Past and Future of Non-Saccharomyces Yeasts: From Spoilage Microorganisms to Biotechnological Tools for Improving Wine Aroma Complexity

REVIEW published: 31 March 2016 doi: 10.3389/fmicb.2016.00411 Edited by: Andrea Gomez-Zavaglia, Center for Research and Development in Food Cryotechnology (CIDCA), Argentina Reviewed by: Maurizio Ciani, Università Politecnica delle Marche, Italy Robert Lawrence Brown, United States Department of Agriculture – Agricultural Research Service, USA *Correspondence: Paloma Manzanares [email protected] Specialty section: This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology Received: 12 January 2016 Accepted: 14 March 2016 Published: 31 March 2016 Citation: Padilla B, Gil JV and Manzanares P (2016) Past and Future of Non-Saccharomyces Yeasts: From Spoilage Microorganisms to Biotechnological Tools for Improving Wine Aroma Complexity. Front. Microbiol. 7:411. doi: 10.3389/fmicb.2016.00411 Past and Future of Non-Saccharomyces Yeasts: From Spoilage Microorganisms to Biotechnological Tools for Improving Wine Aroma Complexity Beatriz Padilla1,JoséV.Gil 2,3 and Paloma Manzanares2* 1Departament de Bioquímica i Biotecnologia, Facultat d’Enologia, Universitat Rovira i Virgili, Tarragona, Spain, 2Departamento de Biotecnología de Alimentos, Instituto de Agroquímica y Tecnología de Alimentos, Consejo Superior de Investigaciones Científicas, Paterna, Spain, 3Departamento de Medicina Preventiva y Salud Pública, Ciencias de la Alimentación, Toxicología y Medicina Legal, Facultad de Farmacia, Universitat de València, Burjassot, Spain It is well established that non-Saccharomyces wine yeasts, considered in the past as undesired or spoilage yeasts, can enhance the analytical composition, and aroma profile of the wine. The contribution of non-Saccharomyces yeasts, including the ability to secret enzymes and produce secondary metabolites, glycerol and ethanol, release of mannoproteins or contributions to color stability, is speciesand strain-specific, pointing out the key importance of a clever strain selection. The use of mixed starters of selected non-Saccharomyces yeasts with strains of Saccharomyces cerevisiae represents an alternative to both spontaneous and inoculated wine fermentations, taking advantage of the potential positive role that non-Saccharomyces wine yeast species play in the organoleptic characteristics of wine. In this context mixed starters can meet the growing demand for new and improved wine yeast strains adapted to different types and styles of wine. With the aim of presenting old and new evidences on the potential of nonSaccharomyces yeasts to address this market trend, we mainly review the studies focused on non-Saccharomyces strain selection and design of mixed starters directed to improve primary and secondary aroma of wines. The ability of non-Saccharomyces wine yeasts to produce enzymes and metabolites of oenological relevance is also discussed. Keywords: non-Saccharomyces yeasts, enzymes, secondary metabolites, primary aroma, secondary aroma, mixed starters, aroma complexity INTRODUCTION Wine fermentation is a complex microbiological process in which yeasts play a fundamental role. Although Saccharomyces cerevisiae is the main microorganism involved in the alcoholic fermentation of grape must, winemaking is a non-sterile process. Many other species of yeasts belonging to various non-Saccharomyces genera occur in grape juice and contribute to the first stages of fermentation and to the organoleptic characteristics of final wine (Fleet, 2008). Frontiers in Microbiology | www.frontiersin.org 1March 2016 | Volume 7 | Article 411 Padilla et al. Non-Saccharomyces Yeasts and Wine Aroma In the past, non-Saccharomyces yeasts were considered to be of secondary significance or undesirable spoilage yeasts; nowadays it is widely accepted that selected strains through appropriate screenings can positively impact on the winemaking process. Thus the growing demand for new and improved wine yeast strains adapted to different types and styles of wines can be met by non-Saccharomyces wine yeasts. Since these yeasts are in general poor fermenters, the design of mixed starters including selected non-Saccharomyces with optimized biotechnological characteristics and S. cerevisiae to ensure a complete fermentation has become one of the main challenges of researchers and oenologists. Moreover, proper mixed starter management during fermentation will allow winemakers to tailor wines to the changing demands of consumers. The production of wines with particular flavor profiles has been one of the main reasons for including non-Saccharomyces yeasts in mixed starters. However, promising approaches to lowering alcohol content of wines, to control wine spoilage or to improve oenological properties are being explored, and undoubtedly they represent new opportunities for exploitation in wine production. Here we revisit the contribution of nonSaccharomyces yeasts to wine aroma complexity. First we review the ability of these yeasts to produce enzymes and metabolites of oenological relevance and finally we discuss the design of mixed starters directed to improve primary and secondary aroma of wines. Special attention was paid to update the information covered in recent reviews on the impact of non-Saccharomyces in wine production. NON-Saccharomyces YEASTS IN WINE PRODUCTION In the second half of the 19th century, Louis Pasteur revealed the role of yeasts during the wine fermentation process, demonstrating that yeast is the primary catalyst responsible for the conversion of grape sugars to alcohol and CO2.He noticed that in fermenting grape musts coexisted a wide variety of microorganisms, including different types of yeasts. His drawings, based on microscopic observations, showed two kinds of yeasts. The first, which was abundant in the early stages of the process, was the small, apically budding, lemon-shaped Saccharomyces apiculatus (now Hanseniaspora uvarum). The second which became the most abundant as alcoholic fermentation progressed, was a larger yeast with round cells, which Pasteur called either Saccharomyces pastorianus or Saccharomyces ellipsoideus (probably the current S. cerevisiae) (Barnett, 2000). Despite the complex wine microbial ecology, S. cerevisiae became the wine yeast par excellence based mainly on its fermentation behavior (Reed and Peppler, 1973;Bely et al., 1990; Fleet, 1993), but also on its important role in the release of aroma precursors (Dubourdieu, 1996;Úbeda and Briones, 2000;Ugliano et al., 2006) and in the formation of secondary aroma (Fleet, 1993; Pretorius, 2003). The other yeast species occurring in musts and wines were considered as a source of potential spoilage problems during wine production. In fact, the presence or overgrowth of some of these species was often related to stuck or sluggish fermentations, or to the production of detrimental compounds to the sensory properties of wine (du Toit and Pretorius, 2000). In the context of this simplistic view of the wine fermentation process, where the most important objective was the inoculation and dominance of S. cerevisiae,theterm‘non-Saccharomyces’ yeasts referred to the wide variety of yeast genera, including more than 20 in both Ascomycota and Basidiomycota phyla, present in grape juice. Yeasts occurring in grape musts at the early stages of fermentation originate from two main sources, the vineyard and the grapes, and the contact surfaces and equipment of the winery (Pretorius et al., 1999). The latter plays a small role as a source of non-Saccharomyces yeasts, while S. cerevisiae is the predominant yeast in such surfaces (Peynaud and Domercq, 1959;Rosini, 1984;Lonvaud-Funel, 1996;Pretorius, 2000). However, it has been recently reported for the first time the implantation in grape must of Hanseniaspora species present in the winery environment (Grangeteau et al., 2015) opening the possibility, still unexplored, that some of the non-Saccharomyces species could persist from 1 year to another in the winery environment and become dominant during fermentation, as usually described for S. cerevisiae (Santamaría et al., 2005;Le Jeune et al., 2006; Mercado et al., 2007). The great quantitative and qualitative variability of nonSaccharomyces species found in the early stages of fermentation can be explained by the large number of factors influencing the grape microbiota such as localization, climatic conditions, cultivar, application of pesticides, and other agronomic practices, stage of ripening, health of the grapes, harvesting procedures and the specific weather conditions in each vintage year (Martini et al., 1980;Rosini et al., 1982;Querol et al., 1990; Regueiro et al., 1993;Epifanio et al., 1999;Jolly et al., 2006; Brilli et al., 2015). In spite of this wide variability of yeast species, during the first 3–4 days of a spontaneous fermentation of grape must, yeast population is numerically dominated by apiculate yeasts, Hanseniaspora/Kloeckera, and Candida species, followed by several species belonging to the genera Metschnikowia and Pichia, and occasionally to Brettanomyces, Kluyveromyces,Schizosaccharomyces,Torulaspora,Rhodotorula, Zygosaccharomyces, and Cryptococcus genera (Goto, 1980;Benda, 1982;Fleet et al., 1984;Heard and Fleet, 1985;Parish and Caroll, 1985;Martínez et al., 1989;Herraiz et al., 1990;Frezier and Dubourdieu, 1992;Schütz and Gafner, 1993;Granchi et al., 1998; Combina et al., 2005;Fleet, 2008). This scenario, with abundance of apiculate yeasts in the 1st days of alcoholic fermentation and varying amounts of other non-Saccharomyces yeasts, followed by the progressive dominance of S. cerevisiae is a common denominator in the process of elaboration of all wines, including those produced by inoculation with selected wine yeast strains (Heard and Fleet, 1985). Industrial wine fermentations are currently conducted by starters of selected wine yeast strains of S. cerevisiae.Thefirst reported use of a selected yeast starter for wine production dates from 1890, when Müller-Thurgau introduced this technology adapting the techniques developed by Christian Hansen for the Carlsberg Brewery (Pretorius, 2000;Barnett and Lichtenthaler, 2001).Nowadays,theuseofactivedryyeastsisoneofthe Frontiers in Microbiology | www.frontiersin.org 2March 2016 | Volume 7 | Article 411 Padilla et al. Non-Saccharomyces Yeasts and Wine Aroma most common practices in winemaking and the market offers a wide variety of yeast strains as dehydrated cultures promising a good implantation, specific skills for different types of wines and a great list of other features such as the ability of enhancing varietal and fermentative aromas, glycerol production, tolerance to alcohol, or specific enzymatic activities. However, the main reason of selected starters is to achieve wines with uniform quality through different years avoiding the variability associated with spontaneous fermentations and the risk of spoilage (Beltran et al., 2002;Santamaría et al., 2005). In such cases, dominant growth of the inoculated strain is required. However many factors might affect the implantation/persistence of individual strains within the total population (Fleet, 2008;Blanco et al., 2012), including the variability that exists from one vintage to another at a given winery (Lange et al., 2014). Despite the advantages of using pure cultures of S. cerevisiae with regard to the easy of control and homogeneity of fermentations, wine produced with pure yeast monocultures lacks the complexity of flavor, stylistic distinction and vintage variability caused by indigenous yeasts (Lambrechts and Pretorius, 2000;Romano et al., 2003). This fact is a neverending debate between researchers and oenologists, and the growth of non-Saccharomyces yeasts can still be seen as an uncontrollable risk or as an opportunity of improving the quality of wine. Nevertheless it is worthwhile to note that the world’s best quality wines are produced after a fermentation process in which, in a greater or lesser extent, various species of nonSaccharomyces yeasts have played a role in the winemaking process and, therefore, have contributed to the final result. It is in this context where the inclusion of non-Saccharomyces wine yeast species as part of mixed starters together with S. cerevisiae to improve wine quality was suggested as a way of taking advantage of spontaneous fermentations without running the risks of stuck fermentations or wine spoilage (Jolly et al., 2003;Rojas et al., 2003;Romano et al., 2003;Ciani et al., 2006). However this practice is linked to new challenges for researchers and oenologists such as the selection of suitable non-Saccharomyces strains, the appropriate modality and time of inoculation, the proportion of yeasts in the culture and the potential microorganism interactions, among others. Figure 1 shows a schematic outline of spontaneous versus inoculated fermentation and the use of mixed starters of selected nonSaccharomyces yeasts with strains of S. cerevisiae as an alternative to both approaches. INFLUENCE OF NON-Saccharomyces YEASTS ON WINE AROMA Undoubtedly, aroma is one of most important characteristics that contribute to the quality of wine. As in many foods, wine aroma is composed by 100s of different compounds with concentrations that can vary between 10−1and 10−10 g/kg (Rapp and Mandery, 1986). The balance and interaction of all of them determine the wine aromatic quality. Wine aroma can be subdivided into three groups: the varietal or primary aroma, determined by the grape variety; the fermentation or secondary aroma; and the bouquet or tertiary aroma resulting from the transformation of aromas during aging. Non-Saccharomyces yeasts can influence both the primary and secondary aroma through the production of enzymes and metabolites, respectively. Influence on Primary Aroma Primary or varietal aroma is formed during the ripening of grapes and its contribution to the final wine aroma is considered an appreciated feature. The production of active compounds of primary wine odor takes place in the exocarp of the grape berry and its final concentration in wine is primarily influenced by the vine variety and secondarily by the state of ripeness and the agronomic and oenological practices (Ewart et al., 1985;Spayd et al., 2002;Hernández-Orte et al., 2008, 2015). Compounds forming primary aroma belong to a limited number of chemical families, including methoxypyrazines, C13-norisoprenoids, volatile sulfur compounds, and terpenes (Ebeler and Thorngate, 2009). Methoxypyrazines are products of amino acid metabolism, and they have been associated to vegetal, green, and herbaceous aromas in certain vine cultivars (reviewed in Sidhu et al., 2015). C13-norisoprenoids derive from carotenoids and particularly β-ionone and β-damascenone are considered impact volatiles of non-floral grapes (Fang and Qian, 2006; Bindon et al., 2007;Pineau et al., 2007; Ristic et al., 2010;Fang and Qian, 2016). Certain organic volatile sulfur compounds such as aromatic thiols make important contributions to Sauvignon Blanc and red cultivars aroma (Darriet et al., 1995;Tominaga et al., 1996, 1998a;Bouchilloux et al., 1998), whereas terpenoids, although present in grapes of all vine varieties, occur in aromatic varieties such as Muscat, Gewürztraminer and Rhine Riesling in the highest concentrations (King and Dickinson, 2000). In grape berries and corresponding wines, approximately seventy terpenoid compounds have been identified (Mateo and Jiménez, 2000). Among them, five monoterpenoid alcohols, namely linalool, geraniol, nerol, citronellol, and α-terpineol are the most abundant and the strongest contributors to wine aroma (Rapp, 1998; Mateo and Jiménez, 2000;Carrau et al., 2005). These compounds provide floral notes and have low odor thresholds (Zalacain et al., 2007). Interestingly most of primary aroma compounds are found in free or bound forms. The latter are not odorant compounds which hydrolysis can occur during fermentation through the action of wine yeasts (Figure 2). Particularly important are aroma precursors linked to sugar molecules, mainly terpenol and C13-norisoprenoid glycosides, and the non-volatile precursor forms of volatile thiols conjugated to cysteine or glutathione. The main yeast enzymes involved in the release of aroma compounds from odorless grape precursors are glycosidases that hydrolyze the non-volatile glycosidic precursors (Gunata et al., 1988), and carbon-sulfur lyases that release volatile thiols from aroma-inactive cysteine-bound conjugates (Tominaga et al., 1998b). Below we focus on the production of these enzymes of oenological relevance by non-Saccharomyces wine yeasts. Table 1 summarizes the yeast species described as producers of glycosidases and carbon-sulfur lyases. Frontiers in Microbiology | www.frontiersin.org 3March 2016 | Volume 7 | Article 411 Padilla et al. Non-Saccharomyces Yeasts and Wine Aroma FIGURE 1 | Use of selected strains of Saccharomyces and non-Saccharomyces yeasts in winemaking. Spontaneous fermentation (a) allows the development of indigenous yeasts from grapes (mainly non-Saccharomyces) and winery (mainly Saccharomyces) leading to wines with a greater aromatic complexity but with less microbiological control. Inoculation with a selected strain of Saccharomyces cerevisiae (c) leads to a greater microbiological control but can reduce the aromatic complexity of wine. The use of mixed cultures of S. cerevisiae and non-Saccharomyces selected strains (b) allows to obtain wines with both greater aromatic complexity and microbiological control of the process. Autochthonous and inoculated selected yeasts are represented in red and blue color, respectively. Glycosidases Since the demonstration that the aromatic components of certain grape varieties are present in the grape berry both in free form and bound to sugars as glycosides (Cordonnier and Bayonove, 1974;Williams et al., 1982), there has been a continuous research to find glycosidases able to release varietal aromas from precursors. The bound aroma fraction comprises glucosides and diglycosides, and compounds such as terpenols, terpene diols, 2-phenylethyl alcohol, benzyl alcohol and C13-norisoprenoids have been shown to be aglycons of such glycosides (Winterhalter and Skouroumounis, 1997). Diglycosides mainly include 6-O-α-Larabinofuranosyl-β-D-glucopyranosides, 6-O-α-L-rhamnopyran -osyl-β-D-glucopyranosides and 6-O-β-D-apiofuranosyl-β-Dglucopyranosides. Due to the important role of monoterpenes in determining the aroma of grapes and wines, hydrolysis of terpene glycosides has been the main focus of research. It is now well established that the enzymatic hydrolysis occurs in two steps (Gunata et al., 1988). During the first step and depending on the conjugate, the glycosidic linkage is cleaved by either an α-L-arabinofuranosidase, an α-L-rhamnosidase or a β-Dapiosidase, and the corresponding monoterpenyl-β-D-glucosides are released. In the second step, monoterpenes are liberated by the action of a β-D-glucosidase. Although oenological yeasts may produce glycosidases, the potential effectiveness of enzymes may be hampered by acidic wine conditions or high ethanol concentrations; another limitation of these enzymes is their weak activity in the presence of glucose in the must or wine, making it especially necessary to analyze their inhibition by these wine components. The potential effectiveness of yeast-derived glycosidases is even further reduced in most cases by the fact that some of the enzymes are intracellular and released only in very small amounts into the culture medium. The degree to which these factors inhibit glycosidase production and activity depends on the species and strains of the organisms involved, pointing out the need of enzyme screenings. Frontiers in Microbiology | www.frontiersin.org 4March 2016 | Volume 7 | Article 411 Padilla et al. Non-Saccharomyces Yeasts and Wine Aroma FIGURE 2 | Release of primary aroma compounds by yeasts. Monoterpenes and volatile thiols occur in grape as odorless precursors which can be released by enzymatic activities of Saccharomyces and non-Saccharomyces yeasts during fermentation. Monoterpene glycosides are mainly glucosides and diglycosides, in which the glucose moiety (G) has been further substituted mainly with arabinose (Ar), apiose (Ap), or rhamnose (R). A two-step enzyme-catalyzed reaction is the main mechanism proposed for the enzymatic hydrolysis of the diglycosides and subsequent release of the free volatile terpene (T) to wine. First a specific glycosidase cleaves the linkage between both sugars and in a second step the released glucoside is hydrolyzed by a β-D-glucosidase, liberating glucose and the corresponding terpene. Volatile thiols are generated from the odourless cysteinylated precursors cysteine-3-mercaptohexan-1-ol (Cys-3MH) and cysteine-4-mercapto-4-methylpentan-2-one (Cys-4MMP) by the action of carbon-sulfur-lyases. Widespread occurrence of β-D-glucosidase activity in nonSaccharomyces yeasts has been revealed in several screenings. Rosi et al. (1994) showed that yeasts of the genera Candida, Debaryomyces,Hanseniaspora/Kloeckera,Kluyveromyces, Metschnikowia,Pichia,Saccharomycodes,Schizosaccharomyces, and Zygosaccharomyces can produce β-D-glucosidases. Later on this capability was confirmed by other authors (Charoenchai et al., 1997;McMahon et al., 1999;Manzanares et al., 2000; Strauss et al., 2001;Spagna et al., 2002;Cordero-Otero et al., 2003;Fernández-González et al., 2003;Rodríguez et al., 2004; González-Pombo et al., 2008;Sabel et al., 2014;López et al., 2015) and was extended also to the genera Torulaspora (HernándezOrte et al., 2008;Cordero-Bueso et al., 2013), Brettanomyces (Cordero-Otero et al., 2003;Fia et al., 2005;Arévalo-Villena et al., 2007), and Trichosporon (Wang et al., 2011). Some of these enzymes, selected as a result of their activity with artificial substrates, proved also effective in hydrolyzing either a grape glycoside extract or in releasing terpenols after addition to must or wine. Debaryomyces hansenii and H. uvarum β-Dglucosidases hydrolyzed terpenic glycosides isolated from grape must (Rosi et al., 1994;Fernández-González et al., 2003). Terpene release was also observed in must and wine treated with β-D-glucosidases from Hanseniaspora sp. and Pichia anomala (Swangkeaw et al., 2011). Moreover, results suggested that the enzyme from Hansenispora sp. was more efficient in releasing desirable aromas during an early stage of alcoholic fermentation while β-D-glucosidase from P. anomala was suitable at the final stage. Several yeast β-D-glucosidases have been purified and characterized. Two Debaryomyces β-D-glucosidases suitable for enhancing wine aroma have been reported. An intracellular D. hansenii β-D-glucosidase, tolerant to ethanol and glucose, efficiently released monoterpenols from the glycosides extracted from Muscat grape must. In addition, when the enzyme was added during Muscat fermentation, a considerably increase in the concentration of mainly nerol and linalool was observed (Yanai and Sato, 1999). By contrast, a Debaryomyces pseudopolymorphus strain (Cordero-Otero et al., 2003) produced an exocellular β-D-glucosidase with acidic optimal pH and not inhibited by glucose or ethanol (Arévalo-Villena et al., 2006), although the effectiveness of the purified enzyme in winemaking was not tested. Enzymatic treatment of wine with a purified ethanol tolerant β-D-glucosidase from Sporidiobolus pararoseus,a member of oenological ecosystems in the southeastern region of Brazil, considerably increased the amount of free terpenes (Baffi et al., 2011, 2013). Several strategies for either improving β-D-glucosidase stability or enzyme yield have been described. An extracellular β-D-glucosidase from Issatchenkia terricola, active in the presence of glucose, ethanol, and metabisulfite was immobilized for improving acidic pH stability. This strategy increased the amount of monoterpenes and norisoprenoids, showing the potential of the immobilized enzyme for aroma development in wines (González-Pombo et al., 2011). With respect to yield enhancement, the usefulness of response surface methodology for optimizing the production of a Trichosporon asahii β-Dglucosidase was reported (Wang et al., 2012). T. asahii βD-glucosidase exhibited better ability than fungal and plant commercial enzymes in hydrolyzing aromatic precursors in young wine. Also, a recombinant S. cerevisiae wine yeast expressing the Candida molischiana bgln gene encoding a β-Dglucosidase able torelease terpenols and alcohols from a glycoside extract has been used to facilitate protein purification (Genovés et al., 2003). Some of the β-D-glucosidase screenings described above also included the search of less common glycosidases, such as α-L-arabinofuranosidase, α-L-rhamnosidase and β-D-xylosidase. From more than 300 wine yeast strains, only one strain of P. anomala showed α-L-arabinofuranosidase activity whereas none of them was positive for α-L-rhamnosidase production (Spagna et al., 2002). Also the potential of certain wine yeasts from the genera Candida,Hanseniaspora, and Pichia to produce β-D-xylosidase activity active at winemaking conditions has been discussed (Manzanares et al., 1999;Yanai and Sato, 2001;Rodríguez et al., 2004;López et al., 2015). Interestingly yeast strains able to display several glycosidase activities have been reported: one strain of Aureobasidium pullulans able to hydrolyze grape glycosides displayed β-D-glucosidase, α-Larabinofuranosidase and α-L-rhamnosidase activities, whereas Candida guilliermondii produced both β-D-glucosidase and αL-rhamnosidase (McMahon et al., 1999). Two H. uvarum, one Hanseniaspora vineae and one P. anomala strains were described as producers of the four glycosidase activities (Mateo et al., 2011), while a Wickerhamomyces anomalus Frontiers in Microbiology | www.frontiersin.org 5March 2016 | Volume 7 | Article 411 Padilla et al. Non-Saccharomyces Yeasts and Wine Aroma TABLE 1 | Non-Saccharomyces yeast species described as producers of enzymes involved in the release of aroma compounds from grape precursors. Enzyme1 Yeast species BGL ARA RHA XYL CSL Reference A. pullulans xxx McMahon et al., 1999 B. anomalus xFia et al., 2005 Brettanomyces spp. x Cordero-Otero et al., 2003;Arévalo-Villena et al., 2007 C. guilliermondii xxxMcMahon et al., 1999;Cordero-Otero et al., 2003;Rodríguez et al., 2004 C. molischiana xFernández-González et al., 2003;Genovés et al., 2003 C. stellata xxxRosi et al., 1994;Strauss et al., 2001;Cordero-Otero et al., 2003 C. utilis xYanai and Sato, 2001 C. zemplinina xAnfang et al., 2009 D. castellii xRosi et al., 1994 D. hansenii xRosi et al., 1994;Yanai and Sato, 1999;Fernández-González et al., 2003 D. polymorphus xRosi et al., 1994;Cordero-Otero et al., 2003;Arévalo-Villena et al., 2007 D. pseudopolymorphus xCordero-Otero et al., 2003;Arévalo-Villena et al., 2006, 2007 D. vanriji xGarcía et al., 2002 Hanseniaspora sp. x x Swangkeaw et al., 2011 H. guilliermondii xManzanares et al., 2000 H. osmophila xxManzanares et al., 1999, 2000 H. vineae xxxx Mateo et al., 2011;Maturano et al., 2012;López et al., 2015 H. uvarum xxxx Rosi et al., 1994;Charoenchai et al., 1997;Manzanares et al., 1999, 2000;Strauss et al., 2001;Fernández-González et al., 2003; Rodríguez et al., 2004;Arévalo-Villena et al., 2007;Mateo et al., 2011;López et al., 2015 I. terricola xGonzález-Pombo et al., 2011 K. thermotolerans xxRosi et al., 1994;Zott et al., 2011 M. pulcherrima/C. pulcherrima xxxRosi et al., 1994;Fernández-González et al., 2003;Rodríguez et al., 2004, 2010a;González-Pombo et al., 2008;Zott et al., 2011 P. a n g u s t a xYanai and Sato, 2000a P. anomala xxxx Rosi et al., 1994;Charoenchai et al., 1997;Manzanares et al., 1999, 2000;Spagna et al., 2002;Mateo et al., 2011;Swangkeaw et al., 2011 P. capsulata xYanai and Sato, 2000b P. guilliermondii xRodríguez et al., 2004, 2010b P. kluyvery xAnfang et al., 2009 P. membranifaciens xxLópez et al., 2015 S. ludwigii xRosi et al., 1994 S. pombe xRosi et al., 1994 S. pararoseus xBaffi et al., 2013 T. delbrueckii xxHernández-Orte et al., 2008;Zott et al., 2011;Maturano et al., 2012;Cordero-Bueso et al., 2013;ˆ Cuˆ s and Jenko, 2013 T. asahii xWang et al., 2011 W. anomalus xx x Sabel et al., 2014;López et al., 2015 Z. bailii xRosi et al., 1994;Cordero-Otero et al., 2003 1BGL, β-D-glucosidase; ARA, α-L-arabinofuranosidase; RHA, α-L-rhamnosidase; XYL, β-D-xylosidase; CSL, carbon-sulfur lyase. (alternative names Hansenula anomala,P. anomala and Candida pelliculosa)strainproducingβ-D-glucosidase, also exhibited αL-arabinofuranosidase and β-D-xylosidase activities (Sabel et al., 2014). However, the effectiveness of purified glycosidases for terpene releasing from precursors has only been reported for the intracellular α-L-rhamnosidases from Pichia angusta (Yanai and Sato, 2000a)andPichia guilliermondii (Rodríguez et al., 2004, 2010b), an α-L-arabinofuranosidase from Pichia capsulata (Yanai and Sato, 2000b)andaβ-D-xylosidase from Candida utilis (Yanai and Sato, 2001). The latter also increased the concentration of terpenes after addition to Moscatel grape must during fermentation (Yanai and Sato, 2001). The role of exo-glucanases in the release of aromatic compounds from glycosidically bound precursors in a single Frontiers in Microbiology | www.frontiersin.org 6March 2016 | Volume 7 | Article 411 Padilla et al. Non-Saccharomyces Yeasts and Wine Aroma enzymatic step has been discussed (Gil et al., 2005). In this regard, a yeast isolate AS1, identified as a W. anomalus strain was selected by its capability to hydrolyze several synthetic and natural glycosides under oenological conditions (Sabel et al., 2014). Later, the enzyme responsible for the hydrolysis of selected glycosides was purified from the culture supernatant of AS1 and characterized as a multifunctional exo-β-1,3-glucanase active under typical wine related conditions (Schwentke et al., 2014). The feasibility of using β-D-glucosidase-producing yeasts in fermentation instead of adding purified enzymes represents an interesting option. Either alone or in a mixed starter with S. cerevisiae, the ability of non-Saccharomyces yeasts to contribute to the aromatic wine profile has been assessed. Different strains of Torulaspora delbrueckii contributed to the aroma profile with flowery and fruity aroma (Maturano et al., 2012;CorderoBueso et al., 2013). Secreted H. vineae and T. delbrueckii β-Dglucosidases were detected throughout the fermentation process, although activity diminished with increasing fermentation time, suggesting the adverse effect of ethanol (Maturano et al., 2012). Also, isolates of Pichia membranifaciens,H. vineae, H. uvarum, and W. anomalus showing β-D-glucosidase activity provoked a moderated overall terpene increase when inoculated to final wines (López et al., 2015). The first mixed starters based on non-Saccharomyces yeasts able to produce β-Dglucosidase activity were described for Debaryomyces vanriji and D. pseudopolymorphus (García et al., 2002;CorderoOtero et al., 2003), and later on for Candida pulcherrima (alternative name Metschnikowia pulcherrima)(Rodríguez et al., 2010a)andT. delbrueckii (ˆ Cuˆ s and Jenko, 2013). Detailed information of these mixed starters will be found in next sections. Carbon-Sulfur Lyases Some sulfur containing compounds, the so-called volatile or varietal thiols, can contribute to positive fragances such as tropical, passion fruit and guava-like nuances. These compounds considered to be impact odorants in Sauvignon Blanc wines are 4-mercapto-4-methylpentan-2-one (4MMP), reminiscent of box tree, passion fruit, broom, and black current bud; and 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA), responsible for the passion fruit, grapefruit, and citrus aroma. Volatile thiols are not unique to Sauvignon Blanc wines. They have been also found to contribute significantly to the aroma profiles of wines made from other varieties such as Riesling, Colombard, Semillon, Cabernet Sauvignon, and Merlot (revised in Coetzee and du Toit, 2012). Volatile thiols are mostly non-existent in grape juice and they are generated during the fermentation process by yeasts from odorless, non-volatile precursors initially present in must (revised in Cordente et al., 2012). It has been shown that 4MMP and 3MH exist in grapes in their nonvolatile precursor form, conjugated to cysteine, or glutathione. The key enzyme for cleaving cysteinylated precursors is the S. cerevisiae β-lyase IRC7, with a substrate preference for cys-4MMP over cys-3MH (Roncoroni et al., 2011). The mechanism by which the glutathionated thiol precursors are degraded has not been fully elucidated, but is likely to involve a multi-step pathway with the production of the cysteinylated form as an intermediate (Grant-Preece et al., 2010). No precursor of 3MHA has been identified in grapes; this compound is formed during fermentation through esterification of 3MH by the alcohol acetyltransferase ATF1 (Swiegers et al., 2006). Undoubtedly the main factor in volatile thiol release during alcoholic fermentation is the yeast strain (Dubourdieu et al., 2006). It was found that S. cerevisiae strains varied significantly in terms of their capabilities to produce volatile thiols and to modulate the varietal characters of Sauvignon Blanc wine (Swiegers et al., 2009). With regard to non-Saccharomyces species, only two screenings have addressed their feasibility to release volatile thiols. The first screening by Anfang et al. (2009) showed that most of the eleven non-Saccharomyces isolates tested were able to produce concentrations of 3MH above the perception threshold, but only two isolates of Pichia kluyvery and Candida zemplinina (alternative names Candida stellata and Starmerella bacillaris) produced concentrations of 3MH and 3MHA comparable with those produced by S. cerevisiae. In contrast to that found for S. cerevisiae,resultsshowedan inverse correlation between the concentrations of 3MH and 3MHA produced by the P. kluyvery and C. zemplinina isolates, suggesting a decreased ability to convert 3MH to 3MHA, or possibly alternate metabolic routes for its formation (Anfang et al., 2009). In a second screening, the potential impact of 15 non-Saccharomyces strains from seven species on 4MMP and 3MH release in model medium and Sauvignon Blanc must was evaluated after partial fermentation (Zott et al., 2011). In general, non-Saccharomyces strains had greater ability to release 3MH than 4MMP in both media. Only M. pulcherrima and H. uvarum strains in model medium and Kluyveromyces thermotolerans in must were able to produce significant amounts of 4MMP. With respect to 3MH release, M. pulcherrima and T. delbrueckii strains released large amounts of this compound in model medium whereas M. pulcherrima and K. thermotolerans stood out as good producers in natural must. C. zemplinina isolates included in the screening did not produce volatile thiols, in contrast to previous results (Anfang et al., 2009). This can be explained by the strain dependent capacity to release 3MH as showed for M. pulcherrima (Zott et al., 2011). Undoubtedly additional screening experiments including numerous nonSaccharomyces strains are required to obtain a clear image of species-associated behavior or strain effects. Mixed fermentations with volatile thiol releasing yeasts will be discussed in later sections. Influence on Secondary Aroma Most of the compounds that determine wine aroma arise from the fermentation process. Their concentrations are mainly dependent on the predominant yeasts and the fermentation conditions (Egli et al., 1998;Henick-Kling et al., 1998;Steger and Lambrechts, 2000). Although ethanol, glycerol, and CO2 are quantitatively the most abundant of these compounds, their contribution to the secondary aroma is relatively limited. Volatile fatty acids, higher alcohols, esters, and, to a lesser extent, aldehydes, have a greater contribution to secondary aroma (Rapp Frontiers in Microbiology | www.frontiersin.org 7March 2016 | Volume 7 | Article 411 Padilla et al. Non-Saccharomyces Yeasts and Wine Aroma and Versini, 1991), although volatiles derived from fatty acids and from nitrogenor sulfur-containing compounds also contribute (Boulton et al., 1996). The biosynthesis of these compounds has been reviewed in greater detail by Lambrechts and Pretorius (2000). It is worthwhile to note that the biosynthesis of these compounds is speciesand strain-dependent, allowing the selection of those strains of biotechnological interest. Moreover, and depending on the concentration reached in wine, those compounds arising from yeast metabolism have a positive or negative impact on wine aroma and quality. Below we describe the contribution of non-Saccharomyces yeast species to wine secondary aroma. Table 2 shows the yeast species described as highor low-producers of secondary aroma compounds. Volatile Fatty Acids Acetic acid is responsible for 90% of the volatile acidity of wines while the remaining fatty acids, such as propanoic and butanoic acid, are present in small quantities (Radler, 1993). Their production is also associated with bacterial growth (Ribereau-Gayon et al., 1998). Acetic acid becomes unpleasant at concentrations near its flavor threshold of 0.7–1.1 g/L and usually values between 0.2 and 0.7 g/L are considered optimal (Lambrechts and Pretorius, 2000). Studies of acetic acid production by non-Saccharomyces yeasts have generated highly variable results. Some non-Saccharomyces genera such as Hanseniaspora and Zygosaccharomyces have been traditionally described as producers of excessive amounts of acetic acid (du Toit and Pretorius, 2000;Loureiro and MalfeitoFerreira, 2003;Romano et al., 2003;Mendoza et al., 2007)and, for this reason, they have been considered for long time as spoilage yeasts. Also the species Schizosaccharomyces pombe is commonly associated with high levels of acetic acid (Gallander, 1977;Snow and Gallander, 1979). However, this compound is produced with a considerably strain variability. For instance, levels of acetic acid ranging from about 0.6 g/L to more than 3.4 g/L have been described for H. uvarum strains (Romano et al., 2003) while a screening of S. pombe allowed the selection of strains producing less than 0.4 g/L of acetic acid (Benito et al., 2014a). TABLE 2 | Secondary aroma compounds produced by non-Saccharomyces wine yeasts. Compound High producers Low producers Reference Volatile fatty acids Acetic acid Hanseniaspora Zygosaccharomyces S. pombe T. delbrueckii K. thermotolerans C. stellata/C. zemplinina Gallander, 1977;Snow and Gallander, 1979;Ciani and Maccarelli, 1998;du Toit and Pretorius, 2000; Soden et al., 2000;Loureiro and Malfeito-Ferreira, 2003;Romano et al., 2003;Kapsopoulou et al., 2005;Mendoza et al., 2007;Renault et al., 2009; Comitini et al., 2011;Rantsiou et al., 2012;Benito et al., 2014a;Englezos et al., 2015 Higher alcohols M. pulcherrima C. zemplinina L. thermotolerans Hanseniaspora Zygosaccharomyces Romano and Suzzi, 1993;Rojas et al., 2003; Clemente-Jiménez et al., 2004;Moreira et al., 2008;Viana et al., 2008;Andorrà et al., 2010; Beckner Whitener et al., 2015 Esters Candida Hansenula Pichia Hanseniaspora Rhodotorula T. delbrueckii K. gamospora Ough et al., 1968;Suomalainen and Lehtonen, 1979;Nykänen, 1986;Mateo et al., 1991; Sponholz, 1993;Romano et al., 1997;Rojas et al., 2001, 2003;Moreira et al., 2005;Viana et al., 2008; Beckner Whitener et al., 2015 Aldehydes Acetaldehyde K. apiculata C. krusei C. stellata H. anomala M. pulcherrima H. uvarum Fleet and Heard, 1993;Romano et al., 2003 Volatile phenols Brettanomyces/Dekkera P. guilliermondii Candida K. lactis T. delbrueckii M. pulcherrima H. guilliermondii H. osmophila P. membranifaciens Lambrechts and Pretorius, 2000;Shinohara et al., 2000;Dias et al., 2003;Viana et al., 2008;Renault et al., 2009;Beckner Whitener et al., 2015 Sulfur compounds Candida Hanseniaspora T. delbrueckii K. gamospora Strauss et al., 2001;Moreira et al., 2008;Viana et al., 2008;Renault et al., 2009;Beckner Whitener et al., 2015 Frontiers in Microbiology | www.frontiersin.org 8March 2016 | Volume 7 | Article 411 Padilla et al. Non-Saccharomyces Yeasts and Wine Aroma By contrast, different screenings of T. delbrueckii strains for desirable oenological properties pointed out differences in fermentative capability but always a low production of volatile acidity when compared to S. cerevisiae (Ciani and Maccarelli, 1998;Renault et al., 2009;Comitini et al., 2011). This feature is also a characteristic of Lachancea thermotolerans (previously known as K. thermotolerans) together with the high production of L-lactic acid (Kapsopoulou et al., 2005). C. stellata/C. zemplinina presents a strong fructophilic character (Soden et al., 2000), which may be an advantage during the fermentation of sweet wines, since this species do not produce excessive levels of acetic acid as a response to the osmotic stress in comparison to S. cerevisiae (Rantsiou et al., 2012). Recently the strong fructophilic character of C. zemplinina and its ability to produce low quantities of ethanol and acetic acid and high amounts of glycerol were confirmed (Englezos et al., 2015). Higher Alcohols They are the largest group of aromatic compounds (Amerine et al., 1980). Higher alcohols contribute to the aromatic complexity of wine at concentrations below 300 mg/L. However when their concentrations exceed 400 mg/L, they areconsideredtohaveanegativeeffectonaroma(Rapp and Mandery, 1986). The importance of higher alcohols is also related to their role as ester precursors (Soles et al., 1982). In general, studies of higher alcohol production in nonSaccharomyces yeasts highlight the influence that these yeasts can have on the chemical composition and quality of wine (Herraiz et al., 1990;Mateo et al., 1991;Gil et al., 1996). In fermented musts, the total production of higher alcohols by pure cultures of Hanseniaspora species is lower than that found with S. cerevisiae (Rojas et al., 2003;Moreira et al., 2008; Viana et al., 2008). Also, Zygosaccharomyces strains isolated from grape musts have been described as producers of low amounts of higher alcohols (Romano and Suzzi, 1993). By contrast, C. zemplinina wines contained huge amounts of higher alcohols, which concentrations clearly exceeded 400 mg/L (Andorrà et al., 2010). Regarding specific alcohols, increased production of 2phenylethyl alcohol, compound associated with pleasant aromas, has been described as a characteristic of M. pulcherrima (Clemente-Jiménez et al., 2004), L. thermotolerans (Beckner Whitener et al., 2015), and C. zemplinina (Andorrà et al., 2010). Esters Esters are the most abundant compounds found in wine, with around 160 identified to date. Although various esters can be formed during fermentation, the most abundant are those derived from acetic acid (ethyl acetate, isoamyl acetate, isobutyl acetate, and 2-phenylethyl acetate) and ethyl esters of saturated fatty acids (ethyl butanoate, ethyl caproate, ethyl caprylate, and ethyl caprate). The main ester in wine is ethyl acetate, and it can impart spoilage character at levels of 150–200 mg/L (Lambrechts and Pretorius, 2000). Non-Saccharomyces wine yeasts, known as good producers of esters, have been traditionally associated with the negative effects of high ethyl acetate formation, whereas the levels of ethyl esters produced by these yeasts are generally much lower than those detected in S. cerevisiae wines (Rojas et al., 2001, 2003). Species belonging to the genera Candida,Hansenula, and Pichia were described as having a greater capacity to produce ethyl acetate than wine strains of S. cerevisiae (Ough et al., 1968;Nykänen, 1986). Also, in a study where ester production was grouped by yeast genera, Hanseniaspora and Pichia stood out by the production of ethyl acetate (Viana et al., 2008). Both genera produced similar ethyl acetate levels, but Hanseniaspora was also a potent producer of specific fruity acetate esters such as 2-phenylethyl acetate and isoamyl acetate (Rojas et al., 2001;Moreira et al., 2005;Viana et al., 2008), whereas the genera Pichia and Rhodotorula produced remarkable levels of isoamyl acetate (Suomalainen and Lehtonen, 1979;Viana et al., 2008). Among Hanseniaspora species, specifically H. uvarum is reported to be a good producer of esters in general (Mateo et al., 1991;Sponholz, 1993; Romano et al., 1997)whereasHanseniaspora guilliermondii and Hanseniaspora osmophila are strong producers of 2phenylethyl acetate (Rojas et al., 2001, 2003;Viana et al., 2008). Regarding ethyl esters, production of ethyl caprylate seems to be a characteristic of T. delbrueckii (Viana et al., 2008). The aroma profile of the newly discovered yeast Kazachstania gamospora showed that this species produced more esters than the S. cerevisiae control strain, but specially phenylethyl propionate, an ester desirable in wine due to its floral aroma (Beckner Whitener et al., 2015). Aldehydes These compounds with apple-like odors are important to the aroma and bouquet of wine due to their low sensory threshold values. Among aldehydes, acetaldehyde constitutes more than 90% of the total content of wines, and its amount can vary from 10 mg/L up to 300 mg/L (Lambrechts and Pretorius, 2000). Saccharomyces cerevisiae strains usually produce higher acetaldehyde levels (5–120 mg/L) than non-Saccharomyces species (up to 40 mg/L) such as Kloeckera apiculata,Candida krusei,C. stellata,H. anomala,andM. pulcherrima (Fleet and Heard, 1993). A mean acetaldehyde concentration of around 25 mg/L was described for H. uvarum strains, although significant differences in production among strains were observed (Romano et al., 2003). Volatile Phenols and Sulfur Compounds Among volatile phenols, the most important are vinylphenols in white wines and ethylphenols in red wines. Their presence is always undesirable, since even at concentrations below the perception threshold they are reported to mask the fruity notes of white wines. These compounds are produced from the non-volatile ferulic and p-coumaric acids. Traditionally, ethylphenol producers have been ascribed to the genus Brettanomyces/Dekkera (Lambrechts and Pretorius, 2000). However, several studies also identified Frontiers in Microbiology | www.frontiersin.org 9March 2016 | Volume 7 | Article 411 Padilla et al. Non-Saccharomyces Yeasts and Wine Aroma Coetzee,C., and du Toit, W. J. (2012). 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No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Microbiology | www.frontiersin.org 20 March 2016 | Volume 7 | Article 411