Wine fermentation is a complex process where the interplay between grape juice and its associated microbial populations, derived from both the vineyard and winery environments, shapes the chemical and sensory profiles of the resulting wine (Belda et al., 2017; de Celis et al., 2022; García-Izquierdo et al., 2024). Yeasts are particularly important in alcoholic fermentation, where they convert sugars into alcohol and carbon dioxide, while also producing various secondary metabolites that contribute to the wine’s flavor and aroma (Maicas and Mateo, 2023; Wang et al., 2023). The indigenous yeast communities that develop during spontaneous grape must fermentations are shaped by annual environmental conditions in the vineyard, agricultural practices, intrinsic factors of the grapevine, —including the physicochemical properties of the grape must, — and winemaking techniques (Varela and Borneman, 2016; Griggs et al., 2021; Liszkowska and Berlowska, 2021; de Celis et al., 2022; Maicas and Mateo, 2023). A consistent pattern of species emerges in the yeast population: non-Saccharomyces yeasts dominate the initial phase of fermentation, while Saccharomyces cerevisiae becomes the predominant species as fermentation progresses (Bezerra-Bussoli et al., 2013; Drumonde-Neves et al., 2016; Cilião Filho et al., 2017; Raymond Eder et al., 2017, 2018; Borren and Tian, 2021; de Celis et al., 2022; Torres-Guardado et al., 2022; Maicas and Mateo, 2023). Non-Saccharomyces yeasts, which are predominant on grape skins, are introduced into the must during grape crushing and play a significant role in the early fermentation stages, producing various secondary metabolites that strongly impact the organoleptic characteristics of wine (Kamilari et al., 2021; Morata et al., 2021; Tufariello et al., 2021; Romano et al., 2022; Torres-Guardado et al., 2022; Maicas and Mateo, 2023; Wang et al., 2023).

Understanding the dynamics of indigenous microbial communities of fermenting grape juices and musts is a central focus in enology, given its significance for both scientific research and industrial applications (Padilla et al., 2016; Varela, 2016; Griggs et al., 2021; de Celis et al., 2022; Maicas and Mateo, 2023; García-Izquierdo et al., 2024). An increasing number of studies point to a relationship between the grape microbiome and terroir, which includes factors such as soil composition, climate, and annual precipitation, suggesting that specific microbial populations may contribute to the regional identity of wines (Pretorius, 2017, 2020; Liu et al., 2020; Griggs et al., 2021; Kamilari et al., 2021; Zhang et al., 2023a). Moreover, it has been suggested that particular grape varieties harbor microbiomes involving specific yeast strains or species, influencing the unique characteristics of fermented grape juices (Baffi et al., 2011; Bezerra-Bussoli et al., 2013; Lederer et al., 2013; Raymond Eder et al., 2017, 2018, 2025; Raymond Eder and Rosa, 2019). We have recently suggested that Vitis-specific microbial communities play a critical role in shaping the identity of grape juice fermentations (Raymond Eder et al., 2025). However, it remains unclear how strongly a grape varietal–specific indigenous microbial community influences the final organoleptic properties of a given wine, how terroir-associated microbial signatures contribute to the wine’s identity and profile, and to what extent alternative microbiotas—such as those from different vintages or experimentally introduced from other Vitis species (Raymond Eder et al., 2025)—can alter the fermentation profile of a given grape varietal.

Although the OIV (International Organization of Vine and Wine) primarily recognizes Vitis vinifera L. as the species designated for vinification, other Vitis species and their hybrids are also used in winemaking, particularly in non-European countries (De la Fuente Lloreda, 2018). These non-vinifera Vitis species represent underexplored microbial ecosystems in enology and may serve as a potential source of yeasts with unique fermentative properties of both academic and industrial relevance (Drumonde-Neves et al., 2016; Raymond Eder et al., 2017, 2018; Raymond Eder and Rosa, 2019). Moreover, we have recently proposed that they can serve as powerful tools for studying the impact of indigenous microbiota on the aromatic profiles of fermenting grape juices (Raymond Eder et al., 2025). Building on the use of this innovative experimental system, in this study we investigate the impact of the indigenous microbiota of Malbec grapes—a conventional V. vinifera L. varietal used in winemaking—versus the native microbiota of Isabella grapes (V. labrusca L.), on the volatile profile at early stages of fermentation of Isabella grape juices.

Varietal or primary wine flavors originate from the grape variety itself. In addition to the volatile compounds naturally present in grape juice, secondary flavors are developed through alcoholic fermentation via yeast metabolism, which produces higher alcohols, esters, and fatty acids (Carrau et al., 2020; Borren and Tian, 2021; Zilelidou and Nisiotou, 2021; Romano et al., 2022; Liang et al., 2023; Wang et al., 2023; Zhang et al., 2023a). In spontaneous fermentations, the complex native microbial community associated to grapes contributes to the final volatile profiles of wines (Padilla et al., 2016; Belda et al., 2017). Indigenous grape-associated yeasts species are particularly relevant contributors to the volatile profiles of fermenting juices at early times of fermentation (Jolly et al., 2014; Pretorius, 2017; Raymond Eder et al., 2025). In addition to yeast, lactic-acid and acetic-acid bacteria, even when present at low concentrations at early times of fermentations, can also contribute to the final volatile compositions of spontaneous fermentations (Barata et al., 2012; Andorrà et al., 2019; Shimizu et al., 2023; Raymond Eder et al., 2025).

We have recently demonstrated that microbial communities associated to different Vitis species condition the volatile profiles of early-stage Malbec grape juice fermentations (Raymond Eder et al., 2025). In this work, we have extended these studies to analyze the volatile profiles of Isabella grape juices fermented using homologous (Isabella; V. labrusca L.) or heterologous (i.e., Malbec; V. vinifera L.) native microbial communities. Our analyses consider the profiles of free volatile compounds in grape juices, excluding glycoside-bound volatiles (Raymond Eder et al., 2025). Although bound volatiles contribute to the full aromatic potential of a grape juice —upon enzymatic or fermentative release— the analyzed free fraction offers a representative expression of varietal identity at the juice stage. Moreover, this approach avoids potential artifacts introduced by exogenous enzymes, yields reproducible data under our standardized conditions, and reveals the aromatic identity of fresh varietal grape juice. This experimental strategy allowed us to perform a joint analysis of the volatile compound datasets obtained from homologous and heterologous fermentations of Malbec (Raymond Eder et al., 2025) and Isabella (this work) grape juices, conducted with the same starter microbial communities and grape juices samples prepared from grapes harvested in vintage of year 2021 (Raymond Eder et al., 2025). Taken together, the results presented in this work show that the microbiota shapes the volatile profiles of the fermented Malbec and Isabella juices, which nonetheless retain the identity of their original grape varietal.

It has been reported that Isabella, a varietal from the American-originated Vitis labrusca L., is rich in aroma compounds both qualitatively and quantitatively (Ghaste et al., 2015). Key alcohols and terpenes, including methyl anthranilate, β-phenyl ethanol, ethyl-3-hydroxybutyrate, ethyl-β-hydroxy hexanoate, furaneol, phenylacetaldehyde, tryptophol, and 2-hexenol, have been detected in Isabella grape juices and crushed grapes originating from Brazil and Italy, respectively (Ghaste et al., 2015; Gomez et al., 2024). Our analysis revealed that Isabella juice from grapes harvested in the geographic region of Córdoba (Argentina) has a substantial proportion of alcohols, esters, and terpenes, including the previously reported methyl anthranilate, β-phenyl ethanol, ethyl-3-hydroxybutyrate, and methyl salicylate (Ghaste et al., 2015; Gomez et al., 2024). Although different techniques of extraction of volatile compounds were used, Isabella juice from Córdoba was found to contain eleven ethyl esters, whereas only two (i.e., ethyl-butanoate and ethyl-metacrylate) and four (i.e., methyl salicylate, methyl anthranilate, methyl-β-hydroxybutyrate, ethyl-3-hydroxyhexanoate) ethyl esters were detected in the Brazilian and Italian studies, respectively (Ghaste et al., 2015; Gomez et al., 2024). Previous research on the volatile profiles of Isabella and Ives commercial wines also emphasized the prominent role of ethyl acetate and esters, contributing to their characteristic fruity aroma descriptors (Arcanjo et al., 2018). In the present study, isoamyl alcohol, phenethyl alcohol, isoamyl acetate, phenethyl acetate, ethyl octanoate, ethyl decanoate, and ethyl dodecanoate emerged as the dominant volatile compounds during the initial stages of fermentation. Interestingly, as it was observed in our recent study of homologous and heterologous fermentation of Malbec grape juice (Raymond Eder et al., 2025), differences were observed in the volatile profiles of the Isabella fermenting grape juice when using the alternative either Malbec or Isabella associated microbial communities as starters. Homologous fermentations (Igj/Imc) show higher concentrations of ethyl octanoate, ethyl decanoate, phenethyl alcohol, phenethyl acetate, and ethyl phenylacetate than the heterologous conditions (Igj/Mmc) (Raymond Eder et al., 2025).

The evolution of culturable yeast populations in homologous and heterologous Isabella grape juice fermentations was studied using culture-dependent methods. Although his strategy limits our study to the identification of only cultivable yeast species from the overall fungal, yeast, and bacterial biodiversity in the samples, it is known that cultivable non-Saccharomyces yeast species significantly contribute to aromatic profiles in early fermentation stages (Andorrà et al., 2010; Borren and Tian, 2021; Zilelidou and Nisiotou, 2021; Romano et al., 2022; de Celis et al., 2024). At T0, Hanseniaspora spp. (H. opuntiae and H. uvarum) and, to a lesser extent, S. bacillaris, predominated in the fermented juices, consistent with previous studies on yeast communities from this region’s spontaneously fermenting musts and reconstituted grape juices (Raymond Eder et al., 2017, 2018, 2025). As fermentation progressed, from T0 to T96, Hanseniaspora spp. dominated the fermentations in Imc, while S. bacillaris increased its contribution in Mmc. It has been recently reported that the dominating yeast species in a fermentation defines its performance and metabolite profile of the resulting wines (de Celis et al., 2024). In this sense, members of the Hanseniaspora genus have been reported to play a significant role in the production of volatile compounds in wine, particularly acetate esters, in a strain-dependent manner (Jolly et al., 2014; Martin et al., 2018). Specifically, H. uvarum significantly increased ethyl and isoamyl esters and reduced acetate ester concentrations in co-fermentations with S. cerevisiae in a strain-specific way (Tristezza et al., 2016; Hu et al., 2018b, 2018a; Zhang et al., 2023b). H. opuntiae has been shown to positively influence volatile profiles and sensory characteristics of fermentations, significantly increasing acetate esters, mainly 2-phenylethyl acetate, and ethyl esters levels (Hu et al., 2020; del Fresno et al., 2022, 2023; Filippousi et al., 2024). Moreover, fermentation studies where Hanseniaspora spp. were the dominating yeasts, were characterized by higher fusel alcohol acetates production (de Celis et al., 2024). (i.e., phenethyl alcohol). On the other hand, S. bacillaris has been reported to overproduce ethyl and other acetate esters, as well as terpenes (Andorrà et al., 2010; Englezos et al., 2015; Raymond Eder and Rosa, 2021). However, elevated levels of these compounds were not detected in the Igj/Mmc fermentation, where this yeast was dominant at T96. Four other non-Saccharomyces species previously recognized in this terroir (i.e., Candida diversa, T. delbrueckii, H. vineae, and P. terricola) were also isolated at these fermentations. Proposed signature V. labrusca L. non-Saccharomyces species (i.e., Candida azymoides, Pichia cecembensis, and Candida californica) (Drumonde-Neves et al., 2016; Raymond Eder et al., 2017, 2018) were not identified among the limited number of isolates characterized in this study. In addition to the contribution to the volatile profiles of the main yeast species, increased yeast diversity has been linked to greater flavor complexity (Carrau and Henschke, 2021). In this context, the minor yeast species present at lower abundances in Igj/Imc may have also contributed to the elevated concentrations of certain volatile compounds. T. delbrueckii, for instance, enhances succinic acid, linalool, acetate esters, medium chain fatty acids, and terpenes in aromatic grapes (Jolly et al., 2014; Varela and Borneman, 2016). Similarly, P. terricola has been reported to release precursors from grape juice, boosting free monoterpenes and norisoprenoids (Jolly et al., 2014). The apiculate yeast H. vineae has been reported to produce terpenes, sesquiterpenes, and high amounts of acetate esters, such as 2-phenylethyl acetate and ethyl acetate, which influence fermentations by producing flavors and increasing sensory complexity (Martin et al., 2018).

Our results show that microbial communities assembled in different Vitis species influence the volatile profiles of early-stage fermentations of different grape juices. Additional data from fermentations using other microbial communities and grape juice varietals would strengthen the conclusion that alternative yeast populations shape the volatile profiles of fermented juice while preserving grape varietal identity. Also, grape fermentations using pure cultures of selected non-Saccharomyces strains, and/or pools of selected strains that resemble native microbial communities, could further clarify the specific contributions of individual yeast species and strains to the final volatile profiles of fermented grape juices. Emerging evidence suggest that non-conventional Vitis ecosystems may carry unique yeast species or strains not found in V. vinifera L., presenting opportunities for the isolation of valuable Saccharomyces and non-Saccharomyces strains of potential relevance for the winemaking industry.