Not your ordinary yeast: non-Saccharomyces yeasts in wine production uncovered

Abstract

Saccharomyces cerevisiae and grape juice are ‘natural companions’ and make a happy wine marriage. However, this relationship can be enriched by allowing ‘wild’ non-Saccharomyces yeast to participate in a sequential manner in the early phases of grape must fermentation. However, such a triangular relationship is complex and can only be taken to ‘the next level’ if there are no spoilage yeast present and if the ‘wine yeast’ – S. cerevisiae – is able to exert its dominance in time to successfully complete the alcoholic fermentation. Winemakers apply various ‘matchmaking’ strategies (e.g. cellar hygiene, pH, SO₂, temperature and nutrient management) to keep ‘spoilers’ (e.g. Dekkera bruxellensis) at bay, and allow ‘compatible’ wild yeast (e.g. Torulaspora delbrueckii, Pichia kluyveri, Lachancea thermotolerans and Candida/Metschnikowia pulcherrima) to harmonize with potent S. cerevisiae wine yeast and bring the best out in wine. Mismatching can lead to a ‘two is company, three is a crowd’ scenario. More than 40 of the 1500 known yeast species have been isolated from grape must. In this article, we review the specific flavour-active characteristics of those non-Saccharomyces species that might play a positive role in both spontaneous and inoculated wine ferments. We seek to present ‘single-species’ and ‘multi-species’ ferments in a new light and a new context, and we raise important questions about the direction of mixed-fermentation research to address market trends regarding so-called ‘natural’ wines. This review also highlights that, despite the fact that most frontier research and technological developments are often focussed primarily on S. cerevisiae, non-Saccharomyces research can benefit from the techniques and knowledge developed by research on the former.

non-Saccharomyces, yeasts, wine, aroma, flavour, fermentation

Natural yeast and natural wine – a rather unnatural tale

When grapes and yeast combine, wine emerges; however, when wine and people mix, opinions diverge. Sometimes these opinions are based on fact, and sometimes not, but turn to the Roman philosopher, Gaius Plinius Secundus – better known as Pliny the Elder – in his First Century encyclopaedic work, Naturalis Historia, and we find ‘in vino veritas’, that is, the truth is in the wine! So, how can we find the truth through the debates of wine bloggers over the past decade or so – first about ‘organic’, then ‘biodynamic’, and now ‘natural’ wines – when some journalists, importers and retailers are turning this ‘naked-as-nature-intended’ approach of wine production into an ideological crusade?

When did wine become ‘unnatural’? It is a question worth asking, given today's debate about ‘natural’ yeasts and ‘natural’ winemaking practices, and claims by some commentators that ‘natural wine’ is now the ‘hottest category’ in the wine industry. Confusing messages leave many producers and consumers baffled. The answer requires a brief review of 7000 years of winemaking history.

The first fermentation, for example, was more likely the result of serendipity rather than design. Spontaneously, ambient yeasts fermented damaged grapes in harvesting pots which mystified hunter-gatherers – who established agriculture and the first great civilization in Mesopotamia around the Tigris-Euphrates river system – and who tasted wine for the first time (Chambers & Pretorius, 2010). Enjoying the taste and psychotropic effects of their discovery – both a pleasurable and storable drink – they went on to harness ‘natural’ events in repeated yearly ‘experiments’. However, even during those early ‘vintages’, it was clear that, without human intervention, the result of ‘naturally’ fermenting grapes is variable, unreliable and can be undrinkable. It did not take long before the ancients realized that the completely ‘natural’ end-result of fermenting grapes is vinegar.

Throughout history, wine has retained a mythic aura – a ‘natural’ product cloaked in mystique. Could this be a contributing factor why some wine enthusiasts are so concerned that today's winemakers – backed by frontier science and rigorous research – have so much influence over the production process and so much opportunity to direct viticulture and vinification to shape wine according to consumer preferences? What is clear, however, that the pressure is on. There is heated argument as to whether today's wine is of higher quality – due to the contribution of scientific knowledge, technology and research – or whether so-called ‘natural’ wine is better. There is a new-found nostalgia for the wine of yesteryear made with a minimalist approach and variable outcomes.

Some traditionalists and proponents of ‘natural’ wine reject, for example, the ‘interventionist’ practice of inoculating grape must with selected cultured yeasts to avoid the risk of stuck ferments and off-flavours or to produce wine according to predetermined definable flavour specifications and styles. These are the hallmarks of ‘industrial’ products, they say, not ‘natural’ wine (for a review see Lewin, 2010, and references therein).

On the other hand, there is a group of inventive winemakers and yeast researchers who are frustrated by such arguments, waiting impatiently to uncork their artistic creativity and artisanal craftsmanship alongside the next generation of technical innovation. As they have done throughout history, wine's innovators are keen to assist in the crafting of unique, stand-out wines that meet ever-shifting consumer expectations while underpinning profitability and sustainability.

The reality is that winemaking is both art and science and always had been. The supposed dichotomy between ‘natural’ and ‘unnatural’ wine is a false one. History taught us that the best outcome for both winemaker and consumer is achieved when the wine industry harnesses what nature, human ingenuity and cutting-edge science offer in harmony with the unique ‘artistic’ nature of wine. Here, we take stock of what nature's treasure trove of ‘wild’ yeasts has on offer and how inventive winemakers can use them in a scientifically controlled manner to craft wine styles that match consumer expectations in a diverse range of market segments.

Non-Saccharomyces yeasts – a double-edged sword worth investigating

Since 1866 when Louis Pasteur first elucidated the bioconversion of grape juice into wine, this complex biochemical process and the role of the yeast therein has been studied continuously (Figs 1 and 2). The role of the primary yeast, Saccharomyces cerevisiae, often simply referred to as the ‘wine yeast’ has received the most attention. This yeast is not only responsible for the metabolism of grape sugar to alcohol and CO₂ but has an equally important role to play in the formation of secondary metabolites, as well as in conversion of grape aroma precursors to varietal wine aromas (Reed & Peppler, 1973; Fleet, 1993, 2008; Darriet et al., 1995; Dubourdieu, 1996; Pretorius et al., 1999; Ribéreau-Gayon et al., 2000; Pretorius, 2003; Howell et al., 2004; Swiegers & Pretorius, 2005; Swiegers et al., 2005). Grape musts naturally contain a mixture of yeast species and wine fermentation is not a ‘single-species’ fermentation (Fleet, 1990). The dominance of S. cerevisiae (inoculated or indigenous) in the fermentation is expected and desired. However, the indigenous non-Saccharomyces yeasts, already present in the must, and often in greater numbers than S. cerevisiae, are adapted to the specific environment and in an active growth state, which gives them a competitive edge (Cray et al., 2013).

1

A schematic outline of the main steps in white wine production. Some steps and the sequence thereof differ between the production of white and red wine (compare Fig. 2).

2

A schematic outline of the main steps in red wine production. Some steps and the sequence thereof differ between the production of red and white wine (compare Fig. 1).

Non-Saccharomyces yeasts were originally seen as responsible for microbial-related problems in wine production due to their isolation from spoiled wines (Van der Walt & Van Kerken, 1958; Amerine & Cruess, 1960; Van Zyl & Du Plessis, 1961; Van Kerken, 1963; Rankine, 1972; Le Roux et al., 1973). Although it was known that some non-Saccharomyces yeasts could form beneficial metabolites for wine quality (Castor, 1954; Amerine & Cruess, 1960; Van Zyl et al., 1963), this was outweighed by the high levels of volatile acidity and other negative compounds produced (Castor, 1954; Amerine & Cruess, 1960; Van Zyl et al., 1963; Amerine et al., 1967, 1972). This caused a blanket distaste for all non-Saccharomyces yeasts.

Authors of earlier publications considered non-Saccharomyces yeasts to be sensitive to SO₂ added during wine production, to control their growth and that of spoilage bacteria (Amerine & Cruess, 1960; Van Zyl & Du Plessis, 1961; Amerine et al., 1972). Non-Saccharomyces yeasts were also known to be poor fermenters of grape must and intolerant to ethanol (Castor, 1954), especially in the presence of SO₂ (Amerine & Cruess, 1960; Amerine et al., 1972). It was therefore accepted that those non-Saccharomyces yeasts, not initially inhibited by the SO₂, died during fermentation due to the combined toxicity of the SO₂ and alcohol.

In contrast, winemakers conducting spontaneous fermentations (comprising mixed and sequential dominance of non-Saccharomyces and Saccharomyces yeasts), viewed indigenous yeasts as integral to the authenticity of their wines by imparting desired and distinct superior regional characteristics (Amerine et al., 1972). Spontaneous fermented wines, although carrying a higher risk of spoilage, are generally regarded as having improved characteristics, such as complexity, mouth-feel (texture) and integration of flavours relative to inoculated wines (Heard & Fleet, 1985; Fleet, 1990; Bisson & Kunkee, 1991; Gil et al., 1996; Lema et al., 1996; Grbin, 1999; Heard, 1999; Soden et al., 2000; Varela et al., 2009).

Later, research highlighted the high numbers (10⁶ to 10⁸ cells mL⁻¹), and sustained presence of non-Saccharomyces yeasts in modern wine fermentations, resulting in wine microbiologists revisiting the role of these yeasts. Consequently, their role in wine production has been debated extensively (Fleet et al., 1984; Heard & Fleet, 1985; Fleet, 1990, 2003; Herraiz et al., 1990; Longo et al., 1991; Romano et al., 1992; Todd, 1995; Gafner et al., 1996; Gil et al., 1996; Lema et al., 1996; Granchi et al., 1998; Henick-Kling et al., 1998; Lambrechts & Pretorius, 2000; Rementeria et al., 2003; Combina et al., 2005; Xufre et al., 2006; Varela et al., 2009; Ciani et al., 2010; Ciani & Comitini, 2011). Non-Saccharomyces yeasts, as the name suggests, refers to all yeast species found in wine production barring S. cerevisiae, with the proviso that this only includes yeast with a positive role in wine production. Recognized spoilage yeasts, such as Dekkera/Brettanomyces, are normally left out of this description. Although most fields of research are often focussed primarily on S. cerevisiae, non-Saccharomyces research can benefit from the techniques and knowledge developed by the S. cerevisiae and other yeast researchers (Cray et al., 2013).

Yeast classification

Non-Saccharomyces yeast is a loose colloquial term used among wine microbiologists and in wine industries, which includes many different yeast species. These yeasts are either ascomycetous or basidiomycetous that have vegetative states which predominantly reproduce by budding or fission and which do not form their sexual states within or on a fruiting body (Kurtzman et al., 2011a). Current taxonomies recognize 149 yeast genera comprising nearly 1500 species (Kurtzman et al., 2011b). Of these, more than 40 species have been isolated from grape must (Jolly et al., 2006; Ciani et al., 2010).

Yeasts may be known by two valid names, the teleomorphic name referring to the sexual state producing ascospores (Kurtzman et al., 2011a), and the anamorphic name referring to the asexual state that does not form ascospores. Yeast classification can be difficult because some yeasts do not sporulate easily and the ability to form ascospores can be lost during long-term storage (Kurtzman et al., 2011c). Delays between isolation and identification can lead to a newly isolated yeast being identified as either teleomorphic or anamorphic if culture-based techniques are being followed. On-going changes in yeast taxonomy (Kreger-van Rij, 1984; Kurtzman & Fell, 1998; Kurtzman et al., 2011b) also results in confusion for nontaxonomists. Especially when citing older literature, it is not always clear what yeasts were actually investigated. Fortunately, DNA-based approaches have largely helped to clarify modern taxonomy. Some of the more commonly encountered teleomorphic yeasts and their anamorphic counterparts in must and wine are given in Table 1.

1

Teleomorphs, anamorphs and synonyms (Kurtzman et al., 2011b) of some of the non-Saccharomyces yeasts in the Ascomycetous genera reported on grapes and in wine fermentations (Jolly et al., 2006; Ciani & Comitini, 2011; Tofalo et al., 2012)

Teleomorphic form	Anamorphic form	Synonyms*
Citeromyces matritensis	Candida globosa
Debaryomyces hansenii	Candida famata	Pichia hansenii
Dekkera bruxellensis	Brettanomyces bruxellensis
Hanseniaspora guilliermondii	Kloeckera apis
Hanseniaspora occidentalis	Kloeckera javanica
Hanseniaspora osmophila	Kloeckera corticis
Hanseniaspora uvarum	Kloeckera apiculata
Hanseniaspora vineae	Kloeckera africana
Lachancea kluyveri	–‡	Saccharomyces kluyveri
Lachancea thermotolerans	–‡	Kluyveromyces thermotolerans; Candida dattlia
Metschnikowia pulcherrima	Candida pulcherrima	Torulopsis pulcherrima
Meyerozyma guilliermondii	Candida guilliermondii	Pichia guilliermondii
Milleronzyma farinosa	–‡	Pichia farinosa
Pichia fermentans	Candida lambica
Pichia kluyveri	–‡	Hansenula kluyveri
Pichia membranifaciens	Candida valida
Pichia occidentalis	Candida sorbosa	Issatchenkia occidentalis
Pichia terricola	–‡	Issatchenkia terricola
Saccharomycodes ludwigii	–‡
Starmerela bombicola	Candida bombicola	Torulopsis bombicola
Torulaspora delbrueckii	Candida colliculosa	Saccharomyces rosei
Wickerhamomyces anomalus	Candida pelliculosa	Pichia anomala; Hansenula anomala
Zygoascus meyerae	Candida hellenica
Zygosaccharomyces bailii	–‡	Saccharomyces bailii
–†	Candida zemplinina	Possibly Candida stellata in older literature
–†	Candida stellata	Torulopsis stellata

*

Names sometimes found in older literature.

†

No teleomorphic form.

‡

No anamorphic form.

The biology of non-Saccharomyces yeast

Origin of non-Saccharomyces yeasts during wine production

Yeasts are found throughout nature typically forming communities within specific habitats (Starmer & Lachance, 2011). Within the winemaking environment (habitat), grape berry surfaces, cellar equipment surfaces and grape can be considered specialized niches where wine-related yeasts form communities (Polsinelli et al., 1996; Goddard & Anfang, 2010; Gayevskiy & Goddard, 2012). These niches differ broadly. The surface of the unripe grape berry presents nutrient limitations that are alleviated as berries ripen and/or are damaged. Due to constant contact with grape must, cellar surfaces can harbour yeasts, but this is highly dependent on the cellar-hygiene practices followed. Although grape must is a rich nutritive environment, low pH, high osmotic pressure and the presence of SO₂ detract from this otherwise ideal yeast niche. Many external factors affect populations both on grapes and in must (Martini et al., 1980, 1996; Rosini et al., 1982; Sharf & Margalith, 1983; Monteil et al., 1987; Gao & Fleet, 1988; Bisson & Kunkee, 1991; Regueiro et al., 1993; Boulton et al., 1996; Cabras et al., 1999; Epifanio et al., 1999; Guerra et al., 1999; Pretorius et al., 1999; Pretorius, 2000; Jawich et al., 2005; Hierro et al., 2006).

During crushing, the non-Saccharomyces yeasts on the grapes, on cellar equipment and in the cellar environment (air- and insectborne) are carried over to the must (Peynaud & Domercq, 1959; Bisson & Kunkee, 1991; Boulton et al., 1996; Lonvaud-Funel, 1996; Török et al., 1996; Constantí et al., 1997; Mortimer & Polsinelli, 1999; Fleet, 2003). However, cellar surfaces play a smaller role than grapes as a source of non-Saccharomyces yeasts, as S. cerevisiae is the predominant yeast inhabiting such surfaces (Peynaud & Domercq, 1959; Rosini, 1984; Lonvaud-Funel, 1996; Pretorius, 2000). Furthermore, hygienic procedures used in most modern cellars should minimize contamination of must by resident cellar flora (Pretorius, 2000). Dominant yeasts in must after crushing should therefore be the same as are found on grapes (Rementeria et al., 2003).

Despite all the variables in grape harvest and wine production, the yeast species generally found on grapes and in wines are similar throughout the world (Amerine et al., 1967; Longo et al., 1991; Yanagida et al., 1992; Constantí et al., 1997; Zahavi et al., 2002; Jolly et al., 2006). However, the proportion or population profile of yeasts in various regions shows distinct differences.

Importance of non-Saccharomyces yeast

The contribution by non-Saccharomyces yeasts to wine flavour will depend on the concentration of metabolites formed. This in turn is affected by how active the non-Saccharomyces yeasts are. The specific environmental conditions in the must, that is, high osmotic pressure; equimolar mixture of glucose and fructose; the presence of SO_2; nonoptimal growth temperature; increasing alcohol concentrations and anaerobic conditions; and decreasing nutrients all play a role in determining what species can survive and grow (Bisson & Kunkee, 1991; Longo et al., 1991). The clarification of white must (centrifugation, enzyme treatments, cold settling) can also reduce the initial population of yeasts (Fleet, 1990; Lonvaud-Funel, 1996; Pretorius, 2000).

The initial belief that all non-Saccharomyces yeasts died soon after the commencement of an alcoholic fermentation due to the rising ethanol concentration and added SO₂ has not been sustained by later research (Fleet et al., 1984; Heard & Fleet, 1985; Fleet, 1990, 2003; Querol et al., 1990; Longo et al., 1991; Todd, 1995; Gafner et al., 1996; Granchi et al., 1998; Zohre & Erten, 2002; Jolly et al., 2003c; Combina et al., 2005; Renault et al., 2009). The higher numbers of non-Saccharomyces yeasts reported in recent literature might be the result of improved cellar technology and hygiene in modern cellars. This has led to a reduction in SO₂ usage, which presumably results in the survival of a greater number and diversity of non-Saccharomyces yeasts. In parallel, the use of modern laboratory techniques has made the detection of non-Saccharomyces yeasts easier.

Non-Saccharomyces yeasts found in grape must and during fermentation can be divided into three groups: (1) yeasts that are largely aerobic, for example, Pichia spp., Debaryomyces spp., Rhodotorula spp., Candida spp., and Cryptococcus albidus; (2) apiculate yeasts with low fermentative activity, for example, Hanseniaspora uvarum (Kloeckera apiculata), Hanseniaspora guilliermondii (Kloeckera apis), Hanseniaspora occidentalis (Kloeckera javanica); and (3) yeasts with fermentative metabolism, for example, Kluyveromyces marxianus (Candida kefyr), Torulaspora delbrueckii (Candida colliculosa), Metschnikowia pulcherrima (Candida pulcherrima) and Zygosaccharomyces bailii (Fleet et al., 1984; Querol et al., 1990; Bisson & Kunkee, 1991; Longo et al., 1991; Lonvaud-Funel, 1996; Lorenzini, 1999; Torija et al., 2001; Combina et al., 2005).

During fermentation, and more evident in spontaneous fermentations, which lack the initial high-density inoculum of S. cerevisiae, there is a sequential succession of yeasts. Initially, species of Hanseniaspora (Kloeckera), Rhodotorula, Pichia, Candida,Metschnikowia and Cryptococcus are found at low levels in fresh must (Parish & Caroll, 1985; Bisson & Kunkee, 1991; Frezier & Dubourdieu, 1992; Granchi et al., 1998; Fleet, 2003; Combina et al., 2005). Of these, H. uvarum is usually present in the highest numbers, followed by various Candida spp. This is usually more apparent in red must than white, possibly due to the higher pH of the former. However, exceptions do occur and Hanseniaspora can also be absent or present at low levels (Van Zyl & Du Plessis, 1961; Parish & Caroll, 1985; Jolly et al., 2003a; Jolly, 2006).

Despite the sustained presence of certain non-Saccharomyces yeasts, the majority do disappear during the early stages of a vigorous fermentation (Fleet et al., 1984; Henick-Kling et al., 1998). This might be due to their slow growth and inhibition by the combined effects of SO₂, low pH, high ethanol and oxygen deficiency (Heard & Fleet, 1988; Combina et al., 2005). This is consistent with their oxidative or weak fermentative metabolism. Nutrient limitation and size or dominance of S. cerevisiae inoculum can also have a suppressive effect, sometimes separate from temperature or ethanol concentration (Granchi et al., 1998). It has been reported that T. delbrueckii and Kluyveromyces thermotolerans (now classified as Lachancea thermotolerans) are less tolerant to low oxygen levels and this, rather than ethanol toxicity, affects their growth and leads to their death during fermentation (Holm Hansen et al., 2001; Lachance & Kurtzman, 2011). It was also shown that a cell–cell contact mechanism in the presence of high concentrations of viable S. cerevisiae yeasts played a role in the inhibition of these two non-Saccharomyces species (Nissen et al., 2003).

The non-Saccharomyces spp. that do survive and are present until the end of fermentation may also have a higher tolerance to ethanol which would account for their sustained presence (Pina et al., 2004; Combina et al., 2005). Other species reported throughout fermentation are Saccharomyces acidifaciens (now classified as Z. bailii; Peynaud & Domercq, 1959) and Pichia sp. (Bisson & Kunkee, 1991). Characteristics of the individual species will affect the extent to which they are present. Growth parameters for one species will not necessarily be the same for others, while strains within a species can also show different growth kinetics. The standard practice of di-ammonium hydrogen phosphate (DAP) addition to grape must, higher pH values and increased temperatures can all lead to increased fermentation ability of non-Saccharomyces yeast (Jolly et al., 2003c).

Besides affecting wine flavour, the metabolism of non-Saccharomyces yeast can also influence the growth and activity of wine bacteria. In the initial phases of fermentation, non-Saccharomyces yeast can deplete essential nutrients that, combined with toxic metabolites formed, can inhibit the growth of lactic acid bacteria essential for the secondary malolactic fermentation in wine (Fornachon, 1968; Costello et al., 2003; Ribéreau-Gayon et al., 2006). Conversely, other by-products formed by non-Saccharomyces yeast can have a stimulating effect on lactic acid bacteria.

Contribution by non-Saccharomyces yeast (specific metabolites)

Ethanol is the main product of alcoholic fermentation. Currently, consumer and market demand for wines containing lower ethanol has shaped research to develop and evaluate strategies to generate reduced- or low-ethanol wines (Kutyna et al., 2010). Several studies have reported lower ethanol yields when using non-Saccharomyces yeast (Ciani & Ferraro, 1996; Ferraro et al., 2000; Soden et al., 2000; Ciani et al., 2006; Comitini et al., 2011; Magyar & Toth, 2011; Di Maio et al., 2012; Sadoudi et al., 2012). Unfortunately, lower ethanol yields are sometimes the result of wines with high residual sugar (> 5 g L⁻¹; Ciani & Ferraro, 1996; Ciani et al., 2006; Magyar & Toth, 2011). Nevertheless, statistically significant differences in ethanol concentration between wines obtained by mixed fermentation and wines produced by S. cerevisiae monocultures ranged from 0.2% v/v to 0.7% v/v (Ferraro et al., 2000; Soden et al., 2000; Comitini et al., 2011; Izquierdo Canas et al., 2011; Di Maio et al., 2012; Sadoudi et al., 2012; Benito et al., 2013; Gobbi et al., 2013). Another alternative to lower ethanol concentration in wine is to exploit the oxidative metabolism observed in some non-Saccharomyces species (Gonzalez et al., 2013). However, only one study has reported the use of aerobic yeast for the production of reduced-alcohol wine (Erten & Campbell, 2001). Wines containing 3% v/v ethanol were obtained after fermentation of grape must by Williopsis saturnus and Pichia subpelliculosa under intensive aerobic conditions. These reduced-alcohol wines were judged to be of an acceptable quality (Erten & Campbell, 2001).

The range of flavour compounds produced by different non-Saccharomyces yeasts is well documented (Castor, 1954; Suomalainen & Lehtonen, 1979; Soles et al., 1982; Nykänen, 1986; Herraiz et al., 1990; Rauhut, 1993; Romano & Suzzi, 1993a; Lema et al., 1996; Lambrechts & Pretorius, 2000; Rojas et al., 2003; Romano et al., 2003; Moreira et al., 2005; Swiegers & Pretorius, 2005; Swiegers et al., 2005). The metabolic products resulting from non-Saccharomyces growth include terpenoids, esters, higher alcohols, glycerol, acetaldehyde, acetic acid and succinic acid (Fleet et al., 1984; Bisson & Kunkee, 1991; Boulton et al., 1996; Lonvaud-Funel, 1996; Heard, 1999; King & Dickson, 2000; Zohre & Erten, 2002; Clemente-Jimenez et al., 2004). Although far less studied, wine colour can also be affected by non-Saccharomyces yeast (Benito et al., 2011; Morata et al., 2012). Sequential fermentation of grape juice enriched with anthocyanins using P. guilliermondii and S. cerevisiae has been shown to increase the formation of vinylphenolic pyranoanthocyanins molecules which show greater colour stability (Benito et al., 2011). The role of other non-Saccharomyces strains on wine colour remains to be established.

The primary flavour of wine is derived from the grapes, while secondary flavours are derived from ester formation by yeasts during wine fermentation (Nykänen, 1986; Lambrechts & Pretorius, 2000). Several flavour and aroma compounds in grapes are present as glycosylated flavourless precursors (Todd, 1995; Pretorius, 2003). These compounds may be hydrolysed by the enzyme β-glucosidase to form free volatiles that can improve the flavour and aroma of wine, but this enzyme is not encoded by the S. cerevisiae genome (Ubeda-Iranzo et al., 1998; Van Rensburg et al., 2005). In contrast, non-Saccharomyces yeasts belonging to the genera Debaryomyces, Hansenula,Candida, Pichia and Kloeckera possess various degrees of β–glucosidase activity and can play a role in releasing volatile compounds from non-volatile precursors (Rosi et al., 1994; Todd, 1995; Spagna et al., 2002; Fernández-González et al., 2003; Rodríguez et al., 2004; Hernandez-Orte et al., 2008). Cofermentation of Chardonnay grape juice with Debaryomyces pseudopolymorphus and S. cerevisiae resulted in an increased concentration of the terpenols: citronellol, nerol and geraniol in wine (Cordero Otero et al., 2003). Similarly, cofermentation of Muscat grape juice with Debaryomyces vanriji and S. cerevisiae produced wines with increased concentration of several terpenols (Garcia et al., 2002). Equally, mixed cultures of Sauvignon Blanc grape juice with C. zemplinina/S. cerevisiae and T. delbrueckii/S. cerevisiae generated wines with high concentrations of terpenols compared to wines fermented with S. cerevisiae (Sadoudi et al., 2012).

Another strategy to increase the release of bound volatile compounds is to exogenously add enzyme preparations that can act on nonvolatile precursors. Several studies have characterized and described the effect of β-glucosidase addition on grape juice or wine, focusing particularly in the inhibition of β-glucosidase activity by sugar, alcohol, pH and/or temperature. An intracellular β-glucosidase from Debaryomyces hansenii, which is not inhibited by glucose and ethanol, was used during fermentation of Muscat grape juice resulting in an increase in concentration of monoterpenols in the wine (Yanai & Sato, 1999). Similarly, intracellular β-glucosidases from Hanseniaspora sp. and Pichia anomala have been shown to increase the concentration of volatile compounds after treatment of Traminette grape juice and Traminette wine, respectively (Swangkeaw et al., 2011). A β-glucosidase from Sporidiobolus pararoseus has also been shown to increase the release of volatile terpenols in white and red wine (Baffi et al., 2011), whereas β-glucosidase from Issatchenkia terricola was able to increase the amount of free monoterpenes and norisoprenoids in white Muscat wine (Gonzalez-Pombo et al., 2011). The concentration of volatile terpenes in Arien, Riesling and Muscat wines was also increased following addition of an enzyme extract from Debaryomyces pseudopolymorphus. Consequently, sensory differences were found between treatments (Arevalo-Villena et al., 2007).

Over 160 esters have been distinguished in wine (Jackson, 2000). These esters can have a positive effect on wine quality, especially in wine from varieties with neutral flavours that are consumed shortly after production (Lambrechts & Pretorius, 2000; Sumby et al., 2010). Non-Saccharomyces can be divided into two groups, viz. neutral yeasts (producing little or no flavour compounds) and flavour-producing species (both desired and undesired; Van Zyl et al., 1963). Flavour-producing yeasts included P. anomala (Hansenula anomala) and K. apiculata. Candida pulcherrima is also known to be a high producer of esters (Bisson & Kunkee, 1991; Clemente-Jimenez et al., 2004). The net accumulation of esters in wine is determined by the balance between the yeast's ester-synthesizing enzymes and esterases (responsible for cleavage and in some cases, formation of ester bonds; Swiegers & Pretorius, 2005). Although extracellular esterases are known to occur in S. cerevisiae (Ubeda-Iranzo et al., 1998), the situation for non-Saccharomyces needs further investigation.

Different non-Saccharomyces yeasts produce different levels of higher alcohols (n-propanol, isobutanol, isoamyl alcohol, active amyl alcohol; Romano et al., 1992; Lambrechts & Pretorius, 2000). This is important during wine production, as high concentrations of higher alcohols are generally not desired, whereas lower values can add to wine complexity (Romano & Suzzi, 1993b). Non-Saccharomyces yeasts often form lower levels of these alcohols than S. cerevisiae, but there is great strain variability (Romano et al., 1992, 1993; Zironi et al., 1993).

Glycerol, the next major yeast metabolite produced during wine fermentation after ethanol, is important in yeast metabolism for regulating redox potential in the cell (Scanes et al., 1998; Prior et al., 2000). Glycerol contributes to smoothness (mouth-feel), sweetness and complexity in wines (Ciani & Maccarelli, 1998), but the grape variety and wine style will determine the extent to which glycerol impacts on these properties. Although the quality of Chardonnay, Sauvignon Blanc and Chenin Blanc is not improved by increased glycerol concentrations (Nieuwoudt et al., 2002), some wines might benefit from increased glycerol levels. Several non-Saccharomyces yeasts, particularly L. thermotolerans and C. zemplinina, can consistently produce high glycerol concentrations during wine fermentation (Ciani & Ferraro, 1998; Soden et al., 2000; Comitini et al., 2011).

Unfortunately, increased glycerol production is usually linked to increased acetic acid production (Prior et al., 2000), which can be detrimental to wine quality. Spontaneously fermented wines have higher glycerol levels, indicating a possible contribution by non-Saccharomyces yeasts (Romano et al., 1997a; Henick-Kling et al., 1998). Nevertheless, the use of some non-Saccharomyces yeast in mixed fermentations with S. cerevisiae can generate wines with decreased volatile acidity and acetic acid concentration (Bely et al., 2008; Comitini et al., 2011; Domizio et al., 2011a).

Some non-Saccharomyces yeasts are able to form succinic acid (Ciani & Maccarelli, 1998; Ferraro et al., 2000). This correlates with high ethanol production and ethanol tolerance. Succinic acid production could positively influence the analytical profile of wines by contributing to the total acidity in wines with insufficient acidity. However, succinic acid has a ‘salt-bitter-acid’ taste (Amerine et al., 1972) and excessive levels will negatively influence wine quality.

Other non-Saccharomyces metabolites can act as intermediaries in aroma metabolic pathways. Acetoin is considered a relatively odourless compound in wine with a threshold value of c. 150 mg L⁻¹ (Romano & Suzzi, 1996). However, diacetyl and 2,3-butanediol (potentially off-flavours in wine) can be derived from acetoin by chemical oxidation and yeast-mediated reduction, respectively. This indicates that acetoin can play a role in off-flavour formation in wines. Indeed, high concentrations of acetoin produced by non-Saccharomyces yeasts can be utilized by S. cerevisiae in mixed and sequential culture fermentations (Zironi et al., 1993). However, Zironi et al. (1993) could not confirm what metabolites were formed from acetoin by S. cerevisiae.

Other compounds that are known to play a role in the sensory quality of wine include volatile fatty acids, carbonyl and sulphur compounds (Nykänen, 1986; Lambrechts & Pretorius, 2000; Moreira et al., 2005). However, as stated by Guth (1997), there are over 680 documented compounds in wine and a large number of these can, depending on concentration, contribute either positively or negatively to wine aroma and flavour. Volatile thiols greatly contribute to the varietal character of some grape varieties, particularly Sauvignon Blanc (Swiegers et al., 2009). Some non-Saccharomyces strains, specifically isolates from C. zemplinina and Pichia kluyveri can produce significant amounts of the volatile thiols 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexan-1-ol acetate (3MHA), respectively, in Sauvignon Blanc wines (Anfang et al., 2009). Similarly, T. delbrueckii,M. pulcherrima and L. thermotolerans have also been described as able to release important quantities of 3MH from its precursor during Sauvignon Blanc fermentation (Zott et al., 2011).

Other non-Saccharomyces extracellular enzymatic activities, such as proteolytic and pectinolytic (polygalacturonase) enzymes, might also be beneficial to winemaking (Strauss et al., 2001). For example, proteolytic activity of some non-Saccharomyces yeast could lead to a reduction in protein levels with accompanying increase in protein stability of the end-product. However, Dizy & Bisson (2000) reported to the contrary that increased yeast proteolytic activity did not lead to a reduction in haze formation in white wine. Species found to produce the greatest number of extracellular enzymes are C. stellata,H. uvarum and M. pulcherrima.

Non-Saccharomyces yeasts have also been reported to affect the concentration of polysaccharides in wine (Domizio et al., 2011a, b). Two-strain mixed cultures of S. cerevisiae and Hanseniaspora osmophila,Pichia fermentans,Saccharomycodes ludwigii,Zygosaccharomyces bailii and/or Zygosaccharomyces florentinus were found to produced wines with increased concentration of polysaccharides (Domizio et al., 2011a, b). Polysaccharides can positively influence wine taste and mouth-feel by increasing the perception of wine ‘viscosity’ and ‘fullness’ on the palate (Vidal et al., 2004).

The early death of some non-Saccharomyces yeasts during fermentation can also be a source of specific nutrients for S. cerevisiae enabling it to ferment optimally. These nutrients include cellular constituents such as cell wall polysaccharides (mannoproteins). For this method of nutrient supply to be effective, any killer or other inhibitory effects by the non-Saccharomyces yeasts against S. cerevisiae should be known (Herraiz et al., 1990; Panon, 1997; Nguyen & Panon, 1998; Fleet, 2003) so that the subsequent S. cerevisiae fermentation is not adversely affected.

The deliberate use of non-Saccharomyces yeast in wine production

Various authors have reported on deliberate inoculation of selected non-Saccharomyces yeasts for wine production. These included Torulaspora,Candida, Hanseniaspora,Zygosaccharomyces,Schizosaccharomyces,Lachancea (formerly Kluyveromyces; Lachance & Kurtzman, 2011) and Pichia species. All those yeasts are poor fermenters; therefore, S. cerevisiae (either indigenous or inoculated) is always needed to complete wine fermentation. Typically, non-Saccharomyces yeasts have been used in sequential fermentation where these yeasts are allowed to grow or ferment between one hour and fifteen days before inoculation with S. cerevisiae (Ciani & Ferraro, 1998; Ferraro et al., 2000; Herraiz et al., 1990; Zironi et al., 1993; Jolly et al., 2003b,c). Many of these trials were conducted on a laboratory-scale utilizing small volumes of grape juice and the results may not necessarily be the same as what could be expected in larger commercial fermentations. Factors such as small amounts of air that can enter small volume fermentations (e.g. during sampling), and rapid sedimentation of yeast cells that can reduce the fermentation rate, can affect the final results (Henschke, 1990).

Torulaspora delbrueckii

Torulaspora delbrueckii (anamorph: C. colliculosa), was one of the first commercial non-Saccharomyces yeast to be released. Torulaspora delbrueckii, formerly classified as Saccharomyces rosei, was previously suggested for vinification of musts low in sugar and acid was used for the commercial production of red and rosé wines in Italy (Castelli, 1955). Recently, pure cultures of T. delbrueckii have been shown to produce lower levels of volatile acidity than S. cerevisiae strains (Moreno et al., 1991; Renault et al., 2009). Thus, T. delbrueckii has been useful in the production of wines from high sugar musts derived from botrytized grapes (Bely et al., 2008). Other metabolites produced by T. delbrueckii include succinic acid (Ciani & Maccarelli, 1998) and, for particular strains, linalool, which is derived from monoterpene alcohols and adds to the varietal aroma of Muscat type wines (King & Dickson, 2000).

As T. delbrueckii affects wine composition it also modulates wine flavour and aroma. Following a coinoculated strategy, with T. delbrueckii and S. cerevisiae, Sauvignon Blanc and Chenin Blanc wines were both judged to be better than their respective S. cerevisiae reference wines five and 18 months after production (Jolly et al., 2003b). Similarly, Amarone wines produced by sequential inoculation with T. delbrueckii and S. cerevisiae were judged to have increased aroma intensity, including ‘ripe red fruit’ aroma, increased sweetness and astringency and decreased intensity for vegetal attributes (Azzolini et al., 2012).

In 2003, the first commercial release of T. delbrueckii was as a component of a yeast blend (Vinoflora^® Melody.nsac and Vinoflora^® Harmony.nsac) with S. cerevisiae and K. thermotolerans (Anonymous, 2004a; CHR Hansen, 2013a, b). Subsequently, the T. delbrueckii component was released on its own (CHR Hansen, 2013a, b). A further two T. delbrueckii strains from other commercial yeast manufacturers are also available (Lallemand, 2012; Laffort, 2013), indicating that some winemakers are eager to experiment with carefully selected and tested non-Saccharomyces yeasts.

Candida pulcherrima

Metschnikowia pulcherrima (anamorph C. pulcherrima) is another yeast commercially available. This commercial strain produces an extra-cellular α-arabinofuranosidase that impacts on the concentration of varietal aromas such as terpenes and volatile thiols (Lallemand, 2012). This yeast species is also known to produce high concentrations of esters (Bisson & Kunkee, 1991; Rodríguez et al., 2010; Sadoudi et al., 2012), especially the pear-associated ester, ethyl octanoate (Lambrechts & Pretorius, 2000; Clemente-Jimenez et al., 2004) and can occur in high numbers in grape must (Schütz & Gafner, 1993; Jolly et al., 2003a). Wines of the grape varieties Sauvignon Blanc, Chenin Blanc and Muscat d'Alexandrie obtained by sequential fermentation with C. pulcherrima and S. cerevisiae showed higher quality scores than control wines (obtained by fermentation with S. cerevisiae; Jolly et al., 2003b; Rodríguez et al., 2010). Similarly, an indigenous C. pulcherrima strain has been reported to increase wine flavour and aroma of Debina wines following sequential inoculation (Parapouli et al., 2010). However, a Chardonnay wine produced by sequential inoculation with C. pulcherrima and S. cerevisiae was judged to be of an inferior quality than the control wine (S. cerevisiae only) implying that specific non-Saccharomyces/grape variety combinations lead to increased wine quality scores (Jolly et al., 2003b).

It has also been reported that C. pulcherrima can have an antagonistic effect on several yeasts including S. cerevisiae which leads to delays in fermentation (Panon, 1997; Nguyen & Panon, 1998). This phenomenon was due to a killer effect, although not the same as the classical S. cerevisiae killer phenomenon, and was linked to pulcherrimin pigment produced by C. pulcherrima. Differing reports on the interactions between C. pulcherrima and other yeasts may be due to different distinct biotypes within the C. pulcherrima species (Pallmann et al., 2001).

Candida zemplinina/Candida stellata

In 2011, specific strains of Candida stellata were reclassified to C. zemplinina (Kurtzman et al., 2011b). It can therefore be surmised that older literature references to C. stellata, may probably be C. zemplinina and that true C. stellata may not be associated with grapes and wine. In this review the original taxonomic names as published, are used.

Candida stellata is known as a high glycerol producer with concentrations reported in wine up to 14 g L⁻¹ (Ciani & Picciotti, 1995; Ciani & Ferraro, 1998; Ciani & Maccarelli, 1998). In contrast, S. cerevisiae has been reported to produce between 4 and 10.4 g L⁻¹ of glycerol (Radler & Schütz, 1982; Ciani & Maccarelli, 1998; Prior et al., 2000). Glycerol concentrations over 5.2 g L⁻¹ can produce a sweet taste (Noble & Bursick, 1984). Glycerol is also thought to contribute to the mouth-feel and complexity of wine flavour at lower levels (Scanes et al., 1998; Prior et al., 2000).

Unlike S. cerevisiae, which favours glucose utilization, C. stellata consumes fructose preferentially to glucose and is therefore considered a fructophilic yeast (Soden et al., 2000; Magyar & Toth, 2011; Di Maio et al., 2012). As S. cerevisiae is a glucophilic yeast, it is not unusual to observe high residual fructose after fermentation of grape musts containing high concentrations of initial sugar. However, after a sequential inoculation strategy of Pinot Grigio grape must containing high sugar concentration (270 g L⁻¹), wines obtained by mixed cultures of C. stellata and S. cerevisiae showed no residual sugar, due to the complementary utilization of fructose and glucose by both strains (Ciani & Ferraro, 1998). Hence, fermentation kinetics were faster, shortening fermentation length. Resulting wines showed increased concentrations of glycerol and succinic acid and reduced concentrations of acetic acid and higher alcohols (Ciani & Ferraro, 1998). Similar findings were observed following a sequential inoculation strategy with C. stellata/S. cerevisiae using Trebbiano Toscano grape juice (Ferraro et al., 2000).

Sauvignon Blanc wines produced by sequential inoculation with C. zemplinina and S. cerevisiae showed very different volatile profiles than wines fermented with S. cerevisiae monocultures (Sadoudi et al., 2012). Specifically, C. zemplinina/S. cerevisiae wines showed significantly increased concentrations of terpenols (linalool, citronellol, geraniol, nerolidol and farnesol) and decreased concentrations of aldehydes and acetate esters (Sadoudi et al., 2012). Conversely, coinoculation of Macabeo grape juice with C. zemplinina and S. cerevisiae produced wines with increased concentration of higher alcohols, ethyl esters and short-chain fatty acids (Andorra et al., 2010), indicating that yeast strain and/or grape variety affect the volatile profile of wines fermented with C. zemplinina.

Wines exhibiting different compositions of volatile compounds will show a different flavour profile; however, the effect of volatile composition, either positive or negative, on wine flavour is not simple to predict. Chardonnay wines produced by both coinoculation and sequential inoculation with C. stellata and S. cerevisiae showed low aroma intensity for ‘desirable’ sensory attributes, or exhibited high intensities for ‘undesirable’ sensory descriptors (Soden et al., 1998, 2000). Compared to wines fermented with S. cerevisiae monoculture, coinoculated wine was scored lower for ‘floral’ and ‘banana’ aromas while other sensory descriptors were similar. Wine produced by sequential fermentation showed lower scores for ‘banana’, ‘floral’ and ‘lime’ aromas, but it was similar in ‘honey’, ‘apricot’ and ‘sauerkraut’ aromas attributed to the C. stellata yeast. This wine also showed a high ‘ethyl acetate’ aroma, had the highest concentrations of glycerol and succinic acid, and a lower concentration of ethanol. Wine produced by monoculture of C. stellata was scored particularly high for ‘apricot’, ‘honey’ and ‘sauerkraut’ aromas. The ‘sauerkraut’ and ‘ethyl acetate’ nuances could be considered to detract from wine quality as they are listed under ‘microbiological’ and ‘oxidized’ according to wine evaluation terminology (Noble et al., 1987).

Similarly, Chardonnay wines produced by sequential inoculation with C. stellata and S. cerevisiae were judged to be of lesser quality than reference wines produced with monocultures of S. cerevisiae, even though reference wines showed lower concentrations of total esters (Jolly et al., 2003b). It seems that the use of C. zemplinina for wine production might involve a role for increasing wine complexity rather than increasing the perception of particular ‘desirable’ sensory attributes.

Hanseniaspora species

The apiculate yeasts Hanseniaspora uvarum (anamorph Kloeckera apiculata) are the non-Saccharomyces yeasts found in the highest numbers in grape must. Therefore, they should be in the best position to make a contribution to wine quality. Hanseniaspora spp. generally show low fermentative power but are important in the production of wine volatile compounds, and the chemical composition of wines made with Hanseniaspora spp./S. cerevisiae combinations differ from reference wines produced with S. cerevisiae monoculture (Herraiz et al., 1990; Mateo et al., 1991; Zironi et al., 1993; Gil et al., 1996). The low frequency of Hanseniaspora spp. during fermentation has also been suggested as a reason for the lack of aroma complexity of Folle Blanche wines in the Basque region in Spain (Rementeria et al., 2003).

Hanseniaspora vineae (formerly H. osmophila) and H. guilliermondii have been reported to produce increased amounts of 2-phenyl-ethyl acetate during fermentation (Rojas et al., 2003; Viana et al., 2009). This acetate ester is associated with ‘rose’, ‘honey’, ‘fruity’ and ‘flowery’ aroma descriptors (Lambrechts & Pretorius, 2000; Swiegers & Pretorius, 2005; Swiegers et al., 2005), and as part of the ‘fermentation bouquet’, it can contribute to the overall flavour of young wines. Cofermentation of Bobal grape must with H. vineae and S. cerevisiae produced wines that not only showed an increased concentration of 2-phenylethyl acetate but also exhibited higher ‘fruity’ sensory scores than wines produced with S. cerevisiae monoculture (Viana et al., 2009). The amount of 2-phenylethyl acetate produced, however, depended on the proportion of H. vineae/S. cerevisiae (Viana et al., 2009). The same authors reported a higher production of 2-phenylethyl acetate in Tempranillo wines produced by sequential inoculation with H. vineae/S. cerevisiae compared with wines produced by cofermentation (Viana et al., 2011).

In addition to 2-phenylethyl acetate, wines produced with H. guilliermondii and S. cerevisiae have shown higher concentrations of hexyl acetate, ethyl acetate and isoamyl acetate than wines produced with S. cerevisiae (Moreira et al., 2008). In these wines, the production of heavy sulphur compounds was also affected by H. guilliermondii. Thus, wines obtained by mixed fermentation showed increased concentrations of 3-(ethylthio)-1-propanol (associated with ‘rancid’ and ‘sweaty’ sensory descriptors), 3-mercapto-1-propanol (associated with ‘sweaty’ and ‘potato’), trans-2-methyltetrahydrothiophen-3-ol (‘onion’, ‘chive-garlic’) and decreased concentrations of 2-(methylthio)-ethanol + 2-methyltetrahydrothiophen-3-one, the former associated with ‘French bean’ and ‘cauliflower’ descriptors, while the latter is described by the attributes ‘metallic’ and ‘natural gas’ (Moreira et al., 2008, 2010). Although some of these compounds are associated with unpleasant sensory descriptors, they might have a role increasing wine complexity.

Hanseniaspora uvarum has also been used in mixed fermentations with S. cerevisiae for wine production. Macabeo wines fermented with H. uvarum/S. cerevisiae showed increased concentrations of higher alcohols, acetate and ethyl esters and medium-chain fatty acids (Andorra et al., 2010), while Douro wines exhibited increased isoamyl acetate and decreased 2-(methylthio)-ethanol + 2-methyltetrahydrothiophen-3-one (Moreira et al., 2008).

Apiculate yeasts are also known as high producers of acetic acid (0.75–2.25 g L⁻¹) and ethyl acetate, making them less attractive for wine production (Ciani & Picciotti, 1995; Caridi & Ramondino, 1999; Rojas et al., 2003). However, high-strain variability exists and some are comparable with S. cerevisiae in levels of volatile acidity produced (Owuama & Saunders, 1990; Romano et al., 1992, 1997b; Ciani & Maccarelli, 1998). Although apiculate yeasts may be associated with the production of undesirable flavour compounds (volatile acidity, sulphur compounds, etc.), they can have a positive influence on the flavour profile of certain wine styles. For example, in one particular study, Sauvignon Blanc wines produced with H. uvarum and S. cerevisiae were preferred over wines produced with S. cerevisiae monoculture (Jolly et al., 2003b). Selected strains of apiculate yeasts might, therefore, favour aroma and flavour enhancement in wines.

Other aspects of the metabolism of Hanseniaspora spp. involve the production of acetoin in grape must (Romano et al., 1993), the formation of unwanted biogenic amines in wine (Caruso et al., 2002) and the desired ability to reduce ochratoxin A levels in synthetic must (Angione et al., 2007). Some reports have observed that the initial growth of Hanseniaspora had a retarding effect on the subsequent growth of S. cerevisiae (Herraiz et al., 1990). This phenomenon could have further implications as a cause for lagging or stuck fermentations. Therefore, a cautionary approach would have to be taken when considering using Hanseniaspora spp. in wine production.

Zygosaccharomyces species

Zygosaccharomyces spp. are considered to be winery contaminants producing high quantities of acetic acid and are especially a problem in wineries producing sweet and sparkling wines (Amerine & Cruess, 1960; Loureiro & Malfeito-Ferreira, 2003). However, it has been suggested yeasts bearing a close resemblance to Zygosaccharomyces were wrongly identified as Zygosaccharomyces species (Romano & Suzzi, 1993a). Studies investigating a positive contribution of Zygosaccharomyces spp. to wine fermentation included a Z. fermentati strain that produced low levels of acetic acid, H₂S and SO₂ and had high fermentation vigour; and a Z. bailii strain that showed malic acid degradation and generally low H₂S production. In addition, both species flocculated (Romano & Suzzi, 1993a). These characteristics could benefit wine production during, for example, re-fermentation of wine. Wines produced by mixed fermentation with combinations of Z. bailii/S. cerevisiae and Z. florentinus/S. cerevisiae have shown increased production of polysaccharides, which can have a positive influence in wine taste (Domizio et al., 2011a, b).

A commercial Zygosaccharomyces yeast was released specifically for re-starting stuck fermentations due to its fructophilic nature (Gafner et al., 2000; Sütterlin et al., 2004; Sütterlin, 2010). This may also be beneficial in fermentations of grape musts from riper grapes (containing high sugar concentrations) where the fructose concentration can exceed that of glucose at the start of fermentation that affects S. cerevisiae growth (Margalith, 1981; Berthels et al., 2004).

Schizosaccharomyces species

Schizosaccharomyces spp. can degrade organic acids such as malic acid and gluconic acid (Gao & Fleet, 1995; Peinado et al., 2004). This ability has been applied on a practical level, where a Schizosaccharomyces malidevorans mutant, that could utilize malic acid more rapidly than the wild-type strain was used for commercial-scale (1000–2500 L) deacidification of grape juice (Thornton & Rodríguez, 1996). After treatment, it was found that Chardonnay, Semillon and Cabernet Sauvignon wines did not show any sensory defects. Finalized wines were used for blending before being sold as varietal wines. Similarly, Schizosaccharomyces pombe was used in mixed fermentations with S. cerevisiae to remove malic acid and total acidity in Arien grape juice (Benito et al., 2013). Although wines obtained by mixed fermentation showed increased concentration of acetaldehyde, propanol and 2,3-butanediol and slightly decreased concentration of esters, they received a more favourable sensory score by the judging panel than wine produced by a S. cerevisiae monoculture (Benito et al., 2013).

Schizosaccharomyces pombe has also been used to partially remove gluconic acid after fermentation of Pedro Ximenez wines (Peinado et al., 2004). However, these wines contained considerably more acetaldehyde, 2,3-butanediol and 1,1-diethoxyethane, responsible for the oxidized characters of Sherry wines, than untreated wines (Peinado et al., 2004). The use of S. pombe in mixed fermentations of grape juice with S. cerevisiae, intended to decrease gluconic acid concentration, has also been associated with increased production of off-flavours in these wines (Peinado et al., 2007).

Lachancea thermotolerans (Kluyveromyces thermotolerans)

Lachancea thermotolerans (formerly K. thermotolerans; Lachance & Kurtzman, 2011) has been described to produce wines with increased concentrations of lactic acid, glycerol and 2-phenylethanol during mixed fermentations of grape musts (Kapsopoulou et al., 2007; Comitini et al., 2011; Gobbi et al., 2013). In addition, commercial-scale fermentations (10 000 L) of Sangiovese grape must with L. thermotolerans/S. cerevisiae produced wines which were scored higher in ‘spicy’ and ‘acidity’ attributes than S. cerevisiae wines (Gobbi et al., 2013). However, the effect of L. thermotolerans on wine chemical composition and therefore on wine flavour depends on the time of inoculation with S. cerevisiae (Kapsopoulou et al., 2007; Gobbi et al., 2013). Thus, later, a L. thermotolerans ferment is inoculated with S. cerevisiae the more lactic acid and glycerol the final wine will contain.

A commercial active dried yeast blend of L. thermotolerans (marketed as K. thermotolerans) and S. cerevisiae (Viniflora^® Symphony.nsac) was previously commercially available (Anonymous, 2004b). This combination was developed for enhancement of aroma and flavour in white (Chardonnay, Pinot Blanc, Pinot Gris and Riesling) and red (Cabernet Sauvignon; Merlot, Shiraz and Pinot Noir) grape varieties. According to the product information sheet, the use of this yeast in simultaneous inoculation could lead to enhancement of floral and tropical fruit aromas and more complex and rounded flavours in white and red wine, respectively. Although the ratio of the L. thermotolerans cell count to that of S. cerevisiae in this particular product was not specified, it appeared to be in the region of 1 : 30 (N. Jolly, unpublished data, 2005). However, Pinot Noir wines produced with Viniflora^® Symphony.nsac showed decreased ‘red fruit’ aroma as compared to the corresponding S. cerevisiae control (Merit.ferm) wine and no differences in ‘spice’ characteristics (Takush & Osborne, 2012). Interestingly, both treatments scored lower in overall aroma intensity, ‘dark fruit’ aroma and ‘jammy/cooked’ aroma compared with S. cerevisiae strain EC1118, suggesting that the choice of S. cerevisiae strain can also influence wine aroma profile in mixed fermentations. In 2012, the L. thermotolerans component of Viniflora^® Symphony.nsac was released on its own as a single-active dried yeast (CHR Hansen, 2013a, b).

Pichia kluyveri

Cofermentation with P. kluyveri has been reported to lead to higher levels of varietal thiols, especially 3-mercaptohexyl acetate (3MHA; Anfang et al., 2009). However, it has also been reported that zymocins (so-called killer toxins) produced by P. kluyveri can inhibit certain S. cerevisiae strains (Middelbeek et al., 1980). A commercial yeast product is available that is reported to extract flavour precursors from grape juice at higher levels than other yeasts tested (CHR Hansen, 2013a). This yeast strain is especially recommended for Riesling, Sauvignon Blanc and Chardonnay wines. This product differs from other commercially active dried yeast products in that it is delivered and stored frozen (−45 °C) and used to directly inoculate the grape must without any rehydration (CHR Hansen, 2013b).

Other non-Saccharomyces species

Pichia fermentans (Candida lambica) was investigated by Clemente-Jimenez et al. (2005) in microvinifications of Macabeo wine. Mixed fermentations with P. fermentans and S. cerevisiae produced wines with increased concentrations of some volatile compounds such as acetaldehyde, ethyl acetate, 1-propanol, n-butanol, 1-hexanol, ethyl octanoate, 2,3-butanediol and glycerol. In addition, wines produced by P. fermentans/S. cerevisiae combinations showed increased concentration of polysaccharides, which can improve wine taste and body (Domizio et al., 2011b).

Hansenula anomala (alternative names: Wickerhamomyces anomalus,Candida pelliculosa,Pichia anomala) is another species that has been studied during mixed fermentation with S. cerevisiae (Domizio et al., 2011a; Izquierdo Canas et al., 2011). In addition to increasing the formation of some higher alcohols, and acetate and ethyl esters (Domizio et al., 2011a), the use of H. anomala during sequential inoculation trials of Arien grape juice produced wines with decreased C6 alcohols, which are related to ‘green’ sensory attributes, and lower thioalcohol levels, which are associated with ‘reductive’ characters (Izquierdo Canas et al., 2011). This translated into higher scores for sensory descriptors such as ‘fruit’, ‘aroma intensity’, ‘fresh fruit’, ‘sweet smell’, ‘aftertaste persistence’ and ‘floral’, depending on the vintage, compared to S. cerevisiae wines. Wines obtained by mixed fermentation were judged to be better and were preferred over wines produced with a S. cerevisiae monoculture (Izquierdo Canas et al., 2011).

Other species studied include: Williopsis saturnus,Candida cantarellii,Issatchenkia orientalis and Saccharomycodes ludwigii. Mixed fermentations of Emir grape juice with W. saturnus and S. cerevisiae produced wines with increased concentrations of acetic acid, propanol, ethyl acetate and isoamyl acetate (Erten & Tanguler, 2010; Tanguler, 2012). The production of these compounds, however, was inversely correlated with the inoculum level of W. saturnus (Tanguler, 2012). In another study, wines obtained by mixed fermentation with W. saturnus and S. cerevisiae did not show significantly differences in sensory descriptors, but only minor flavour differences compared with S. cerevisiae wines (Lee et al., 2012).

Candida cantarellii (Torulopsis cantarellii) has been shown to produce Syrah wines with increased concentrations of glycerol, acetoin, propanol and succinic acid after mixed fermentation with S. cerevisiae (Toro & Vazquez, 2002). Unfortunately, no sensory analysis has been reported for wines produced by this species. Issatchenkia orientalis has been used in cofermentation of grape juice with S. cerevisiae for reducing the concentration of malic acid in wine (Kim et al., 2008). In addition, mixed fermented wines also showed a reduction in acetaldehyde, propanol, 2-butanol and isoamyl alcohol and were evaluated with the highest scores for ‘colour’, ‘flavour’ and ‘taste’ (Kim et al., 2008). Saccharomycodes ludwigii has been studied during monoculture fermentations of Trebiano grape must (Romano et al., 1999). These wines showed increased concentrations of higher alcohols and acetic acid compared to S. cerevisiae monocultures. While increased formation of polysaccharides, isobutanol, and amyl alcohol and decreased ethyl lactate concentration have been observed in wines fermented with S. ludwigii and S. cerevisiae (Domizio et al., 2011b).

Combinations of non-Saccharomyces yeasts and interactions with other yeasts and bacteria

Combinations of more than one species of non-Saccharomyces yeasts have also been investigated. Torulaspora delbrueckii,H. uvarum (reported as K. apiculata) and S. cerevisiae were used in sequential fermentations of grape must (Herraiz et al., 1990). Hanseniaspora uvarum was inoculated first followed three days later by T. delbrueckii and finally S. cerevisiae was inoculated after eight days. The wines produced had volatile compositions different from the S. cerevisiae wines, but were not evaluated sensorially (Herraiz et al., 1990). In a similar fashion, Izquierdo Canas et al. (2011) studied the sequential inoculation of grape must with Hansenula anomala,T. delbrueckii and S. cerevisiae. Although wines produced by this combination showed some differences in chemical composition to wines produced with S. cerevisiae monoculture, they were similar sensorially and were equally preferred by an expert panel (Izquierdo Canas et al., 2011). Interestingly, the combinations H. anomala/S. cerevisiae and T. delbrueckii/S. cerevisiae produced wines that showed more flavour complexity. Andorra et al. (2010) studied cofermentation of grape juice with combinations of Candida zemplinina,H. uvarum and S. cerevisiae. Wines fermented with the three strains showed higher concentrations of acetic acid, higher alcohols and ethyl esters than S. cerevisiae wines, but similar to the dual combinations C. zemplinina/S. cerevisiae and H. uvarum/S. cerevisiae. Interestingly, the combination C. zemplinina/H. uvarum/S. cerevisiae produced wines with the lowest concentrations of short-chain acids and medium-chain fatty acids (Andorra et al., 2010). Unfortunately, sensory evaluations were not performed.

The first commercial release of two yeast blends was in 2003. These contained a mixture of T. delbrueckii,K. thermotolerans and S. cerevisiae in different proportions (Anonymous, 2004a, b; CHR Hansen, 2013a, b). According to the manufacturer's technical data sheet, this combination (simultaneous inoculation) of yeasts leads to wines with a ‘richer’ and ‘rounder’ flavour with enhanced ‘fruity’ notes. Improvements in wine quality have been observed for a number of white (Chardonnay, Pinot Blanc, Pinot Gris; Riesling) and red grape varieties (Cabernet Sauvignon, Pinot noir, Shiraz, Merlot). The proportion of the non-Saccharomyces yeasts to the S. cerevisiae appeared to be in the region of 1 : 14 (Jolly, unpublished data, 2005). Only one of the yeast blends is currently commercially available (CHR Hansen, 2013a, b).

Genomics of non-Saccharomyces yeast

Saccharomyces cerevisiae was the first eukaryote whose genome was completely sequenced (Goffeau et al., 1996). Since then, several S. cerevisiae industrial strains and particularly wine yeast strains have also been sequenced (Borneman et al., 2013). Genomics in an industrial context has the potential to provide valuable information for strain development programmes and for mapping of quantitative trait loci (QTL) of yeast phenotypic characteristics relevant to a particular process (Borneman et al., 2013).

Similarly, the availability of non-Saccharomyces genome sequences will help in the characterization of commercially relevant strains and aid future selection programmes. Most of the non-Saccharomyces genomes that have been or are being sequenced correspond to type-strains, and not necessarily to yeast strains that can be found in fermenting grape must. Nevertheless, these genomes will provide invaluable information for industrial strains and particularly for wine yeast strains. The genome of the strains Candida glabrata,Debaryomyces hansenii,Lachancea kluyveri,Lachancea thermotolerans,Millerozyma farinosa,S. pombe,Torulaspora delbrueckii and Zygosaccharomyces rouxii have been fully sequenced, while several have been submitted to NCBI recently (Table 2).

2

Non-Saccharomyces yeast species whose genomes have been sequenced

Species	Strain	Status/organization	Web site	Reference or initial data
Candida glabrata	CBS138	Complete genome Génolevures Consortium	http://genolevures.org
Candida lusitaniae	ATCC42720	Quality draft Broad Institute	http://www.broadinstitute.org/annotation/genome/candida_lusitaniae/MultiHome.html
Debaryomyces hansenii	CBS767	Complete genome Génolevures Consortium	http://genolevures.org	Lepingle et al., 2000
Hanseniaspora uvarum	DSM2768	Genome submitted University of Osnabrück	http://www.ncbi.nlm.nih.gov/bioproject/178141
Kluyveromyces marxianus	CBS712	Partial genome Génolevures Consortium	http://genolevures.org
Lachancea kluyveri	CBS3082	Complete genome The genome Institute at Washington University	http://genome.wustl.edu/genomes/	Neuveglise et al., 2000
Lachancea thermotolerans	CBS6340	Complete genome Génolevures Consortium	http://genolevures.org	Malpertuy et al., 2000
Millerozyma farinosa	CBS 7064	Complete genome Génolevures Consortium	http://www.ncbi.nlm.nih.gov/bioproject/188687	Louis et al., 2012
Pichia guilliermondii	ATCC6260	Quality draft genome Broad Institute	http://www.broadinstitute.org/annotation/genome/candida_guilliermondii/MultiHome.html
Pichia membranifaciens	NRRL Y-2026	Genome submitted DOE Joint Genome Institute	http://www.ncbi.nlm.nih.gov/bioproject/52937
Saccharomycodes ludwigii	NBRC 1722	Genome submitted EMBL	http://www.ncbi.nlm.nih.gov/bioproject/28063
Schizosaccharomyces pombe	972h	Complete genome Sanger Institute	http://www.pombase.org/	Wood et al., 2002
Torulaspora delbrueckii	CBS1146	Chromosome with gaps Smurfit Institute of Genetics	http://www.ncbi.nlm.nih.gov/bioproject/79345	Gordon et al., 2011
Wickerhamomyces anomalus	NRRL Y-366-8	Genome submitted DOE Joint Genome Institute	http://www.ncbi.nlm.nih.gov/bioproject/60493
Zygoascus meyerae	E23	Genome submitted Chulalongkorn University	http://www.ncbi.nlm.nih.gov/bioproject/186506
Zygosaccharomyces bailii	ISA1307	Genome submitted Helmholtz Zentrum Munchen	http://www.ncbi.nlm.nih.gov/bioproject/210974
	CLIB213	Genome submitted INRA Montpellier	http://www.ncbi.nlm.nih.gov/bioproject/211734
Zygosaccharomyces rouxii	CBS732	Complete genome Génolevures Consortium	http://genolevures.org

Candida glabrata has 13 chromosomes with a total size of 12.3 Mb not including ribosomal DNA (rDNA), which is organized into two distinct loci, on Chromosomes 12 and 13. There are c. 5283 coding genes, and 207 tRNA genes (Sherman et al., 2009). Debaryomyces hansenii possesses seven chromosomes totalling 12.2 Mb not including rDNA. This strain seems to have the highest coding capacity among yeasts with a putative number of 6906 coding genes and uses an alternative genetic code in which the CUG codon (encoding for the amino acid leucine) is used as a serine codon and is read by a special tRNA-Ser (CAG), as in Candida albicans (Sherman et al., 2009). Lachancea kluyveri is a diploid strain with eight pairs of homologous chromosomes (Neuveglise et al., 2000), ranged from 0.95 Mb to 3 Mb (Sherman et al., 2009). The nuclear genome is c. 11.3 Mb long with a predicted total of 5321 protein-encoding genes (Sherman et al., 2009), while the mitochondrial genome was reported to be 49 kb long (Piskur et al., 1998). Lachancea thermotolerans is diploid, harbouring eight pairs of chromosomes for a total haploid size of 10.4 Mb (excluding the rDNA repeats). The total number of annotated genes is 5350, including 5104 protein-encoding genes and 246 noncoding RNA genes (Sherman et al., 2009). The mitochondrial genome of 23.5 kb in length has also been completely sequenced and annotated (Talla et al., 2005). The genome of M. farinosa comprises seven pairs of chromosomes with a total genome size of 21.5 Mb, and it contains 5736 protein-encoding genes, represented by two different allelic copies (3205 genes), two identical copies (2311 genes) or a unique copy (220 genes; Louis et al., 2012). Schizosaccharomyces pombe has three chromosomes ranging from 3.5 Mb to 5.7 Mb and a 20 kb mitochondrial genome for a total genome size of 13.8 Mb. Schizosaccharomyces pombe contains the smallest number of protein-encoding genes recorded so far for a eukaryote: 4,824 (Wood et al., 2002). The T. delbrueckii genome consists of eight chromosomes with a total of 9.22 Mb, with 4972 predicted protein-encoding genes and 204 genes encoding rDNA (Gordon et al., 2011). Zygosaccharomyces rouxii has seven chromosomes ranged from 1.1 Mb to 1.8 Mb with a total size of 10.4 Mb. Zygosaccharomyces rouxii nuclear genome contains c. 4998 protein-encoding genes and 272 tRNA genes (Sherman et al., 2009). Additionally, Z. rouxii carries a natural plasmid of 6.2 kb, pSR1 which contains three ORFs (Araki et al., 1985).

Research trends

The potential benefits of non-Saccharomyces yeast in wine production are now known. However, the wealth of yeast biodiversity with still hidden potential presents many opportunities for exploitation in wine production (Pretorius et al., 1999; Pretorius, 2000; Fleet, 2008). Cellular aggregation (biofilm and flocculation) by non-Saccharomyces yeast in the winery environment still needs investigation. Biofilm formation can be initiated by S. cerevisiae, a characteristic previously thought to be restricted to bacteria (Reynolds & Fink, 2001; Parsek & Greenberg, 2005; Vallejo et al., 2013). This has implications for wine fermentation and storage of wines. The ability of wine-related non-Saccharomyces to form biofilms is not well researched, but it has been suggested that yeast cells on the surfaces of grape berries may interact in a biofilm system (Renouf et al., 2005).

Metabolites produced by non-Saccharomyces strains that can act against spoilage yeast is another area receiving attention (Masih et al., 2001; Weiler & Schmitt, 2003; Comitini et al., 2004; Ciani & Comitini, 2011). This has potential application during wine maturation and storage. The respiration of sugars by non-Saccharomyces as an approach to lowering alcohol content has also garnered interest (Erten & Campbell, 2001; Gonzalez et al., 2013).

However, to exploit further benefits of non-Saccharomyces yeasts in wine production, the yeast populations on grapes and in must, as well as the effect of winemaking practices on these yeasts, must be known. How the various yeast species and their metabolites interact with each other, and lactic acid bacteria also requires in-depth study. This knowledge will help realize the predictions of Heard (1999) concerning the use of mixed starter cultures tailored to reflect the characteristics of a given wine region. The use of indigenous yeast species with modern technology to produce novel grape-based beverages has further applications for other fruit wines (Sadineni et al., 2012).

Strain selection is of key importance, as not all strains within a species will necessarily show the same desirable characteristics (Fleet, 2008). The accepted list of desirable characteristics as pertaining to the wine yeast S. cerevisiae (Yap, 1987; Henschke, 1997; Pretorius, 2000) will not necessarily apply to non-Saccharomyces yeasts. High fermentation efficiency, high sulphite tolerance and zymocidal (killer) properties, for example, might not be needed in the new technology of wine production. Non-Saccharomyces wine yeasts will necessarily have a different list of desired characteristics. Thorough briefings and assistance of wine producers will have to accompany any new non-Saccharomyces technology for wine production. However, the goals as set out by Pretorius (2000, 2003), Pretorius & Bauer (2002), Pretorius et al. (2012) and others a regarding efficient sugar utilization, enhanced production of desirable volatile esters, enhanced liberation of grape terpenoids and production of glycerol to improve wine flavour and other sensory properties, can be met by selected non-Saccharomyces wine yeasts. This path may bypass current controversies regarding the genetic modification of the ‘wine yeast’ S. cerevisiae. When genetically modified organisms (GMO) are accepted by wine consumers and industries, genetic modification of selected non-Saccharomyces yeasts can further enhance their performance and role in wine production.

Concluding remarks

The diverse array of yeast available to a winemaker through the cellar environment, in the air, on the grape or through inoculation remains a crucial element to the production of wines with a wide range of complex flavours and aromas. Harnessing the performance of fermentation for a desired outcome tantalizes and challenges. Research undertaken in S. cerevisiae can make great contributions to understanding the role and uses of non-Saccharomyces yeast in ‘spontaneous’ and ‘inoculated multispecies’ ferments. The management of ‘mixed ferments’ is more complex than ‘single-species’ ferments because so many things can go wrong. Therefore, a modern approach to ‘multispecies’ wine ferments backed by frontier science and rigorous research is essential to help winemakers achieve their primary objective of achieving a better than 98% conversion of grape sugar to alcohol and carbon dioxide, at a controlled rate and without the development of off-flavours. Therein lays wine's magic blend of art and science.

The art and science of winemaking is analogous to an orchestra: the ‘maestro winemaker’ conducts the symphony (i.e. choosing and managing the participating, desirable non-Saccharomyces yeast) and consumers face the music (i.e. the resulting wine)… but nothing in the fermentation vessel is over until the fat lady (i.e. S. cerevisiae) has sung (i.e. fermented grape sugar to ‘dryness’ and successfully completed the alcoholic fermentation). With the knowledge created, different winemakers can continue to produce different symphonies (i.e. wine styles) with ‘single-species’ and ‘multispecies’ ferments, and different audiences (i.e. consumers) will continue to appreciate their ‘music of choice’. Some will choose the pleasing ‘city-hall-filling’ sounds of a large philharmonic orchestra comprising a great range of diverse instrumentalists (as is the case with wine created from spontaneous fermentation); some will prefer to listen to a smaller ensemble or a quartet or a chamber choir (analogous to wine produced by a selected group of non-Saccharomyces and Saccharomyces yeast); and others will keep purchasing their tickets to be entertained by a well-known and reliable superstar soprano (i.e. S. cerevisiae).

Acknowledgement

Neil Jolly from the ARC Infruitec-Nietvoorbij is financially supported by a combination of funds from the South African Government and wine industry (Winetech). Cristian Varela from The Australian Wine Research Institute is supported by Australia's grape growers and winemakers through their investment body the Grape and Wine Research Development Corporation with matching funding from the Australian Government. Isak Pretorius is supported by an internal grant from Macquarie University.

Author notes

Editor: Jens Nielsen