International Journal of Food Microbiology 252 (2017) 1–9

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Sensory profile and volatile aroma composition of reduced alcohol Merlot MARK
wines fermented with Metschnikowia pulcherrima and Saccharomyces uvarum
C. Varela⁎, A. Barker, T. Tran1, A. Borneman, C. Curtin2
The Australian Wine Research Institute, PO Box 197, Glen Osmond, Adelaide, South Australia 5064, Australia

A R T I C L E I N F O A B S T R A C T

Keywords: Strategies for production of wines containing lower alcohol concentrations are in strong demand, for reasons of
Wine quality, health, and taxation. Development and application of wine yeasts that are less efficient at transforming
Yeast grape sugars into ethanol has the potential to allow winemakers the freedom to make lower alcohol wines from
Non-Saccharomyces grapes harvested at optimal ripeness, without the need for post-fermentation processes aimed at removing
Low alcohol
ethanol. We have recently shown that two non-conventional wine yeast species Metschnikowia pulcherrima and
Flavour
Metagenomics
Saccharomyces uvarum were both able to produce wine with reduced alcohol concentration. Both species
produced laboratory-scale wines with markedly different volatile aroma compound composition relative to
Saccharomyces cerevisiae. This work describes the volatile composition and sensory profiles of reduced-alcohol
pilot-scale Merlot wines produced with M. pulcherrima and S. uvarum. Wines fermented with M. pulcherrima
contained 1.0% v/v less ethanol than S. cerevisiae fermented wines, while those fermented with S. uvarum
showed a 1.7% v/v reduction in ethanol. Compared to S. cerevisiae ferments, wines produced with M. pulcherrima
showed higher concentrations of ethyl acetate, total esters, total higher alcohols and total sulfur compounds,
while wines fermented with S. uvarum were characterised by the highest total concentration of higher alcohols.
Sensorially, M. pulcherrima wines received relatively high scores for sensory descriptors such as red fruit and fruit
flavour and overall exhibited a sensory profile similar to that of wine made with S. cerevisiae, whereas the main
sensory descriptors associated with wines fermented with S. uvarum were barnyard and meat. This work
demonstrates the successful application of M. pulcherrima AWRI3050 for the production of pilot-scale red wines
with reduced alcohol concentration and highlights the need for rigorous evaluation of non-conventional yeasts
with regard to their sensory impacts.

1. Introduction in ethanol concentration can be achieved by post-fermentation pro-

The ethanol content of wine is largely determined by the ripeness,
and hence sugar content, of grapes. Market demand for wines contain-
ing lower ethanol concentrations (Rowley, 2013) has seen a number of
studies into the feasibility of pre- and post-fermentation strategies that
reduce the concentration of sugar available for fermentation into
ethanol, and reduce the concentration of ethanol in finished wine
(Belisario-Sanchez et al., 2009; Bindon et al., 2013; Schmidtke et al.,
2012; Stoll et al., 2010; Varela et al., 2015).
Ideally, these strategies should reduce ethanol levels without
compromising wine flavour, quality, consumer acceptance or increasing
the cost of production. Earlier harvest of grapes shows promise for
moderate reductions in final ethanol concentration while not impacting
on consumer acceptability (Bindon et al., 2014). Substantial decreases
cesses, however these can substantially alter wine volatile composition
and sensory profile (Belisario-Sanchez et al., 2009; King and Heymann,
2014) and also impact on consumer preferences (King and Heymann,
2014; Meillon et al., 2010a, b). Using wine yeast to produce wine with
reduced alcohol content remains one of the simplest and least expensive
approaches for winemakers to employ, and could be used in combina-
tion with other strategies.
Saccharomyces cerevisiae, the principal yeast species used in wine-
making, is very efficient at producing ethanol from sugars under most
environmental conditions, and although some natural variability can be
found among wild isolates of this species, existing S. cerevisiae wine
strains generate comparable alcohol concentrations when fermenting
the same must (Ciani et al., 2016). Research has therefore focused on
generating new S. cerevisiae strains that produce less alcohol than

⁎
Corresponding author.
E-mail address: Cristian.Varela@awri.com.au (C. Varela).
1
Current address: AB Mauri, 1 Richardson Place, North Ryde, NSW 2113, Australia.
2
Current address: College of Agricultural Sciences, Oregon State University, Wiegand Hall, 3051 SW Campus Way, Corvallis, OR 97331, USA.

http://dx.doi.org/10.1016/j.ijfoodmicro.2017.04.002
Received 2 March 2017; Received in revised form 11 April 2017; Accepted 11 April 2017
Available online 12 April 2017
0168-1605/ © 2017 Elsevier B.V. All rights reserved.
C. Varela et al.
traditional wine yeast during fermentation (Ehsani et al., 2009; Kutyna
et al., 2010; Tilloy et al., 2014; Varela et al., 2012); and isolating non-
conventional yeasts that metabolise sugar without generating ethanol
or do so with less efficiency (Ciani et al., 2016; Varela, 2016).
Non-conventional yeast, which include non-Saccharomyces and non-
cerevisiae yeast, are part of the natural microbiota present on grapes,
and harvesting and winemaking equipment, and are present at least
during the early stages of fermentation (Fleet and Heard, 1993; Renouf
et al., 2006; Renouf and Lonvaud-Funel, 2007). The use of non-
conventional yeast is increasingly popular particularly for their effects
on wine composition, flavour, aroma and colour (Jolly et al., 2014;
Varela, 2016) and for their potential to produce reduced-alcohol wines
(Ciani et al., 2016; Varela, 2016). Some of the mechanisms responsible
for reduced ethanol yields include altered biomass synthesis, by-
product formation and/or alternative regulation of respiration.
While some non-Saccharomyces yeast require moderate application
of oxygen during fermentation to grow and impact on wine composition
(Gonzalez et al., 2013), and indeed to produce wine with reduced
ethanol concentration (Contreras et al., 2015a; Quiros et al., 2014),
Contreras et al. (2014) identified a Metschnikowia pulcherrima strain
able to produce wine with reduced ethanol concentration when
sequentially inoculated with S. cerevisiae without aeration. Further-
more, when studying yeast population dynamics during Shiraz fermen-
tations, an indigenous Saccharomyces uvarum yeast strain, which was
also able to produce wine with reduced ethanol concentration, was also
isolated (Contreras et al., 2015b). When used in combination, these
strains of M. pulcherrima and S. uvarum behaved additively, reducing
final ethanol concentrations in both white and red wines to a greater
extent than either strain alone (Varela et al., 2016). Preliminary data on
volatile aroma composition suggested that some treatments may affect
the sensory properties of wine.
Here we describe the sensory profile and volatile aroma composi-
tion of pilot-scale Merlot wines produced with M. pulcherrima
AWRI3050 and S. uvarum AWRI2846. While wines fermented with
both yeast strains showed reduced ethanol concentration compared to
S. cerevisiae wines, volatile and sensory profiles differed substantially
between wines produced with non-conventional yeasts. This work
shows for the first time the use of amplicon-based ITS phylotyping for
validating the role of non-conventional yeasts in pilot-scale wine
fermentation.
2. Materials and methods
2.1. Microorganisms and media
Saccharomyces cerevisiae AWRI838, Metschnikowia pulcherrima
AWRI3050 and Saccharomyces uvarum AWRI2846 were obtained from
the Australian Wine Research Institute (AWRI) Wine Microorganism
Culture Collection. M. pulcherrima AWRI3050 is a non-flocculant
derivative obtained from M. pulcherrima AWRI1149. Briefly, a 80 mL
sample from the end of fermentation of sterile Shiraz (200 mL) was
allowed to settle at room temperature for 20 min. A 1 mL sample was
then taken from the top of the settled culture and used to inoculate a
flask containing 200 mL of sterile Shiraz. After a third selection step,
samples from the top of settled cultures were plated and single colonies
isolated. Colonies were identified as M. pulcherrima following PCR and
RFLP of the 5.8S rRNA gene as described previously (Contreras et al.,
2015b). M. pulcherrima AWRI3050 showed no significant differences in
wine basic composition to M. pulcherrima AWRI1149 (Table S1).
Cryogenically preserved (− 80 °C) strains were cultured and main-
tained on YM plates (3 g/L malt extract, 3 g/L yeast extract, 5 g/L
peptone, 10 g/L glucose, 16 g/L agar) and stored at 4 °C.
2.2. Pilot-scale fermentations
Unlike S. uvarum AWRI2846, M. pulcherrima AWRI3050 is not able
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International Journal of Food Microbiology 252 (2017) 1–9
to finish wine fermentation unaided and requires co- or sequential-
inoculation with S. cerevisiae. Therefore, four treatments were per-
formed to evaluate M. pulcherrima AWRI3050 and S. uvarum AWRI2846
in Merlot must: a control S. cerevisiae inoculation (AWRI838,
1 × 106 cells/mL); a M. pulcherrima/S. cerevisiae co-inoculated ferment
(M. pulcherrima 1 × 106 cells/mL, S. cerevisiae 1 × 105 cells/mL); a S.
uvarum inoculation (1 × 106 cells/mL); and an uninoculated control
fermentation. Merlot must was prepared from grapes obtained from the
Riverland region (South Australia) and contained 240 g/L of sugar
(equal amounts of glucose and fructose), 190 mg N/L of yeast assimil-
able nitrogen (YAN) and 6.2 g/L titratable acidity (determined to
pH 8.2) with a pH of 3.2. Fermentations were performed in triplicate
at 22 °C in fermentation vessels containing 50 kg of randomised grapes
and 50 mg/kg potassium metabisulfite, which were stored at 10 °C
overnight. Twenty-four hours before inoculation Merlot must was
treated with 125 mg/L dimethyl dicarbonate (DMDC) to reduce the
population of native microorganisms.
Starter cultures of all yeast strains were grown overnight in YM
medium under aerobic conditions at 28 °C, shaking at 120 rpm. These
cultures were then used to inoculate 1 L of sterile Merlot, diluted 1:1
with water, in 5 L Erlenmeyer flasks. Flasks were incubated overnight at
22 °C with shaking (120 rpm) under aerobic conditions and then used
to inoculate Merlot must. Ferments were incubated at 22 °C and the
solids cap was plunged twice daily. Samples were taken during
fermentation to determine yeast populations and basic chemical
composition. After sugar was completely consumed Merlot wines were
inoculated for malolactic fermentation with Oenococcus oeni (Lalvin
VP41, Lallemand) as recommended by the manufacturer.
2.3. Analytical techniques
Ethanol, glucose, fructose, glycerol and organic acids were quanti-
fied by high-performance liquid chromatography (HPLC) using a
BioRad HPX87H column as described previously (Varela et al., 2004).
Analysis of higher alcohols, acetate-, and ethyl esters was performed by
Metabolomics Australia (Adelaide) using gas chromatography–mass
spectrometry (GCMS) using a stable isotope dilution analysis (SIDA)
as previously described (Bizaj et al., 2012). Yeast-derived sulfur-
containing volatiles (carbon disulfide, diethyl disulfide, dimethyl
sulfide, ethanethiol, ethyl thioacetate, hydrogen sulfide, methanethiol
and methyl thioacetate) were determined by using headspace cool-on-
column gas chromatography coupled with sulfur chemiluminescence
detection (GC-SCD), with ethylmethyl sulfide and propyl thioacetate as
internal standards (Siebert et al., 2010). 4-Ethylphenol and 4-ethyl-
guaiacol were analysed according the method of Pollnitz et al. (2000).
2.4. Determination of microbial populations
Total DNA was isolated from each sample using the PowerFood
Microbial DNA isolation kit (Mobio, California, USA). Proportions of
each microbial strain were assessed using amplicon-based ITS phylo-
typing as described previously (Sternes et al., 2017). Briefly, 1.0 ng of
total DNA from each sample was subjected to a two-step PCR process
that amplifies a portion of the fungal ribosomal ITS region (Bokulich
and Mills, 2013) while adding both custom in-line barcodes and
sequences necessary for Illumina sequencing (including compatible
Illumina dual-indexes). Following sequencing, raw reads were first
quality and adaptor trimmed (Trimmomatic (Bolger et al., 2014);
Cutadapt (Martin, 2011)), with paired-end reads overlapped (Magoc
and Salzberg, 2011) to form a single contiguous ITS synthetic read.
Reads were then assigned to samples and timepoints using a combina-
tion of both the in-line and Illumina barcodes using custom python
scripts. Operational taxonomic units (OTUs) were clustered de novo
using Swarm v2.0 (Mahe et al., 2014) and taxonomies assigned using
the “assign_taxonomy.py” functionality of QIIME (Caporaso et al., 2010)
in conjunction with a modified version of the QIIME UNITE fungal ITS
C. Varela et al.
database (Sternes et al., 2017). Sequences and lengths for all OUTs
identified in this study are included in Table S2.
2.5. Sensory descriptive analysis
A panel of nine assessors (one male, eight females) with an average
age of 46 years (SD = 10.1) was convened to evaluate the Merlot
wines. A consensus-based descriptive methodology was used as de-
scribed previously (Bindon et al., 2014). Details of the attributes
selected by the panel and the reference standards are listed in Table
S3. Wines (30 mL) were presented to panelists in 3-digit-coded,
covered, ISO standard wine glasses at 22–24 °C, in isolated booths
under daylight-type lighting, with randomised presentation order. All
samples were expectorated. Twelve wines were assessed, comprising
each of the four treatments. Wines were evaluated in triplicate in three
formal sessions that were held on different days. All samples were
presented in a modified Williams Latin Square incomplete random
block design generated by Fizz sensory acquisition software (version
2.46, Biosystemes, Couternon, France). Wines were assessed in sets of
three wines and assessors were forced to have a 30 s rest between
samples and a minimum ten-minute rest between trays. Each presenta-
tion replicate was poured from a separate bottle. The intensity of each
attribute was rated using an unstructured 15 cm line scale numbered
from 0 to 10, with indented anchor points of ‘low’ and ‘high’ placed at
10% and 90% respectively. Data was acquired using Fizz sensory
software. Panel performance was assessed using Fizz, Senstools (OP & P,
The Netherlands) and PanelCheck (Matforsk) software. Sensory attri-
butes used by the panel and their significance are listed in Table S4.
2.6. Statistical analysis
Differences between measurements were determined using two-way
ANOVA and Tukey's multiple comparisons test with the software
GraphPad Prism v6.03. For sensory data ANOVA and Fisher's least
significant difference (LSD) test were carried out using Minitab
(Minitab Inc., Sydney, NSW). The effects of yeast strain, judge,
fermentation replicate nested in yeast, presentation replicate nested
in yeast and fermentation replicate as well as the interactions between
judge and yeast, and between judge and fermentation replicate nested
in yeast were assessed. Differences were considered significant when p
values were lower than 0.05. Principal component analysis (PCA) using
Unscrambler × 10.3 (CAMO ASA, Oslo, Norway) was carried out for
reducing the dimensionality of data and for finding the best differentia-
tion among samples.
3. Results
It had previously been shown at laboratory scale that the two non-
conventional yeast strains, M. pulcherrima AWRI1149 and S. uvarum
AWRI2846, produced wines containing less ethanol than those using
with S. cerevisiae (Varela et al., 2016). However, M. pulcherrima
AWRI1149 flocculates in red grape must (data not shown) making
quantification of cell density difficult and potentially affecting fermen-
tation kinetics in absence of agitation, e.g. pilot-scale or industrial scale
fermentations. To avoid these issues M. pulcherrima AWRI3050, a non-
flocculant derivative of AWRI1149, was isolated and used in this study.
Shiraz wines fermented sequentially with M. pulcherrima AWR3050 and
S. cerevisiae showed the same basic chemical composition than wines
produced with M. pulcherrima AWRI1149/S. cerevisiae (Table S1). Four
treatments were carried out to evaluate wine flavour profile in Merlot
wines inoculated with either S. cerevisiae, M. pulcherrima/S. cerevisiae or
S. uvarum, in addition to an uninoculated control fermentation, where
the indigenous microflora is the primary driver of fermentation.
3
International Journal of Food Microbiology 252 (2017) 1–9
Fig. 1. Sugar utilisation profiles during fermentation of Merlot must inoculated with S.
cerevisiae AWRI838 (blue), M. pulcherrima AWRI3050/S. cerevisiae AWRI838 (orange), S.
uvarum AWRI2846 (red), and uninoculated controls (green). (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this
article.)
3.1. Sugar consumption and yeast population dynamics during wine
fermentation
Fermentations inoculated with S. cerevisiae and M. pulcherrima/S.
cerevisiae showed similar sugar consumption kinetics and completed
alcoholic fermentation in 11 days (Fig. 1). Ferments inoculated with S.
uvarum showed comparable sugar utilisation kinetics to S. cerevisiae
fermentations up to day 5, and after this period they slowed signifi-
cantly and subsequently required an additional four days to complete
fermentation. Uninoculated fermentations exhibited the slowest sugar
utilisation kinetics during the first third of the fermentation, which is
likely due to the reduction in total native microorganisms due to DMDC
treatment of the must. However, after day 5, sugar consumption
increased and all ferments completed alcoholic fermentation in 15 days
(Fig. 1).
All ferments showed similar microbial population composition
immediately after inoculation (day 0), with the genera Aureobasidium,
Metschnikowia and Penicillium in high abundance and the genera
Botrytis, Hanseniaspora, Cryptococcus and Davidiella in minor propor-
tions (Fig. 2). However, subsequent yeast population dynamics were
markedly different and displayed a clear relationship to the inoculation
treatment. In addition to the genera present in all ferments, fermenta-
tions inoculated with S. cerevisiae showed high abundance for this
species immediately after inoculation (Fig. 2). Two days later, S.
cerevisiae dominated yeast populations in two of the three fermentation
replicates and from day 3 to the end of fermentation this species
dominated all fermentation replicates. Two operational taxonomic units
(OTUs) were observed for the genus Metschnikowia, the second of these
OTUs was only found in the ferments co-inoculated with M. pulcherrima
and S. cerevisiae, such that this OTU was assigned to M. pulcherrima
AWRI3050. After inoculation, the M. pulcherrima/S. cerevisiae ferments
showed high abundances for the species M. pulcherrima and S. cerevisiae
in addition to the genera found in the uninoculated controls (Fig. 2). M.
pulcherrima populations decreased considerably after two days with S.
cerevisiae dominating all fermentation replicates from this time point to
the end of fermentation. Ferments inoculated with S. uvarum contained
minor proportions of this species after inoculation (Fig. 2). These
proportions increased in all fermentation replicates two days later
and then dominated yeast populations from day 3 to the end of
fermentation. Uninoculated fermentations exhibited similar yeast po-
pulation composition during the first two days in all fermentation
replicates. After three days S. cerevisiae increased in proportion and
then dominated fermentation from day 5 until all sugar was consumed.
C. Varela et al. International Journal of Food Microbiology 252 (2017) 1–9

Day 0 Day 2 Day 3 Day 5 Day 7 Day 13

1.00

0.75

S. cerevisiae
0.50

0.25

0.00

1.00

0.75

M. pulcherrima
ID
g__Alternaria
0.50 g__Aureobasidium
g__Botrytis
g__Cladosporium
proportional abundance

0.25 g__Cryptococcus
g__Davidiella
g__Discostroma
g__Hanseniaspora
0.00 g__Kazachstania
g__Lachancea
1.00 g__Metschnikowia
s__Metschnikowia pulcherrima
g__Paraconiothyrium
g__Penicillium
0.75 g__Pleospora
g__Rhodotorula

S. uvarum
g__Torulaspora
0.50 s__Saccharomyces cerevisiae
s__Saccharomyces uvarum
Unassigned

0.25

0.00

1.00

0.75

0.50 Uninoculated

0.25

0.00
A B C A B C A B C A B C A B C A B C
ferment replicate

Fig. 2. Microbial population dynamics for replicate fermentations of Merlot must inoculated with either S. cerevisiae AWRI838 (SC), M. pulcherrima AWRI3050/S. cerevisiae AWRI838
(MP), S. uvarum AWRI2846 (SU), in addition to the uninoculated control (UN).

3.2. Wine chemical and volatile composition
All treatments resulted in reduced ethanol concentration relative to
wines made with S. cerevisiae. Wines fermented with M. pulcherrima/S.
cerevisiae had 1.0% v/v lower ethanol concentration, while wines
produced with S. uvarum and uninoculated wines were 1.7% v/v and
0.7% v/v lower, respectively (Table 1). Although significantly different,
there were minimal residual sugar concentration differences between
treatments. Compared to S. cerevisiae wines all treatments had higher
concentrations of glycerol and succinic acid with the highest concen-
trations found in wines produced with S. uvarum. The concentration of
acetic acid was lowest for S. uvarum wines, while malic acid concentra-
tion was not significantly different between treatments, as a result of
performing malolactic fermentation. Although titratable acidity was
higher for all treatments when compared to S. cerevisiae wines, wines
produced with S. uvarum had the highest concentration and the lowest
pH values of the wines (Table 1).
Major differences in the concentration of volatile compounds were
also found among treatments. While ethyl acetate concentrations were
highest in the wines produced with M. pulcherrima/S. cerevisiae, in
addition to the uninoculated ferments, the total concentration of esters
(excluding ethyl acetate) was significantly different only for M.
pulcherrima/S. cerevisiae wines (Table 1). The latter result was mainly
driven by the higher concentrations of 2-methylbutyl acetate found in
M. pulcherrima/S. cerevisiae wines compared to the other treatments. All
treatments showed increased concentrations of higher alcohols than the
S. cerevisiae wines and particularly in 2-methyl propanol and 2 & 3-
methyl butanol. Wines produced with S. uvarum had the highest total
concentration of higher alcohols. Uninoculated ferments showed the
highest total concentration of sulfur compounds, with total levels
eighteen times higher than the control S. cerevisiae wines being
recorded. Wines produced with M. pulcherrima/S. cerevisiae had five
times higher total concentration of sulfur compounds than S. cerevisiae
wines. The high total concentration of sulfur compounds observed in
both the M. pulcherrima/S. cerevisiae and uninoculated wines was the
result of increased concentrations of dimethyl sulfide, ethanethiol,
hydrogen sulfide and methanethiol (Table 1). The concentrations of
4-ethylphenol and 4-ethylguaiacol were below the detection threshold
(< 10 μg/L) for all wines.
Principal component analysis (PCA) was used to visualise the
differences in volatile composition among treatments and to identify
the volatile fermentation products that provided greatest discrimination
between treatments (Fig. 3). The first principal component (PC1)
accounted for 57% of the total variation, while PC2 explained a further
23%. Wines clustered tightly according to inoculation treatment. Wines
made by inoculation with S. cerevisiae had higher concentration of the

4
C. Varela et al. International Journal of Food Microbiology 252 (2017) 1–9

Table 1
Chemical composition of the Merlot wines fermented with S. cerevisiae AWRI838, M. pulcherrima AWRI3050, S. uvarum AWRI2846, and uninoculated fermentations.

S. cerevisiae M. pulcherrima/S. cerevisiae S. uvarum Uninoculated

Residual sugar [g/L] 0.25 ± 0.01a 0.23 ± 0.03ab 0.28 ± 0.03a 0.19 ± 0.03b
Ethanol [%v/v] 14.81 ± 0.01a 13.81 ± 0.01c 13.13 ± 0.06d 14.13 ± 0.12b
Glycerol [g/L] 6.72 ± 0.03d 7.83 ± 0.06b 11.76 ± 0.13a 7.42 ± 0.18c
Acetic acid [g/L] 0.15 ± 0.02b 0.15 ± 0.01b 0.05 ± 0.01c 0.23 ± 0.04a
Malic acid [g/L] 0.22 ± 0.09a 0.19 ± 0.02a 0.37 ± 0.15a 0.19 ± 0.01a
Succinic acid [g/L] 0.53 ± 0.01c 0.79 ± 0.04b 2.02 ± 0.16a 0.75 ± 0.07b
Titratable acidity pH 7 [g/L] 5.16 ± 0.06c 5.50 ± 0.10b 7.97 ± 0.12a 5.57 ± 0.12b
pH 3.46 ± 0.01a 3.44 ± 0.01a 3.36 ± 0.01c 3.40 ± 0.02b
Esters
Ethyl acetate [mg/L] 38.54 ± 0.94c 52.25 ± 1.66a 44.55 ± 0.62bc 49.30 ± 3.24ab
Ethyl propanoate [mg/L] 0.21 ± 0.01b 0.15 ± 0.01c 0.43 ± 0.01a 0.17 ± 0.02c
Ethyl 2-methyl propanoate [μg/L] 13 ± 6c 20 ± 0bc 103 ± 6a 30 ± 0b
2-Methylpropyl acetate [mg/L] 0.03 ± 0.00b 0.06 ± 0.00a 0.06 ± 0.00a 0.05 ± 0.00a
Ethyl butanoate [mg/L] 0.26 ± 0.02a 0.23 ± 0.02ab 0.19 ± 0.01b 0.23 ± 0.02ab
Ethyl 2-methyl butanoate [μg/L] 0 ± 0b 0 ± 0b 20 ± 0a 7 ± 6b
Ethyl 3-methyl butanoate [μg/L] 10 ± 1b 10 ± 1b 51 ± 1a 10 ± 1b
2-Methylbutyl acetate [mg/L] 1.57 ± 0.07d 2.69 ± 0.10a 2.36 ± 0.07b 2.00 ± 0.01c
Ethyl hexanoate [mg/L] 0.70 ± 0.06a 0.59 ± 0.02b 0.19 ± 0.01c 0.52 ± 0.05b
Hexyl acetate [mg/L] 0.03 ± 0.01a 0.04 ± 0.01a 0.02 ± 0.01a 0.03 ± 0.01a
Ethyl octanoate [mg/L] 0.71 ± 0.03a 0.58 ± 0.01b 0.11 ± 0.01d 0.47 ± 0.04c
Ethyl decanoate [mg/L] 0.17 ± 0.01b 0.21 ± 0.02a 0.12 ± 0.01c 0.15 ± 0.01b
Total esters [mg/L]§ 3.69 ± 0.13b 4.59 ± 0.13a 3.65 ± 0.07b 3.65 ± 0.13b
Higher alcohols
Butanol [mg/L] 0.83 ± 0.01c 0.82 ± 0.01c 1.10 ± 0.03a 0.92 ± 0.02b
2-Methyl propanol [mg/L] 31.28 ± 1.91c 48.10 ± 0.65b 68.35 ± 0.66a 62.79 ± 8.65a
2 & 3-Methyl butanol [mg/L] 160.85 ± 7.39c 181.08 ± 1.26b 268.78 ± 1.66a 191.56 ± 8.67b
Hexanol [mg/L] 2.16 ± 0.08b 2.54 ± 0.07a 2.43 ± 0.10ab 2.24 ± 0.16b
Total higher alcohols [mg/L] 195.13 ± 9.16c 232.54 ± 0.76b 340.67 ± 1.90a 257.50 ± 17.28b
Sulfur compounds
Carbon disulfide [μg/L] 1.97 ± 0.87ab 1.95 ± 0.06ab 0.90 ± 0.01b 2.13 ± 0.06a
Dimethyl sulfide [μg/L] 15.67 ± 0.58b 33.67 ± 1.53a 9.63 ± 0.46b 39.67 ± 10.69a
Ethanethiol [μg/L] 0.00 ± 0.00b 5.73 ± 0.98a 0.00 ± 0.00b 6.30 ± 0.01a
Hydrogen sulfide [μg/L] 2.97 ± 0.95c 65.67 ± 12.34b 2.90 ± 0.72c 308.67 ± 20.50a
Methanethiol [μg/] 3.63 ± 0.83ab 7.37 ± 1.03a 0.00 ± 0.00b 7.53 ± 5.00a
Methyl thioacetate [μg/] 0.00 ± 0.01b 0.00 ± 0.01b 8.90 ± 0.36a 9.77 ± 1.16a
Total sulfur compounds [μg/L] 24.87 ± 2.84b 135.30 ± 44.85b 23.57 ± 2.20b 466.97 ± 169.24a

Values are means ± standard deviation of three independent replicates.

Shared superscript letters (a, b, c) in the same row indicate no significant difference (LSD test, p = 0.05).
§
Total excludes ethyl acetate.

ethyl esters, ethyl butanoate, ethyl hexanoate and ethyl octanoate,
whereas wines co-inoculated with M. pulcherrima and S. cerevisiae were
higher in carbon disulfide, methanethiol, dimethyl sulfide and hexyl
acetate. Uninoculated wines exhibited a relatively high concentrations
of hydrogen sulfide and clustered closely with M. pulcherrima wines.
Wines produced with S. uvarum were markedly different from the other
samples and were characterised by higher concentrations of branched
chain esters (ethyl-2-methyl propanoate, ethyl-2-methyl butanoate,
ethyl-3-methyl butanoate), in addition to the higher alcohols butanol
and 2 & 3-methyl butanol.
3.3. Sensory analysis of reduced alcohol wines
In addition to possessing distinct chemical profiles, differences in
appearance, aroma and flavour were also found between treatments
(Fig. 4). The M. pulcherrima/S. cerevisiae and uninoculated wines were
found to have similar sensory profiles, not differing significantly in any
attribute, and together were characterised by relatively high scores for
the sensory attributes ‘purple tint’, ‘overall fruit aroma’, ‘overall fruit
flavour’, ‘red fruit aroma’, and ‘red fruit flavour’; and relatively low
scores for the attributes ‘brown tint’, ‘vegetal aroma’, ‘meat aroma’ and
‘barnyard aroma’. The control wines produced with S. cerevisiae alone
were similar to the M. pulcherrima/S. cerevisiae and uninoculated wines,
but were rated significantly higher in ‘brown tint’ and significantly
lower in ‘red fruit aroma’. As was observed for the chemical analyses,
the wines produced using S. uvarum showed the most distinct sensory
profiles, driven by high ratings for ‘cloudiness’, ‘brown tint’, ‘vegetal
aroma’, ‘meat aroma’ and ‘barnyard aroma’; and low in ‘purple tint’,
‘overall fruit aroma’, ‘mint aroma’ and ‘red fruit aroma’.
4. Discussion
The work described here extends previous research that investigated
the use of non-conventional yeasts to produce wine with reduced
ethanol concentration (Contreras et al., 2015b; Contreras et al., 2014;
Varela et al., 2016). We have previously shown that M. pulcherrima
AWRI1149 and S. uvarum AWRI2846 produced reduced-alcohol Char-
donnay and Shiraz wines at laboratory scale (Varela et al., 2016). The
volatile aroma composition of those wines differed significantly from
one another, suggesting that the non-Saccharomyces yeasts used may
affect wine sensory properties. In fact, this has been observed in
different grape varieties and with different non-Saccharomyces species
(Benito et al., 2015; Gonzalez-Royo et al., 2015). In this study, we
sought to determine whether a non-flocculant derivative of AWRI1149,
M. pulcherrima AWRI3050, and S. uvarum AWRI2846 could produce
lower-ethanol wine at pilot-scale, and to what extent the wines they
produced were sensorially different from wine fermented with S.
cerevisiae.
Since we have previously observed some degree of inhibition of M.
pulcherrima growth by other microorganisms such as H. uvarum, T.
delbrueckii and/or P. kluyveri (Contreras et al., 2015b), Merlot must was
treated with dimethyl dicarbonate (DMDC) to inhibit the native
microbiota (Delfini et al., 2002). Thus, uninoculated fermentations
showed the slowest sugar utilisation kinetics particularly during the

5
C. Varela et al.
Fig. 3. Principal component analysis of yeast volatile fermentation products in Merlot
wines. Scores and loadings plots for the first two principal components. Results are shown
for fermentations inoculated with S. cerevisiae AWRI838 (blue), M. pulcherrima
AWRI3050/S. cerevisiae AWRI838 (orange), S. uvarum AWRI2846 (red), in addition to
uninoculated controls (green). 2M propanol, 2-methylpropanol; 2 & 3M butanol, 2 & 3-
methylbutanol; 2Mpropyl acetate, 2-methylpropyl acetate; 2Mbutyl acetate, 2-methylbu-
tyl acetate; butanol, butanol; C disulfide, carbon disulfide; DMS, dimethyl sulfide;
Ethanediol, ethanediol; E acetate, ethyl acetate; E propanoate, ethyl propanoate; E2M
propanoate, ethyl 2-methylpropanoate; E butanoate, ethyl butanoate; E2M butanoate,
ethyl 2-methylbutanoate; E3M butanoate, ethyl 3-methylbutanoate; E hexanoate, ethyl
hexanoate; E octanoate, ethyl octanoate; E decanoate, ethyl decanoate; H2S, hydrogen
sulfide; hexanol, hexanol; H acetate, hexyl acetate; Methanethiol, methanethiol; M
thioacetate, methyl thioacetate. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
first four days of fermentation, indicating a successful inhibition of
native microorganisms as a result of DMDC addition. After DMDC
treatment (day 0), the genera Aureobasidium, Metschnikowia, Penicillium,
Botrytis, Hanseniaspora, Cryptococcus and Davidiella, which have been
found associated with vines and grapes (Bokulich et al., 2014;
Morrison-Whittle and Goddard, 2015; Taylor et al., 2014), and/or
during wine fermentation (Fleet et al., 2002; Heard, 1999; Romano
et al., 2003), dominated microbial populations.
For the first time we have shown the use of amplicon-based ITS
phylotyping for validating the persistence of non-conventional yeasts
during pilot-scale wine fermentation. For all ferments there was a clear
relationship between fermentation kinetics and yeast population dy-
namics, specifically the point at which Saccharomyces spp. became
dominant. Thus, sugar utilisation in uninoculated ferments increased
6
International Journal of Food Microbiology 252 (2017) 1–9
from day 5 when S. cerevisiae dominated yeast populations. S. cerevisiae
and M. pulcherrima/S. cerevisiae ferments showed the fastest fermenta-
tion kinetics. The fast sugar utilisation kinetics observed for M.
pulcherrima/S. cerevisiae fermentations correlated with the dominance
of S. cerevisiae during fermentation. One of the two operational
taxonomic units (OTUs) observed for the genus Metschnikowia was only
found in the ferments inoculated with M. pulcherrima AWRI3050,
enabling the identification of this species from the native
Metschnikowia population. Thus, at inoculation M. pulcherrima
AWRI3050 dominated these ferments. In contrast to other fermenta-
tions, S. uvarum ferments were dominated by this species and not by S.
cerevisiae, confirming the ability of this species to complete wine
fermentation as observed previously in Chardonnay and Shiraz
(Varela et al., 2016), and in Riesling (Ultee et al., 2013).
Wines fermented with M. pulcherrima AWRI3050 contained 1.0% v/
v less ethanol than S. cerevisiae wines, whereas wines fermented with S.
uvarum AWRI2846 were 1.7% v/v lower than control wines. In addition
to ethanol yield and fermentation kinetics, it is fundamental to establish
the effect of yeast on wine flavour and aroma to determine if a ‘low-
ethanol’ wine yeast might be suitable for commercial winemaking.
While non-conventional wine yeast can impart novel and desirable
characters to wine they can also produce metabolites that impact
negatively on wine flavour profiles (Magyar and Toth, 2011; Rojas
et al., 2001; Viana et al., 2008). Wines produced with different yeast
strains in the present study showed significant differences in the
concentration of key volatile compounds, particularly noteworthy the
increased concentrations of ethyl acetate, total esters, total higher
alcohols and total sulfur compounds found in M. pulcherrima wines.
This yeast species is known to produce high concentrations of esters
during wine fermentation (Bisson and Kunkee, 1991; Clemente-Jimenez
et al., 2004; Rodriguez et al., 2010; Sadoudi et al., 2012) and it has also
been described as able to release important quantities of the thiol 3-
mercaptohexan-1-ol (3MH) from its precursor during Sauvignon Blanc
fermentation (Zott et al., 2011). However, M. pulcherrima production of
other sulfur-containing volatile compounds has not been reported
previously.
The distinctive volatile composition profiles observed among fer-
ments resulted in wines clustered tightly according to inoculation
treatment after principal component analysis. Interestingly, although
S. cerevisiae dominated most of the fermentation for the M. pulcherrima/
S. cerevisiae wines, these wines were most similar to uninoculated
ferments, whereas wines inoculated with S. cerevisiae alone and wines
produced with S. uvarum clustered separately and away from the other
wines. This indicates that the different yeast population dynamics
observed during fermentation generated wines with characteristic
volatile compositional profiles. Consequently, sensory profiles were
similar between M. pulcherrima/S. cerevisiae and uninoculated ferments,
while wines fermented with either S. cerevisiae or S. uvarum exhibited
distinctly different sensory profiles.
M. pulcherrima and uninoculated wines showed high scores for
sensory descriptors that most would consider positive and desirable,
and low scores for negative sensory attributes, despite relatively high
concentrations of ethyl acetate and total sulfur-containing compounds.
Although these compounds are usually associated with negative sensory
descriptors, they can impart positive attributes or add complexity to
wine aroma at low concentrations (Amerine and Roessler, 1983;
Escudero et al., 2007; Jackson, 2009; Siebert et al., 2009; Spedding
and Raut, 1982). Uninoculated ferments are not necessarily associated
with negative sensory attributes and/or off-flavours (Dillon et al.,
2013), and in fact, spontaneous fermentation has been shown to
increase some ‘fruity’ and ‘fresh green’ sensory attributes and enhance
flavour diversity in Chardonnay wines (Medina et al., 2013). Similarly,
there are several studies describing the impact of M. pulcherrima on
wine quality scores and wine sensory profile. When compared to
control S. cerevisiae, wines produced with M. pulcherrima have shown
increased quality scores in Sauvignon blanc and Chenin blanc (Jolly
C. Varela et al.
Fig. 4. Mean scores for significant appearance, aroma and palate attributes for Merlot wines fermented with S. cerevisiae AWRI838 (blue), M. pulcherrima AWRI3050/S. cerevisiae
AWRI838 (orange), S. uvarum AWRI2846 (red), and uninoculated controls (green). The Least Significant Difference value (LSD, p = 0.05) is also shown. Differences were considered
significant when p values were lower than 0.05. Significance levels are as follows: *p < 0.05; **p < 0.01; ***p < 0.001. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
et al., 2003), and in Muscat d'Alexandrie (Rodriguez et al., 2010). M.
pulcherrima has also been shown to increase wine flavour and aroma in
Debina wines (Parapouli et al., 2010), and/or enhance positive sensory
attributes, such as ‘citrus/grape fruit’, ‘pear’ and ‘flowery’ in Riesling
(Benito et al., 2015) and in base wine for sparkling wine production
(Gonzalez-Royo et al., 2015). Only one report has shown a negative
impact of M. pulcherrima, decreasing quality scores in a Chardonnay
wine compared to control S. cerevisiae wines, the opposite found in
Sauvignon blanc and Chenin blanc with the same strain (Jolly et al.,
2003). This could indicate a potential influence of grape variety over M.
pulcherrima metabolism and therefore, on wine sensory profile.
In contrast, wines fermented with S. uvarum showed a sensory
profile mostly dominated by unusual and negative sensory attributes
that would conventionally be consider off-flavours. Interestingly, 4-
ethylphenol and 4-ethylguaiacol the most common volatile compounds
associated with the attribute ‘barnyard’ were not detected in any of the
wines, indicating that other compounds are responsible for the high
rating of this sensory attribute for S. uvarum wines. In the literature, the
effect of S. uvarum on wine sensory profiles include decreasing ‘fruity’
and ‘citrus’ attributes and increasing characters such as ‘nutty’ and
‘aldehyde’ in Chardonnay wines (Eglinton et al., 2000); decreasing
colour in Shiraz wines (Holt et al., 2013); and also enhancing sensory
scores for desirable attributes in Malvasia delle Lipari wines (Muratore
et al., 2007). Although results presented here and in literature reports
suggest a negative role of S. uvarum on wine sensory profile, unusual
characters can be pursued in certain wine varieties particularly for
7
International Journal of Food Microbiology 252 (2017) 1–9
increasing wine complexity, i.e. using wine with these characters as
blending material. Before evaluating strategies to improve the sensorial
characteristics of S. uvarum AWRI2846 it is necessary to investigate the
sensory profile of wines from different grape varieties fermented with
this strain, and to acknowledge that this study comprises a single proof-
of-concept trial in one Merlot must.
Previous studies with white (King and Heymann, 2014) and red
wines (Bindon et al., 2014; Casassa et al., 2013) have shown a
correlation between ethanol concentration and the sensory attributes
‘hotness’ and ‘viscosity’. Interestingly, there was no significant differ-
ences for these attributes among wines in this study. It is possible that
the increased concentration of higher alcohols found in wines produced
with M. pulcherrima/S. cerevisiae, S. uvarum and without inoculation
could influence the sensory perception of this attribute. Similarly, the
increased glycerol concentrations found in the wines mentioned above
could have had an effect on the perception of ‘viscosity’.
In summary, the work described in this paper shows that reduced-
alcohol wines produced with M. pulcherrima AWRI3050 resulted in high
scores for desirable sensory descriptors and low scores for negative
attributes. In contrast, reduced-alcohol wines produced with S. uvarum
AWRI2846 were characterised by unusual and negative sensory attri-
butes. This work thus demonstrates the successful application of M.
pulcherrima AWRI3050 for the production of pilot-scale Merlot wines
with reduced alcohol concentration and good quality sensory profile.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.ijfoodmicro.2017.04.002.
C. Varela et al.
Acknowledgements
The authors would like to thank the sensory panelists, Wes Pearson
and Dr. Leigh Francis for the sensory data. The AWRI, a member of the
Wine Innovation Cluster in Adelaide, is supported by Australia's
grapegrowers and winemakers through their investment body the
Grape and Wine Research Development Corporation with matching
funds from the Australian Government.
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