* ethanol, depending on the strain and presence of aeration [85]. Malo-alcoholic fermentation [86] and the glycerol–pyruvic pathway [87] observed in *S. pombe* inoculations have di fferent impacts on the final ethanol content of the wines. Therefore, while some studies reported an ethanol reduction with inoculations involving *S. pombe* [87], others have reported no di fference or even an increase [88–91].

The use of *S. pombe* (strain 938) for white winemaking was investigated by Benito et al. [87], with sole, mixed, and sequential fermentations in conjunction with *S. cerevisiae (Cru Blanc).* All the strains in the study were able to ferment the wines to dryness. The sole fermentation of *S. pombe* showed 0.65% *v*/*v* lower ethanol compared to *S. cerevisiae* alone, values being 13.18% *v*/*v* and 12.53% *<sup>v</sup>*/*<sup>v</sup>*, respectively. Similar results were obtained with the sequential inoculation, with 0.4% *v*/*v* lower ethanol value in comparison to the control treatment [87]. Loira et al. [88] studied the e ffect on Syrah wine sensory quality of *S. pombe* strains (938, V1 and 4.2) in mixed and sequential fermentations with *S. cerevisiae* strain 7VA. *S. pombe* was not used as a sole inoculum in this study. Ethanol reduction did not occur in the mixed or sequential fermentations with *S. pombe* and *S. cerevisiae* compared to treatments with a sole inoculum of *S. cerevisiae*. In this study, slightly higher ethanol values that were not statistically significant were reported for mixed and sequential fermentations with *S. pombe* strains (13.2% to 13.5% *v*/*v*) compared to *S. cerevisiae* (13.2% *v*/*v*) [88]. Although the same strains of *S. pombe* (938) were used in these studies, di fferent delay times were applied for the sequential inoculation of *S. cerevisiae* (48 h versus 7 days), and di fferent strains of *S. cerevisiae* were used for the comparison.

Increased ethanol levels have been reported in studies that used *S. pombe* where juices had high malic acid content. *S. pombe* (Y0119) was used in a sequential inoculation with *S. cerevisiae* (NT116) to ferment Kei-apple (*Dovyalis ca*ff*ra L.*) juice, which contained high malic acid (45 g/L). An increase in ethanol level (6.08% *v*/*v*) was reported compared to sole inoculation with *S. cerevisiae* (4.67% *v*/*v*) [91]. Similarly, slightly higher ethanol values (approximately 0.2% to 0.5% *v*/*v*) were reported in a study where *S. pombe* was used in sequential inoculation with *S. cerevisiae* for fermenting plum juice (*Prunus domestica L.*), compared to *S. cerevisiae* as a control [90]. Studies that included *S. pombe* that measured glycerol reported that the yeas<sup>t</sup> produced lower levels of glycerol than the other yeasts in the study (Table 2) [20].

## **5. Metschnikowia Pulcherrima**

*Metschnikowia pulcherrima* is a non-*Saccharomyces* yeas<sup>t</sup> that is commercially available from many manufacturers [10,19]. Its ability to enhance varietal aroma compounds [50,92] and reduce the ethanol content of wines has raised interest in its commercial use [93,94]. Recently, the impact of *M. pulcherrima* in winemaking has been reviewed [95].

The ability of *M. pulcherrima* for withstanding ethanol concentrations of up to 7% *v*/*v* has been reported by Combina et al. [96]. Recently, four strains of *M. pulcherrima* were identified as being able to actively grow at a higher ethanol concentration (9% *v*/*v*) [97]. Sixty-two of the 65 strains isolated from the Douro region of Portugal in this study were able to tolerate a 6% *v*/*v* ethanol level. On the other hand, its ability to ferment was reported up to levels of 4% *v*/*v* in micro-fermentations conducted in pasteurised grape must [13]. Consequently, different co-inoculation and sequential inoculation strategies have been studied [14,57,93].

Contreras et al. [49] identified a *M. pulcherrima* strain that can reduce the ethanol content of wine through part of a sequential inoculation with *S. cerevisiae*. Lower ethanol levels of 0.9% and 1.6% *v*/*v* were achieved in Chardonnay and Shiraz wines, respectively, compared to a sole inoculum of *S. cerevisiae,* which found ethanol levels of 15.1% *v*/*v* in Chardonnay and 13.8% *v*/*v* in Shiraz wines. A similar result of 0.9% *v*/*v* ethanol reduction was reported in a study carried out with the sequential inoculation of *M. pulcherrima* with *S. cerevisiae* compared to a sole inoculum of *S. cerevisiae* that produced ethanol levels of 13.2% *v*/*v* [93]. Furthermore, a mixed inoculum of *M. pulcherrima* and *S. uvarum* with sequential inoculation of *S. cerevisiae* was conducted. Two different *M. pulcherrima*/*S. uvarum* inoculum ratios were applied (1 × 10<sup>6</sup> cells/mL to 1 × 10<sup>5</sup> cells/mL, and 1 × 10<sup>6</sup> cells/mL to 1 × 10<sup>4</sup> cells/mL). Both resulted in an ethanol reduction of 1.7% *<sup>v</sup>*/*<sup>v</sup>*, along with higher concentrations of succinic acid and glycerol, compared to wine fermented with *S. cerevisiae* [93]. Varela et al. [50] studied *M. pulcherrima* and *S. uvarum,* both of which were found to be able to produce wines with reduced ethanol content. Along with the ethanol reduction, the sensory profile and volatile aromatic composition of Merlot wines were studied. Fermentation was conducted via co-inoculation using *M. pulcherrima* (1 × 10<sup>6</sup> cells/mL) and *S. cerevisiae* (1 × 10<sup>5</sup> cells/mL). An ethanol reduction of 1.0% *<sup>v</sup>*/*<sup>v</sup>*, along with higher concentrations of ethyl acetate, total esters, total higher alcohols, and total sulfur compounds were detected in wines fermented with a co-inoculation of *M. pulcherrima* and *S. cerevisiae,* compared to wines fermented with *S. cerevisiae*. Although a higher production of ethyl acetate and total sulfur-containing compounds was detected, the sensory panel did not detect associated negative attributes in the wines [50].

The effect of different aeration regimes and immobilisation on the ethanol reduction with selected strains of *M. pulcherrima* has been studied [98]. An ethanol reduction of 1.38% *v*/*v* was achieved in first 72 h of fermentation of Verdicchio must with *M. pulcherrima* under an aeration flow of 20 mL/L/min, compared to the control that used *S. cerevisiae* [98]. A blend of Malvasia and Viura (Macabeo) must was fermented in a study by Morales et al. [57] using a mixed culture of *M. pulcherrima* and *S. cerevisiae*, and using different aeration regimes (sparged with air or nitrogen). The lowest ethanol values were reported for the treatments sparged with air and fermented with the mixed culture of *M. pulcherrima* and *S. cerevisiae*, regardless of the inoculum level of *S. cerevisiae* (1% or 10%). In these treatments, 11% *v*/*v* ethanol was obtained, compared to 14.7% *v*/*v* and 12.9% *v*/*v* ethanol in the treatments fermented with *S. cerevisiae* sparged with nitrogen and air, respectively. However, high acetic acid values (higher than 0.65 g/L) obtained under air-sparged treatment produced wines that would have been unacceptable for consumers and do not meet market regulations were reported. In contrast, treatments sparged

with nitrogen and fermented with the mixed culture of *M. pulcherrima* and *S. cerevisiae* had an ethanol reduction of 0.8% *v*/*v* compared to those fermented with *S. cerevisiae*, with acceptable levels of acetic acid (lower than 0.1 g/L) [57]. The same level of ethanol reduction was achieved (0.8% *v*/*v*) using a sequential fermentation with Viura/Macabeo and Malvasia varieties using *M. pulcherrima* with *S. cerevisiae* compared to the control (*S. cerevisiae*) under non-aerated conditions [99].

The ability of selected immobilised non-*Saccharomyces* yeasts (*Starmerella bombicola* (formerly named *Candida stellata*), *Metschnikowia pulcherrima*, *Hanseniaspora osmophila*, and *Hanseniaspora uvarum*) to reduce the ethanol content in wine via sequential fermentation has been also studied [14]. In synthetic grape juice, the sequential inoculation of *M. pulcherrima* had 1.14% to 1.35% *<sup>v</sup>*/*<sup>v</sup>*, with 48 and 72 h delays in inoculation of *S. cerevisiae,* respectively. Ethanol concentration was reduced by 1.10% to 1.46% *v*/*v* in natural grape juice in the same study [14]. Similarly, Röcker et al. [47] studied five non-*Saccharomyces* yeas<sup>t</sup> strains including *M. pulcherrima* for a sequential inoculation with *S. cerevisiae* var. *bayanus* strain under three different aeration conditions (aeration with sterile pressured air during 15 and 5 days, and under regulated oxygen content of 20% dissolved oxygen), to ferment Riesling must. Ethanol concentration was reduced by up to 3.8% *v*/*v* within 3 days of aeration, although the authors reported an increase in vinegar (associated with acetic acid) and oxidation sensory attributes [47].

Three non-*Saccharomyces* yeas<sup>t</sup> strains (*M. pulcherrima, T. delbrueckii,* and *Zygosaccharomyces bailii*) fermented Chardonnay using sequential inoculation with *S. cerevisiae* under three different aeration conditions (no air addition, 5 mL/min aeration (0.025 VVM), 10 mL/min aeration (0.05 VVM)). The authors reported that the sequential inoculation with *M. pulcherrima* reduced alcohol by 1.6% *<sup>v</sup>*/*<sup>v</sup>*, which was the highest ethanol reduction among all the non-*Saccharomyces* yeas<sup>t</sup> strains in the study, compared to *S. cerevisiae* [43]. In this study, applied aeration conditions did not cause an increase in the acetic acid production. However, in the wines produced with a sequential inoculation of *M. pulcherrima* and *S. cerevisiae*, an over-production of ethyl acetate (280 mg/L) was detected under the aeration regime of 0.05 VVM. This indicates an oxygen threshold for the over-production of this compound [43], which causes unpleasant odors such as nail polish remover and vinegar [100].

The ability of *M. pulcherrima* to reduce the final ethanol content via its respiratory characteristic has been shown with various studies (Table 1). Recently, *M. pulcherrima* has been reported to produce lower levels of glycerol under semi-anaerobic conditions than when the air flow into the fermentations was 1 mL/L/min and 20 mL/L/min (Table 2). Therefore, taken together in respect to ethanol and glycerol content, future research with *M. pulcherrima* (solo, sequential, or co-fermentations) should include a range of aeration strategies, a range of inoculum rates, and an investigation into the length of time before inoculation of *S. cerevisiae* yeast, to render its application feasible at a commercial winemaking scale to achieve ethanol reduction without compromising or enhancing sensory characteristics [14,43,47,59,93,97].

#### **6. Lachancea Thermotolerans**

*Lachancea thermotolerans* (previously *Kluyveromyces thermotolerans*) is available commercially from yeas<sup>t</sup> manufacturers [19,101]. This yeas<sup>t</sup> has specific oenological traits including a positive influence on wines' sensory profile [102] and total acidity [13,103]. The main characteristics of *L. thermotolerans* and its effects on winemaking were recently reviewed [101,104–106].

*L. thermotolerans* in pure culture was able to reach 10.46% *v*/*v* ethanol levels in micro-fermentations of pasteurised commercial white wine must, conducted at 25 ◦C, by leaving more than 50 g/<sup>L</sup> residual sugar. The control wine inoculated with a pure culture of *S. cerevisiae* EC-1118 fermented the must to dryness (less than 6 g/<sup>L</sup> residual sugar), reaching an ethanol level of 13.04% *v*/*v* [102]. The study continued at industrial scale, and an ethanol reduction of 0.7% *v*/*v* was achieved with a Sangiovese must [102]. In the study by Del Fresno et al. [89], a higher ethanol reduction of 1.2% *v*/*v* was achieved. This was compared to a different *S. cerevisiae* strain (7VA)*,* which was sequentially inoculated later than the previous study (6 days), with a higher inoculum amount (both at 10<sup>8</sup> cell/mL). The laboratory-scale

fermentation using Tempranillo must conducted at 25 ◦C and 12.66% *v*/*v* ethanol level used sequential inoculation, while sole inoculation of *S. cerevisiae* 7VA was 13.84% *v*/*v* [89].

As well as its ability to decrease the pH of wine, the metabolic pathway of *L. thermotolerans* that can turn sugars into lactic acid is also described as a way to reduce the level of alcohol in wines [18]. A study conducted using micro-vinifications of Airén must conducted at 25 ◦C [80] with *L. thermotolerans* 617 (100 mL containing 2.27 × 10<sup>7</sup> CFU/mL) followed by the sequential inoculation of *S. cerevisiae* 87 (10<sup>7</sup> CFU/mL of) with 96 h of delay, an ethanol reduction of 0.4% *v*/*v* was achieved, compared to the sole inoculation of *S. cerevisiae* 87. This sequential inoculation also showed a higher lactic acid production (3.18 g/L) and lower pH (3.52, compared to 3.74 in control wine). The sensory panel perceived this treatment to have better sensorial properties, with higher scores for sweetness, despite similar levels of residual sugar compared to control. This was explained by the possible effect of higher L-lactic acid concentration produced by the effect of *L. thermotolerans.*

Other beneficial compositional effects reported in *L. thermotolerans* treated wines include increases in glycerol at concentrations high enough (>5 g/L) that they could be of sensory relevance. As shown by Kapsopoulou et al. [107] for grape must containing 160 g/<sup>L</sup> sugar (with 7.4 g/<sup>L</sup> titratable acidity, at pH 3.5), glycerol formation was significantly higher (5.75 g/L) when *S. cerevisiae* TH941 (5 × 10<sup>5</sup> cell/mL) was inoculated 3 days after the inoculation of *L. thermotolerans* SCM952 (5×10<sup>5</sup> cell/mL). This was compared to sole *S. cerevisiae* fermentations (4.82 g/L) and other sequential inoculations (after 1 and 2 days). The authors attributed this to the increased survival of the *L. thermotolerans* strain in the co-fermentation treatment in comparison to the other treatments. Likewise, a very high glycerol level (11.22 g/L) was reported in Sangiovese red wines obtained at a commercial scale by the sequential inoculation of *S. cerevisiae* EC1118 (10<sup>6</sup> cell/mL) 2 days after that of *L. thermotolerans* 101 (10<sup>7</sup> cell/mL) [102]. This value was significantly higher compared to the control (9.02 g/L) and the co-inoculated *L. thermotolerans* and *S. cerevisiae* treatments (9.68 g/L). Comitini et al. [13] inoculated non-*Saccharomyces* yeasts in combination with the *S. cerevisiae* EC1118 strain as a starter. All non-*Saccharomyces* strains were inoculated at 10<sup>7</sup> cell/mL, while the EC1118 starter strain was inoculated at three different concentrations: 107,105, and 10<sup>3</sup> cell/mL. Of all the inoculations tested, those of *L. thermotolerans* with *S. cerevisiae* produced higher levels of glycerol (6.95–7.58 g/L) at all three ratios than when *S. cerevisiae* was inoculated on its own (6.23–6.56 g/L). Contrary to the *C. zemplinina* and *M. pulcherrima* inoculations, no significant differences were found between the three different inoculum ratios (1:1, 100:1, 10,000:1) of *L. thermotolerans* with *S. cerevisiae*. This suggests that glycerol production may not correlate with cell concentration and persistence of the *L. thermotolerans* yeast, at least according to this study. Where the effects of sequential and co-inoculations of *L. thermotolerans* with *S. cerevisiae* yeasts on glycerol concentration were evaluated, the glycerol content in sequentially inoculated fermentations (7.55 g/L) was higher than those observed in the co-inoculated treatments (7.18 g/L) [80].

These findings sugges<sup>t</sup> that sequential fermentation with *L. thermotolerans* and *S. cerevisiae* remains a viable option for winemakers. Further investigations on the sensory quality of the wines could help identify if these variations in glycerol are discernable by consumers and preserve the wines from the formation of off-flavours.

#### **7. Candida Stellata**/**Starmerella Bombicola**

*Candida stellata* is a widely studied yeas<sup>t</sup> due to its positive contributions to wine, including its capacity to produce desirable metabolites such as glycerol [72], and its ability to carry out enzymatic activities that have positive effects on wine sensory attributes. An extensive review of *C. stellata* was carried out by García et al. [108]. Recent studies uncovered the mistake of referring to *C. zemplinina* instead of *C. stellata*, which may explain the disputable characterisations of the oenological traits of these species [109]. For instance, recently, a commonly used strain of *C. stellata,* DBVPG 3827, was reclassified as *Starmella bombicola* [108].

Traditionally dominating overripe, infected, or botrytised grape berries, *C. stellata* is reported to tolerate at least 9% *v*/*v* ethanol concentration, while at 15 ◦C, its growth was recorded at 11% *v*/*v* ethanol concentration, with decreased tolerance at both 10 ◦C and 30 ◦C [22,108,110].

Soden et al. [72] studied the effect of inoculations with *C. stellata* and *S. cerevisiae* on Chardonnay juice. The treatments included sole yeas<sup>t</sup> fermentations of *C. stellata* and *S. cerevisiae*, as well as co-inoculation and sequential inoculation conducted at a room temperature of 18 ◦C. *C. stellata* in sole fermentation was not able to consume all the sugar, reaching an ethanol concentration of only 5.8% *<sup>v</sup>*/*<sup>v</sup>*. Therefore, *S. cerevisiae* was used for the sequential inoculation, and added after the fermentation activity of *C. stellata* had ceased, with an inoculation density of 5 × 10<sup>6</sup> cells/mL for both yeasts. The resulting wine was dry and had a significantly lower ethanol concentration (11.8% *v*/*v*) in comparison to the control with a sole inoculation of *S. cerevisiae* (12.5% *v*/*v*) [72]. The same authors also highlighted in their study the increases in glycerol concentration in the wines fermented with *C. stellata* in comparison to sole *S. cerevisiae* fermentations. The glycerol levels varied from 5.2 g/<sup>L</sup> in the co-inoculation treatment up to 15.7 g/<sup>L</sup> in the sequential inoculation trial. Similar results for ethanol reduction were found by Ferraro et al. [111] but using immobilised cells of *C. stellata* and *S. cerevisiae* on Trebbiano Toscano grape must fermented at 20 ◦C. Inoculum of *S. cerevisiae* at 5 × 10<sup>6</sup> cells/mL was added after 3 days of fermentation. The sequential fermentation of immobilised cells of *C. stellata* and *S. cerevisiae* reached an ethanol level of 10.6% *v*/*v* compared to the control (11.24% *v*/*v*), which was inoculated with *S. cerevisiae* only [111]. The ethanol reduction was explained by the significant increase (approximately 70%) in glycerol as a consequence of low fermentation rate and reduced production of ethanol with immobilised cells of *C. stellata* [112].

Immobilised cells of *Starmerella bombicola* (formerly referred to as *Candida stellata)* were used in sequential inoculation with *S. cerevisiae* on Verdicchio grape must. The fermentation was conducted at 25 ◦C and the inoculation of *S. cerevisiae* (1 × 10<sup>6</sup> cell/mL) with 72 h of delay. The effect of the removal of the immobilised *S. bombicola* cells was investigated [14]. The 1.07% and 1.64% *v*/*v* less ethanol achieved with and without the removal of *S. bombicola* beads, respectively, was notable compared to inoculation with *S. cerevisiae.* A significant increase was seen in the concentration of by-products such as glycerol and succinic acid. The ethanol reduction was explained by the production of by-products from glycerol–pyruvic fermentation or other metabolic pathways [14].

Both growing in similar environmental conditions, including high sugar-containing musts, and possessing similar taxonomic and oenological profile characteristics, *C. zemplinina* (synonym *Starmerella bacillaris* [113]) and *C. stellata* have produced contrasting results in previous literature. Magyar and Tóth [114] evaluated the oenological characteristics of some yeas<sup>t</sup> strains, including four strains of both *C. stellata* and *C. zemplinina*. While both *C. stellata* and *C. zemplinina* showed a strong fructophilic character, *C. stellata* showed higher ethanol and glycerol yield and the same level of volatile acidity compared to *C. zemplinina* [114].

The role of *Candida zemplinina* (synonym *Starmerella bacillaris*) as a tool to produce wines with less ethanol levels but higher glycerol concentrations has been extensively studied and recently reviewed [115]. *Candida* isolates were obtained from Sicilian musts by Di Maio et al. [116] and sequential inoculations with three different *C. zemplinina* strains and *S. cerevisiae* (NDA21) were conducted on Nero d'Avola must. The highest ethanol reduction was 0.3% *v*/*v* obtained with the sequential inoculation of *C. zemplinina Cz3* strain, compared to a pure inoculation of *S. cerevisiae* (NDA21), along with higher glycerol content [116]. A similar level of ethanol reduction (0.3% *v*/*v*) and higher glycerol production was recorded in the study conducted by Rolle et al. [117] on Barbera must. In this study, two different *C. zemplinina* strains were used (FC54 and C.z03). The inoculation of *C. zemplinina* (10<sup>6</sup> cells/mL) was followed by the inoculation of *S. cerevisiae* Uvaferm BC (10<sup>6</sup> cells/mL) after 2 days. Fermentations of natural grape must by sequential inoculations of *C. zemplinina* with *S. cerevisiae* EC1118 increased the level of glycerol (5.45–6.30 g/<sup>L</sup> in the final wine), and remarkably produced less acetaldehyde and total SO2 compared to the other yeasts [118].

## **8. Torulaspora Delbrueckii**

*Torulaspora delbrueckii* was one of the first commercially available non-*Saccharomyces* yeasts and is currently available in dry or frozen form from many yeas<sup>t</sup> manufacturers and suppliers [3,10,19]. Some of the oenological traits of *T. delbrueckii* reported in studies include optimising wine quality parameters i.e., enhancing aroma composition, and positively impacting the foam properties for traditional methods of sparkling wine [119–122]. Studies have been conducted using this yeas<sup>t</sup> on different wine styles, and its effect on the winemaking practices was recently reviewed [123–125].

Significant ethanol reductions due to the utilisation of *T. delbrueckii* in sequential fermentations have been reported when compared to pure inoculations with *S. cerevisiae* (Table 1). Nevertheless, some studies reported slight ethanol reductions (lower than 0.2% *v*/*v*) [125] or none [88]. Additional, higher ethanol reduction levels were achieved when aeration was integrated during the fermentation process, which stimulates aerobic metabolism [56]. Contreras et al. [56] carried out a sequential inoculation with *T. delbrueckii* AWRI1152, followed by inoculation of *S. cerevisiae* AWRI1631 when 50% of sugar was consumed. Four different aeration regimes were applied (air or nitrogen), and fermentations were conducted at 22 ◦C with agitation (200 rpm) in a chemically defined grape juice medium. With the aeration at 5 mL/min (0.025 VVM) for the first 24 h of the fermentation, an ethanol reduction of 1.5% *v*/*v* was achieved, compared to the control of *S. cerevisiae* under anaerobic conditions. No increase in acetic acid levels occurred; however, the impact on the flavour profile of the wine requires further investigation [56]. To answer this question, Canonico et al. [43] studied three non-*Saccharomyces* yeas<sup>t</sup> strains (*M. pulcherrima, T. delbrueckii,* and *Zygosaccharomyces bailii*) and their ability to reduce ethanol under limited aeration conditions. In this study, sequential inoculation with *T. delbrueckii* resulted in 0.9% to 1% *v*/*v* lower ethanol, depending on the aeration strategy (0.025 VVM and 0.05 VVM) compared to control of *S. cerevisiae* under anaerobic conditions. Furthermore, wines fermented with *T. delbrueckii* under aerobic conditions showed a favorable balance between ethanol reduction and volatile profile [43].

Recent studies showed that the nutrient supplementation has a positive correlation on the ethanol yield of *T. delbrueckii* [126,127]. Additionally, Mecca et al. [127] studied three commercially available *T. delbrueckii* strains and compared some oenological characteristics. Significant differences were reported in ethanol yields, as well as in the volatile aroma compounds [127]. Concerning the glycerol content of wines made using *T. delbrueckii,* a range of 4.1–8.9 g/<sup>L</sup> has been reported in wines (Table 2) [125,128,129]. It has been suggested that *T. delbrueckii* has a more developed glycerol–pyruvic pathway than other yeasts, although some studies have found no differences in glycerol production [91,124,130].

#### **9. Other Non-Saccharomyces and Saccharomyces Non-cerevisiae Yeasts**

In addition to the yeasts mentioned in the sections above, new yeasts are constantly being investigated for their ability to ferment wines and their role in uninoculated fermentations. Contreras et al. [56] studied *Zygosaccharomyces bailii* in sequential inoculation under different aeration conditions in chemically defined grape juice medium. With the aeration at 5 mL/min (0.025 VVM) throughout the fermentation, an ethanol reduction of 2% (*v*/*v*) was achieved, as well as a significantly lower acetic acid compared to the control of *S. cerevisiae* under anaerobic conditions. Using the same *Z. bailii* strain in sequential inoculation, in Chardonnay must, Canonico et al. [43] reported a significant ethanol reduction (1% *v*/*v*) under aeration (0.05 VVM). In this study, a lower ethanol reduction (0.8% *v*/*v*) was achieved with a lower aeration (0.025 VVM); however, the resultant wine had a promising volatile profile including individual esters, higher alcohols, and volatile acidity.

Various studies have reported ethanol reduction with non-*Saccharomyces* yeasts from different genus, including *Pichia* and *Hanseniaspora* (Table 1). Maturano et al. [11] studied two non-*Saccharomyces* yeasts, *H. uvarum* and *C. membranaefaciens*, with the sequential inoculation with *S. cerevisiae* on Malbec must. Taking a step further, three fermentation factors—inoculum size, time prior to inoculation of *Saccharomyces cerevisiae*, and fermentation temperature—were optimised using a Box–Behnken

experimental design [11]. By applying the optimised factors, the highest ethanol reduction with *H. uvarum* was achieved with an inoculum size of 5 × 10<sup>6</sup> cells/mL and a delay of 48 h 37 min before *S. cerevisiae* inoculation with the fermentation temperature of 25 ◦C. However, for *C. membranaefaciens*, the optimised factors were di fferent: an inoculum size of 2.72 × 10<sup>6</sup> cells/mL, delay of 24 h 15 min before *S. cerevisiae* inoculation, and fermentation temperature of 25 ◦C was used. The study showed that the time before the inoculation of *S. cerevisiae* affected the ethanol production of the non-*Saccharomyces* yeasts. The high sugar consumption ability of *H. uvarum* via oxidative metabolism was reported to be the reason for this [11].

The application of sequential inoculations with long delays prior to *S. cerevisiae* inoculation in winery environments could be challenging. Competitive native or wild *S. cerevisiae* species present in the winery environment can dominate the fermentation before achieving the expected effect from the inoculated non-*Saccharomyces* yeasts [14]. A recent study by Canonico et al. [43] reported the volatile profile of reduced ethanol wines. Finding an acceptable balance between ethanol reduction, volatile aroma profile, and sensory characteristics of the wines is crucial. Therefore, studies conducted on a pilot scale that includes sensory analysis of the reduced alcohol wines made from non-*Saccharomyces* yeasts is lacking. Nitrogen managemen<sup>t</sup> is an important factor to achieve ethanol reduction, which has been highlighted by authors in previous studies [126,127,131]. The specific nutrient needs of non-*Saccharomyces* yeasts used for ethanol reduction purposes should be further studied.




**Table 1.** *Cont*.


**Table 1.** *Cont*.

Recent studies were conducted with the aim of determining the combinations of two non-*Saccharomyces* yeas<sup>t</sup> species (*S. pombe* and *L. thermotolerans*) [138]. The combination of these yeasts was studied at a laboratory scale using micro-vinifications of Tempranillo grape must [79,89,139,140]. Higher levels of glycerol concentrations were reported in mixed fermentations with *L. thermotolerans* (CONCERTO™) and *S. pombe* (inoculated simultaneously at 10<sup>6</sup> cfu/mL), compared to a control with *S. cerevisiae* [79,139,140]. Glycerol increase varied between 0.27 and 0.71 g/L, which may be related to the strain of *S. pombe* used (V2 and 4.5) and control *S. cerevisiae* (88 and CECT 87). A different strain of *S. pombe* (938) was studied in combination with *L. thermotolerans* (CONCERTO™) [89]. Two inoculum ratios of *S. pombe*/*L. thermotolerans* (1:1 and 1:3) were used for the laboratory-scale fermentations with Tempranillo must. The authors reported significantly lower glycerol values in mixed fermentation treatments (5.02 g/<sup>L</sup> and 6.78 g/L) compared to the control fermentation with *S. cerevisiae* 7VA (7.42 g/L) [89]. Therefore, more studies need to be conducted to understand the effect of the interaction between these two non-*Saccharomyces* yeasts.

Combinations of non-*Saccharomyces* and *Saccharomyces* non-*cerevisiae* yeasts for wine fermentations have been investigated, including *M. pulcherrima* and *S. uvarum* [93,141]. *M. pulcherrima* and *S. uvarum* were used in mixed inoculum in the study [93]. Glycerol levels of 12.30 and 12.48 g/<sup>L</sup> were reported from two different inoculum ratios of *M. pulcherrima*/*S. uvarum* (1 × 10<sup>6</sup> cells/mL to 1 × 10<sup>5</sup> cells/mL, and 1 × 10<sup>6</sup> cells/mL to 1 × 10<sup>4</sup> cells/mL, respectively) in laboratory-scale fermentations of Shiraz must. These values were significantly higher compared to the control wine fermented with *S. cerevisiae,* where 7.91 g/<sup>L</sup> glycerol concentration was reported. Although the highest level of glycerol (14.55 g/L) was reported in sequential inoculation with *S. uvarum* and *S. cerevisiae*, considering the values of ethanol reduction, a mixed inoculum of *M. pulcherrima* and *S. uvarum* could be a promising combination [93]. The same treatments were applied to Chardonnay must in the study by Varela et al. [141]. Similarly, significantly higher glycerol levels were reported in both inoculum ratios of mixed fermentation of *M. pulcherrima*/*S. uvarum* (11.90 g/<sup>L</sup> and 12.63 g/L) compared to control wine fermented with *S. cerevisiae* (8.20 g/L). Unlike the study of Contreras et al. [93], the reported values in the mixed fermentations were not significantly different from the sequential inoculation of *S. uvarum* and *S. cerevisiae,* in which a

glycerol level of 11.19 g/<sup>L</sup> was reported [141]. Considering the ethanol decrease and glycerol levels together, the combined inoculation of *M. pulcherrima* and *S. uvarum* shows promising results.

Applying di fferent non-*Saccharomyces* and *Saccharomyces* non-*cerevisiae* yeasts, a wide range of glycerol concentrations has been reported in studies (Table 2) of still and sparkling wines, as well as white and red wines (3.5 g/<sup>L</sup> to 15.9 g/L). Glycerol levels above 5.2 g/<sup>L</sup> may directly a ffect the style of the wine by influencing sensory characteristics including the sweetness, body, and structure of the wines [56]. Therefore, the selection criteria for the use of these yeasts should always consider the sensory characteristics of the final wines. In the case of sparkling wines, glycerol levels have an impact on the viscosity, volatile aroma compounds, and foaming [142]. High levels of glycerol produced by some *Saccharomyces* non-*cerevisiae* yeasts, such as *Saccharomyces kudriavzevii* [143] or *Schizosaccharomyces japonicus* [132] during base wine fermentation, may not make them suitable candidates for achieving a complete second alcoholic fermentation in sparkling winemaking [8]. Additionally, studies have reported an increase of acetic acid when glycerol is produced by some yeasts [8,73].


**Table 2.** Glycerol concentrations in wines from non-*Saccharomyces* and *Saccharomyces* non-*cerevisiae* yeas<sup>t</sup> studies.


**Table 2.** *Cont*.

\* Two or more products of the same yeas<sup>t</sup> strain used in the study. ‡ *Saccharomyces* non-*cerevisiae* yeast.
