MINIREVIEW
Not your ordinary yeast: non-Saccharomyces yeasts in wine
production uncovered
Neil P. Jolly1, Cristian Varela2 & Isak S. Pretorius3
1
ARC Infruitec-Nietvoorbij, Stellenbosch, South Africa; 2The Australian Wine Research Institute, Adelaide, SA, Australia; and
Macquarie University, Sydney, NSW, Australia
3
Correspondence: Isak S. Pretorius,
Macquarie University, Balaclava Road, North
Ryde, Sydney, NSW 2109, Australia.
Tel.: +61 2 9850 8645;
fax: +61 2 9850 8799;
e-mail: [email protected]
Received 27 August 2013; accepted 7
October 2013.
Final version published online 11 November
2013.
DOI: 10.1111/1567-1364.12111
Editor: Jens Nielsen
YEAST RESEARCH
Keywords
non-Saccharomyces; yeasts; wine; aroma;
flavour; fermentation.
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.
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
FEMS Yeast Res 14 (2014) 215–237
‘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
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
216
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 newfound 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
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
N.P. Jolly et al.
wine styles that match consumer expectations in a diverse
range of market segments.
Non-Saccharomyces yeasts – a doubleedged 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; Ribereau-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).
Grape growing
Grape harvesting
Consumption
Sorting & triage
Packaging
Labelling
Cooling
Bottling
Destemming
Membrane filtration
WHITE
Cold stabilisation
Crushing
Fining
WINE
Blending
Pressing
MAKING
Battonage
Clarification
Racking
Clarification
Maceration
Malolactic fermentation
(only for certain wine styles)
Fermentation
Natural yeast
or yeast addition
Fig. 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).
FEMS Yeast Res 14 (2014) 215–237
217
Non-Saccharomyces wine yeasts
Grape growing
Grape harvesting
Consumption
Sorting & triage
Packaging
Labelling
Cooling
Bottling
Destemming
Membrane filtration
RED
Cold stabilisation
Crushing
WINE
Fining
MAKING
Blending
Fermentation
Battonage
Racking
Maceration
Pumping over
Clarification
Punch-down
Malolactic Draining of gross lees Natural yeast
or yeast addition
fermentation
Pressing
Maceration
Clarification
Fig. 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
FEMS Yeast Res 14 (2014) 215–237
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
8
10 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
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
218
N.P. Jolly et al.
Table 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
Citeromyces matritensis
Debaryomyces hansenii
Dekkera bruxellensis
Hanseniaspora guilliermondii
Hanseniaspora occidentalis
Hanseniaspora osmophila
Hanseniaspora uvarum
Hanseniaspora vineae
Lachancea kluyveri
Lachancea thermotolerans
Candida globosa
Candida famata
Brettanomyces bruxellensis
Kloeckera apis
Kloeckera javanica
Kloeckera corticis
Kloeckera apiculata
Kloeckera africana
–‡
–‡
Metschnikowia pulcherrima
Meyerozyma guilliermondii
Milleronzyma farinosa
Pichia fermentans
Pichia kluyveri
Pichia membranifaciens
Pichia occidentalis
Pichia terricola
Saccharomycodes ludwigii
Starmerela bombicola
Torulaspora delbrueckii
Wickerhamomyces anomalus
Candida
Candida
–‡
Candida
–‡
Candida
Candida
–‡
–‡
Candida
Candida
Candida
Zygoascus meyerae
Zygosaccharomyces bailii
–†
Candida hellenica
–‡
Candida zemplinina
–†
Candida stellata
pulcherrima
guilliermondii
Synonyms*
Pichia hansenii
Saccharomyces kluyveri
Kluyveromyces thermotolerans;
Candida dattlia
Torulopsis pulcherrima
Pichia guilliermondii
Pichia farinosa
lambica
Hansenula kluyveri
valida
sorbosa
Issatchenkia occidentalis
Issatchenkia terricola
bombicola
colliculosa
pelliculosa
Torulopsis bombicola
Saccharomyces rosei
Pichia anomala; Hansenula
anomala
Saccharomyces bailii
Possibly Candida stellata in
older literature
Torulopsis stellata
*Names sometimes found in older literature.
No teleomorphic form.
‡
No anamorphic form.
†
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.
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,
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
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).
FEMS Yeast Res 14 (2014) 215–237
Non-Saccharomyces wine yeasts
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; T€
or€
ok et al.,
1996; Constantı 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; LonvaudFunel, 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;
Constantı 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 nonSaccharomyces yeasts are. The specific environmental
conditions in the must, that is, high osmotic pressure;
equimolar mixture of glucose and fructose; the presence
of SO2; 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; LonvaudFunel, 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
FEMS Yeast Res 14 (2014) 215–237
219
non-Saccharomyces yeasts. In parallel, the use of modern
laboratory techniques has made the detection of nonSaccharomyces 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; LonvaudFunel, 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
ª 2013 Federation of European Microbiological Societies.
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220
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 nonSaccharomyces yeast (Jolly et al., 2003c).
Besides affecting wine flavour, the metabolism of nonSaccharomyces 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; Ribereau-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
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
N.P. Jolly et al.
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 reducedalcohol 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 reducedalcohol 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;
Nyk€anen, 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 nonSaccharomyces 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 (Nyk€anen,
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
b-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, nonSaccharomyces yeasts belonging to the genera Debaryomyces, Hansenula, Candida, Pichia and Kloeckera possess various degrees of b–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;
Fernandez-Gonzalez et al., 2003; Rodrıguez 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
FEMS Yeast Res 14 (2014) 215–237
Non-Saccharomyces wine yeasts
(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
b-glucosidase addition on grape juice or wine, focusing
particularly in the inhibition of b-glucosidase activity by
sugar, alcohol, pH and/or temperature. An intracellular
b-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 b-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 b-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 b-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). NonSaccharomyces 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; ClementeJimenez 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
FEMS Yeast Res 14 (2014) 215–237
221
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
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
222
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 offflavour 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 (Nyk€anen, 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
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
N.P. Jolly et al.
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 nonSaccharomyces 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 rose 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
FEMS Yeast Res 14 (2014) 215–237
223
Non-Saccharomyces wine yeasts
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 nonSaccharomyces yeasts.
Candida pulcherrima
Metschnikowia pulcherrima (anamorph C. pulcherrima) is
another yeast commercially available. This commercial
strain produces an extra-cellular a-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; Rodrıguez 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 (Sch€
utz & 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; Rodrıguez 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).
FEMS Yeast Res 14 (2014) 215–237
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 & Sch€
utz, 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
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
224
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
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
N.P. Jolly et al.
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
FEMS Yeast Res 14 (2014) 215–237
225
Non-Saccharomyces wine yeasts
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
FEMS Yeast Res 14 (2014) 215–237
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; S€
utterlin et al.,
2004; S€
utterlin, 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 &
Rodrıguez, 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, 2,3butanediol 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
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Published by John Wiley & Sons Ltd. All rights reserved
226
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
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
N.P. Jolly et al.
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).
FEMS Yeast Res 14 (2014) 215–237
Non-Saccharomyces wine yeasts
227
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).
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).
Combinations of non-Saccharomyces
yeasts and interactions with other
yeasts and bacteria
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 typestrains, 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).
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
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/
FEMS Yeast Res 14 (2014) 215–237
Genomics of non-Saccharomyces yeast
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
228
N.P. Jolly et al.
Table 2. Non-Saccharomyces yeast species whose genomes have been sequenced
Species
Strain
Status/organization
Web site
Candida
glabrata
Candida
lusitaniae
Debaryomyces
hansenii
Hanseniaspora
uvarum
Kluyveromyces
marxianus
Lachancea
kluyveri
CBS138
http://genolevures.org
Lachancea
thermotolerans
Millerozyma
farinosa
Pichia
guilliermondii
Pichia
membranifaciens
Saccharomycodes
ludwigii
Schizosaccharomyces
pombe
Torulaspora
delbrueckii
Wickerhamomyces
anomalus
Zygoascus
meyerae
Zygosaccharomyces
bailii
CBS6340
Complete genome
Genolevures Consortium
Quality draft
Broad Institute
Complete genome
Genolevures Consortium
Genome submitted
€ck
University of Osnabru
Partial genome
Genolevures Consortium
Complete genome
The genome Institute at
Washington University
Complete genome
Genolevures Consortium
Complete genome
Genolevures Consortium
Quality draft genome
Broad Institute
Genome submitted
DOE Joint Genome Institute
Genome submitted
EMBL
Complete genome
Sanger Institute
Chromosome with gaps
Smurfit Institute of Genetics
Genome submitted
DOE Joint Genome Institute
Genome submitted
Chulalongkorn University
Genome submitted
Helmholtz Zentrum Munchen
Genome submitted
INRA Montpellier
Complete genome
Genolevures Consortium
ATCC42720
CBS767
DSM2768
CBS712
CBS3082
CBS 7064
ATCC6260
NRRL Y-2026
NBRC 1722
972h
CBS1146
NRRL Y-366-8
E23
ISA1307
CLIB213
Zygosaccharomyces
rouxii
CBS732
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
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
Reference or
initial data
http://www.broadinstitute.org/annotation/
genome/candida_lusitaniae/MultiHome.html
http://genolevures.org
Lepingle
et al., 2000
http://www.ncbi.nlm.nih.gov/bioproject/178141
http://genolevures.org
http://genome.wustl.edu/genomes/
Neuveglise
et al., 2000
http://genolevures.org
Malpertuy
et al., 2000
Louis et al.,
2012
http://www.ncbi.nlm.nih.gov/bioproject/188687
http://www.broadinstitute.org/annotation/genome/
candida_guilliermondii/MultiHome.html
http://www.ncbi.nlm.nih.gov/bioproject/52937
http://www.ncbi.nlm.nih.gov/bioproject/28063
http://www.pombase.org/
http://www.ncbi.nlm.nih.gov/bioproject/79345
Wood et al.,
2002
Gordon et al.,
2011
http://www.ncbi.nlm.nih.gov/bioproject/60493
http://www.ncbi.nlm.nih.gov/bioproject/186506
http://www.ncbi.nlm.nih.gov/bioproject/210974
http://www.ncbi.nlm.nih.gov/bioproject/211734
http://genolevures.org
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:
FEMS Yeast Res 14 (2014) 215–237
229
Non-Saccharomyces wine yeasts
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 winerelated 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
FEMS Yeast Res 14 (2014) 215–237
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 nonSaccharomyces 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
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
230
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).
Acknowledgements
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.
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