Icewine is a type of dessert wine that is fermented from juice squeezed from naturally frozen grapes (Lan et al., 2019). Ice grape juice generally contains high soluble solids level (>35°Bx). Icewine is characterized by high levels of titratable acid (>6.5 g/L, represented as tartaric acid), residual sugar (>125 g/L), as well as aromatic compounds (Bowen and Reynolds, 2015). Because of the specific climatic conditions required for planting and harvesting ice grapes, icewine is only produced in a few countries, such as Canada, Germany, Austria, China, and the United States (Alessandria et al., 2013; Bowen and Reynolds, 2015). In recent years, the Chinese icewine industry has developed rapidly, and China has become an icewine producer that cannot be ignored (Huang et al., 2018). In China, Huanren is a major region that produces icewine, and Vidal blanc is the main variety of grapes for icewine manufacturing in this region. Vidal blanc grapes are resistant to cold in winter because of their thick skin. Vidal blanc icewine is considered the highest quality icewine because of its appealing aroma and attractive flavor (Crandles et al., 2015). In addition to Vidal blanc, Riesling, Chenin Blanc, Chardonnay, Beibinghong are other common grape varieties used in icewine production (Lan et al., 2019).

Aroma is one of the most important aspect of wine quality, and the majority of aromatic compounds are formed during the fermentation process. Alcoholic fermentation of grape juice is a complex biochemical process carried out by dynamic microbiota, in which yeast plays an important role (Li et al., 2019). In the process of conversion of ice grape juice into icewine, the composition changes dramatically under the action of both Saccharomyces and non-Saccharomyces yeasts (Li et al., 2019). Several studies have shown that non-Saccharomyces yeasts positively contribute to the aroma and flavor of wine by secreting certain enzymes (such as esterases, β-glucosidase, β-xylosidase, and pectinase; Maturano et al., 2012; Hong et al., 2019). Therefore, in recent years, mixed culture of non-Saccharomyces yeast during fermentation is an emerging strategy for improving the wine aroma complexity, which has also gradually attracted attention from winemakers and enhanced market competitiveness (Lencioni et al., 2016; Padilla et al., 2016; Englezos et al., 2018). However, the level of current knowledge on the impact of non-Saccharomyces yeast on wine quality is still far from satisfactory. Indeed, some non-Saccharomyces species with application potential isolated from spontaneous fermentation of wine have not been studied enough, and knowledge about strain heterogeneity within a single non-Saccharomyces species and the effect of strain-specificity on wine aromas is still limited (Azzolini et al., 2015; Hong et al., 2021). At present, the studies regarding icewine mainly have focused on the aroma compounds and flavor characteristics of traditional icewine inoculated singly with Saccharomyces cerevisiae (Bowen and Reynolds, 2015; Huang et al., 2018; Lan et al., 2019). But, there are little information on the application of non-Saccharomyces yeast in icewine fermentation and the effects of the multiculture of non-Saccharomyces and S. cerevisiae strains on aroma profiles of icewine. The fermentation conditions of icewine are different from those of general wine, which are characterized by high sugar concentrations, high acid concentrations and low fermentation temperature (Zhang et al., 2018).

Torulaspora delbrueckii is a non-Saccharomyces species with excellent fermentation capacity, which is often studied and used in winemaking in recent years (Azzolini et al., 2015; Zhang et al., 2018). These studies revealed that mixed culture of T. delbrueckii during wine fermentation can decrease volatile acidity and acetaldehyde content, and increase fruity esters, higher alcohols and other positive aroma components (Loira et al., 2014; Velázquez et al., 2015; Belda et al., 2017). Moreover, T. delbrueckii was the first non-Saccharomyces species to be produced into active dry yeasts for winemaking, but the knowledge about T. delbrueckii strain heterogeneity and the impact of strain-specificity on wine aromas is also very little (Azzolini et al., 2015). In addition, the use of T. delbrueckii strains during icewine fermentation has not been found.

In recent years, the use of indigenous yeasts such as Candida zemplinina, Hanseniaspora uvarum, Metschnikowia pulcherrima to increase the complexity of wine aroma has gradually become a trend of winemaking with regional identities (Liu et al., 2016; Tofalo et al., 2016). In our previous study, we investigated that extensive yeast strains were isolated from Vidal blanc icewine during the spontaneous fermentation (Li et al., 2019), and then tolerance, and aroma-related enzymes activities of these yeast strains were studied in order to select the indigenous strains (including T. delbrueckii strains used in this study) with the potential to produce characteristic icewine (Hong et al., 2019). In this paper, based on the abilities of indigenous T. delbrueckii strains to adapt to the icewine micro-environment and their fermentation performances, we used them during fermentation and analyzed the aroma of the icewine. The main objective of this study was to reveal the effects of mixed culture of indigenous T. delbrueckii strains and S. cerevisiae (contrasted with pure culture of S. cerevisiae) during fermentation on aromatic profile of Vidal blanc icewine under laboratory-scale conditions.

In this study, we demonstrated the growth dynamic changes of T. delbrueckii and the effect of mixed culture of T. delbrueckii and S. cerevisiae during fermentation on aroma compounds of Vidal blanc icewine, pure S. cerevisiae fermentation was used as the control. During mixed culture fermentations, T. delbrueckii gradually decreased and disappeared at different times, which might be ascribed to the competition for nutrients or cell–cell contact mechanisms during fermentations (Albergaria and Arneborg, 2016), or the ability of different S. cerevisiae strains to produce alcohol and the tolerance of specific T. delbrueckii strains toward ethanol (Hong et al., 2019).

Due to the inoculation of T. delbrueckii during fermentation, the basic chemical compositions of the icewines were affected, among which acetic acid was the most important to the quality of icewine. Acetic acid concentration was decreased, which is also consistent with the conclusion of Bely et al. (2008) and Comitini et al. (2011). Azzolini et al. (2015) pointed out that the reduction of acetic acid concentration can be extremely important to the results of sensory analysis of sweet wine. Some authors also reported T. delbrueckii as a low acetic acid producer compared with most non-Saccharomyces yeasts (Renault et al., 2009; Belda et al., 2015). Meanwhile, ethanol concentration was also decreased, which is consistent with the result of Belda et al. (2015). The reduction of acetic acid and ethanol concentration may be due to the consumption of sugar by T. delbrueckii for production of glycerol or pyruvic acid, or to an increase in yeast biomass, thus limiting the later S. cerevisiae fermentation activity (Kutyna et al., 2010; Belda et al., 2015; Lino et al., 2021). Nowadays, for consumer health and economic factors, as well as wine quality reasons, the wine sector is increasingly demanding technology to facilitate the production of wines with lower ethanol content (Kutyna et al., 2010; Contreras et al., 2014); the application of non-Saccharomyces yeasts during wine fermentation is one of the strategies. Several previous studies have shown that non-Saccharomyces yeasts usually have the effect of reducing ethanol content, reporting reductions higher than 1% in final ethanol concentration (Rantsiou et al., 2012; Contreras et al., 2014; Giaramida et al., 2016); in this study, it was reduced by about 2%. In order to combat the osmotic stress caused by the ice grape must, yeast cells may allocate carbon resources from sugar metabolism to produce metabolites necessary for adaptation and survival, of which glycerol is one (Heit et al., 2018; Lino et al., 2021). Glycerol is an important product of yeast fermentation, and its concentration in dry wine is usually 4–10 g/L, and higher glycerol levels are considered to improve wine quality (Heit et al., 2018). Regarding the increase in glycerol concentration, this is due to T. delbrueckii has a highly active glycerol-pyruvic pathway (Renault et al., 2009). In addition, differences in the fermentation kinetics among pure and mixed fermentations were evident (Supplementary Figure S1). Compared to the control fermentations, the lower fermentation kinetics of mixed culture fermentations were observed, which illustrated the fermentation kinetics appeared to be driven by the presence of S. cerevisiae.

In terms of higher alcohols, isoamylol and 2-phenylethyl alcohol are produced by yeasts during fermentation through conversions of leucine and phenylalanine via the Ehrlich pathway (Hazelwood et al., 2008). 2-Phenylethyl alcohol has pleasant notes with “honey,” “spice,” “rose,” and “lilac”; there was a relative increase of 2-phenylethyl alcohol in mixed fermented icewines, which is consistent with studies by Sun et al. (2014) in cherry wine, Chen and Liu (2016) in lychee wine and Zhang et al. (2018) in red wine. Higher isoamylol and 2-phenylethyl alcohol concentrations may be due to T. delbrueckii have preferences for leucine and phenylalanine consumption. 1-hexanol was associated with “vegetal” and “herbaceous” odor, which usually weaken the pleasant aroma quality of wines (Englezos et al., 2018); Sadoudi et al. (2012) and Zhang et al. (2018) demonstrated that 1-hexanol in wine fermented by S. cerevisiae was lower level than that in the wine of mixed culture fermented with T. delbrueckii and S. cerevisiae, which is consistent with the results of this study. However, 1-propanol, which usually has a positive influence on wine aroma with a ripe fruit note (Cai et al., 2014), was higher level in pure fermented icewines than in mixed culture fermented icewines. These results revealed that there may be some certain interaction between S. cerevisiae and T. delbrueckii for these alcohols, most probably a synergistic interaction (Sun et al., 2014). Moreover, 1-pentanol was associated with “balsamic” and “bitter almond” odor, and 1-heptanol has “oily” odor (Sánchez-Palomo et al., 2010); these two alcohols were apparently produced by T. delbrueckii participating during the icewine fermentation process. Besides, higher alcohols detected in mixed fermented icewines indicated that in addition to the diversity of T. delbrueckii strain, S. cerevisiae strain is an important factor in determining the formation of higher alcohols.

Esters are considered to provide positive contributions to wine’s fruity and flowery notes, which are produced by esterification of alcohols and acids at low pH (Saerens et al., 2010). Acetate esters are formed by condensation of higher alcohols with acetyl-coA, and catalyzed by ATF1 and ATF2 of alcohol acyl-transferases (AAT) genes in yeast cells (Peddie, 1990); they were thought to have a greater influence on wine aroma than ethyl esters (Lilly et al., 2000). In this study, we found that the inoculation of T. delbrueckii during mixed culture fermentation could promote the production of isoamyl acetate, which is consistent with studies by Renault et al. (2015). However, contradictory results were reported by Puertas et al. (2018). This may be due to the fact that T. delbrueckii was not totally displaced by S. cerevisiae in the study of Puertas et al. (2018), while T. delbrueckii was completely replaced by S. cerevisiae at the middle and later fermentative stages in the current study (Figure 1), and T. delbrueckii possessed a great hydrolytic activity of isoamyl acetate via esterase (Velázquez et al., 2015). Moreover, hexyl acetate has “apple,” “cherry,” and “pear” odor, propyl acetate has “celery” odor, and isobutyl acetate was associated with flowery fragrance (Hong et al., 2021). Ethyl acetate contributes to wine aroma with fruity note when the concentrations below 150 mg/L, otherwise it may negatively impart wine to nail polish remover odor (Swiegers et al., 2005). The effect of T. delbrueckii on these important volatile acetate esters seems to be pronounced, as demonstrated by the strong decline in ethyl acetate, isobutyl acetate and hexyl acetate in mixed fermented icewines. Similar results were previously reported (Sadoudi et al., 2012; Azzolini et al., 2015). This is mainly due to the reduction of acetic acid level by inoculation with T. delbrueckii (Lu et al., 2017). As for ethyl esters, they are formed by the reaction of ethanol with volatile fatty acid during the process of lipid biosynthesis (Saerens et al., 2010). Most ethyl esters in mixed culture fermented icewines were lower levels than those in pure fermented icewine; this is in agreement with the literature (Renault et al., 2015; Puertas et al., 2018).

In terms of fatty acids, the contribution of T. delbrueckii on wine aroma was clearly evident. The level of octanoic acid and decanoic acid in TD1 icewines were higher than those in icewines inoculated with pure S. cerevisiae, while the inoculation of TD2 could reduce the level of decanoic acid, and octanoic acid could not even be detected, which may be related to the production of their respective esters (Lu et al., 2017). Azzolini et al. (2015) pointed out that reductions in fatty acids can be considered positive as they are generally responsible for negatively affecting the overall wine aroma. Therefore, T. delbrueckii and S. cerevisiae mixed culture fermentation may be a strategy to modulate the production of ethyl esters and fatty acids in icewine.

As far as aldehydes are concerned, the sensory quality of decanal and dodecanal is generally considered to have no significant effect on wines (Bakker and Clarke, 2004). Dodecanal were not found in mixed fermented icewines, which might be due to their oxidation to corresponding acids or reduction to corresponding alcohols, and then conversion to related esters (Lu et al., 2017). Notably, 2, 4-dimethylbenzaldehyde was detected in icewine fermented by T. delbrueckii and S. cerevisiae for the first time.

Linalool is associated with flowery fragrance, and D-limonene has “citrus” and “fresh orange” odor. Linalool was increased in the icewines of sequential inoculation T. delbrueckii/S. cerevisiae, which is in agreement with recent studies (Azzolini et al., 2012; Zhang et al., 2018). The formation and levels of terpenes in mixed fermented icewines were evidently different, which may be related to the β-glucosidase activity of T. delbrueckii strains and terpene bioconversion rate (Sadoudi et al., 2012).

Notably, β-damascenone, which was described as providing sweet, exotic flower notes and honey flavor, has been reported as a key odorant in the aroma characteristics of Vidal icewine; small level changed of this metabolite can have a significant effect on sensory evaluation of icewine (Hong et al., 2021).

The results of pure fermentation showed that SC2 had different aromatic characteristic compared to SC1, for example, 1-hexanol, diethyl succinate and hotrienol were detected in SC1 pure fermented icewine, but not in SC2 pure fermented icewine; as expected, mixed culture of T. delbrueckii with SC1 or SC2 achieved distinct profiles of aromatic compounds. Meanwhile, there were significantly different levels in some aroma metabolites between the icewines fermented with TD1 and the icewines fermented with TD2, such as decanoic acid, D-limonene and cineole (Table 2; Figure 2); this result indicated that the strain-specificity of T. delbrueckii caused the different effects on icewine aroma metabolites.

In addition, the metabolic pathways of the main metabolites in icewine, such as ethanol, glycerol, organic acids, and terpenes, are more closely related to carbon sources. But, nitrogen is the main factor for yeast growth and fermentation activity, this parameter would be considered in our future research.

To the best of our knowledge, this is the first study on the application of indigenous T. delbrueckii in Vidal blanc icewine fermentation, and volatile aroma compounds in the icewine fermented by T. delbrueckii and S. cerevisiae. The aromatic profiles of icewines under different fermentation strategies were distinct, which was significant for knowing more about the influences of T. delbrueckii on icewine quality. The inoculation of T. delbrueckii in icewine fermentation could decrease the concentration of acetic acid and ethanol, and increase the glycerol concentration; and produce more 2-phenylethyl alcohol, isoamyl acetate, linalool, D-limonene, p-cymene and cineole; and intensify the fruity, flowery, and sweet characteristic of icewine. Thus, the utilization of indigenous T. delbrueckii strains in mixed fermentation provided a potential way to improve aromatic complexity of icewines product and impart their unique regional flavor. Furthermore, the relevance of strain-specificity within T. delbrueckii to aroma compound differences was shown, which would provide insights for further investigations on the utilization of indigenous non-Saccharomyces yeasts strains in icewine making.

The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author.

JL designed the experiments and wrote the manuscript. BQ analyzed the experimental data. MH conducted the experiments. All authors contributed to the article and approved the submitted version.

The conducted research received funding from the Department of Education of Liaoning Province, under grant agreement no. JYTQN2020033.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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