fermentation

Article
Impact on Sensory and Aromatic Profile of Low
Ethanol Malbec Wines Fermented by Sequential
Culture of Hanseniaspora uvarum and
Saccharomyces cerevisiae Native Yeasts
María Victoria Mestre 1,2,† , Yolanda Paola Maturano 1,2, *,† , Candelaria Gallardo 1,2 ,
Mariana Combina 2,3 , Laura Mercado 3 , María Eugenia Toro 1 , Francisco Carrau 4 ,
Fabio Vazquez 1 and Eduardo Dellacassa 4
1 Instituto de Biotecnología, UNSJ, Av. San Martín 1109 (O), San Juan 5400, Argentina
2 Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET),
Godoy Cruz 2290 C1425FQB CABA, Argentina
3 Estación Experimental Agropecuaria Luján de Cuyo (INTA), Av. San Martin 3853 Luján de Cuyo,
Mendoza M5507, Argentina
4 Laboratorio de Biotecnología de Aromas, Facultad de Química, UdelaR. Av. Gral. Flores 2124,
Montevideo 11800, Uruguay
* Correspondence: paolamaturano@yahoo.com.ar Tel.: +54-0264-4211700/+54-0264-4619625
† These authors contributed equally.




Received: 25 June 2019; Accepted: 8 July 2019; Published: 11 July 2019


Abstract: It is well known that high ethanol levels in wines adversely affect the perception of new
wine consumers. Moreover, numerous issues, such as civil restrictions, health risk and trade barriers,
are associated with high ethanol concentrations. Several strategies have been proposed to produce
wines with lower alcoholic content, one simple and inexpensive approach being the use of new wine
native yeasts with less efficiency in sugar to ethanol conversion. Nevertheless, it is also necessary that
these yeasts do not impair the quality of wine. In this work, we tested the effect of sequential culture
between Hanseniaspora uvarum BHu9 and Saccharomyces cerevisiae BSc114 on ethanol production.
Then, the wines produced were analyzed by GC-MS and tested by a sensorial panel. Co-culture
had a positive impact on ethanol reduction and sensory profile when compared to the S. cerevisiae
monoculture. Wines with lower alcohol content were related to fruity aroma; moreover, color intensity
was associated. The wines obtained with S. cerevisiae BSc114 in pure conditions were described by
parameters linked with high ethanol levels, such as hotness and astringency. Moreover, floral profile
was related to this treatment. Based on these findings, this work provides a contribution to answer the
current consumers’ preferences and addresses the main challenges faced by the enological industry.

Keywords: low-ethanol wines; sequential culture; Hanseniaspora uvarum yeast; aromatic/sensorial

profiles

1. Introduction
Well-structured and full-body wines have become the preferences of many new wine consumers.
In order to obtain these characteristics, it is necessary to ensure optimal phenolic maturity of grapes,
which requires longer grape ripening times [1]. However, in the context of global warming, this practice
results in a significant increase in the berry sugar content at the moment of harvesting, and consequently
higher alcohol levels in the wine [2]. Numerous issues are associated with high ethanol levels in wine
such as consumers’ rejection, civil restrictions, health risk, and trade barriers [1,3,4]. The sensorial

Fermentation 2019, 5, 65; doi:10.3390/fermentation5030065 www.mdpi.com/journal/fermentation

Fermentation 2019, 5, 65 2 of 15

quality of wines is also significantly affected because of an increase in the perception of bitterness,
sweetness, astringency and hotness, and masking of volatile aromatic compounds [5,6]. In this context,
different technological solutions have been evaluated: harvest of unripe berries, increase in crop load,
shading bunches, choosing proper irrigation techniques, and modulation of source–sink relationships
by removing leaves or topping shoots [7–10]. Other authors have tried partial dealcoholization with
physical methods [11–13].
More recently, microbiological solutions have been proposed by using selected non-Saccharomyces
and Saccharomyces cerevisiae yeast strains in simultaneous or sequential fermentations [4,14–16]. The use
of non-Saccharomyces yeasts has become a common trend in the main wine regions, particularly
because of their effects on the composition, flavor and color of the wine [17,18]. In addition to the
aforementioned effects, this yeast group is also known to be less efficient in the production of ethanol
from consumed sugars when compared with S. cerevisiae yeasts [19].
Hanseniaspora genera as a whole and particularly Hanseniaspora uvarum species are
non-Saccharomyces yeasts commonly encountered at high concentrations on the grape surface and
throughout the fermentation process [20]. Recently, 28 H. uvarum isolates were evaluated by our
research group and they demonstrated interesting enological characteristics such us: ability to grow
at high sugar, ethanol and SO2 contents; to produce high concentrations of glycerol; low acetic acid
and hydrogen sulfide levels; and the release of proteolytic enzymes [21]. Moreover, it is important to
highlight that H. uvarum was also found to be a potential candidate to produce less ethanol because it
requires more than 19 g/L of consumed sugar to produce 1% v/v of ethanol [21]. In a more recent study,
a selected H. uvarum yeast strain was assessed in sequential inoculations with S. cerevisiae yeasts under
optimized fermentation conditions [22]. The authors found that the ethanol levels were significantly
reduced compared with fermentations carried out with S. cerevisiae monocultures. Nevertheless,
and in order to achieve holistic knowledge, the aim of the present work was to assess the aromatic
impact of an optimized inoculum of H. uvarum/S. cerevisiae yeasts in fresh must and compare the
findings with a S. cerevisiae monoculture. It is also relevant to establish how ethanol reduction affects
sensorial perception. The results would allow the design of a comprehensive microbiological strategy
in order to answer the current consumers’ preferences and address the main challenges faced by the
enological industry.

2. Materials and Methods

2.1. Microorganisms
Hanseniaspora uvarum BHu9 and Saccharomyces cerevisiae BSc114 were used in the present study.
Both strains of yeasts were previously selected based on their oxidative and fermentative metabolism
in order to obtain reduced ethanol wines [21]. Strains were obtained from the Culture Collection of
Autochthonous Microorganisms (Institute of Biotechnology, School of Engineering—UNSJ, San Juan,
Argentina) and preserved at −80 ◦ C until use.

2.2. Yeast Inoculum Preparation

Each strain was grown on YEPD agar for 48 h and the biomasses were transferred to YEPD broth
(130 rpm during 4 h) [22]. Then, strains were transferred to grape must (13◦ Brix, pH 3.8) supplemented
with 0.1% yeast extract and 0.4% peptone, and incubated at 25 ◦ C during 24 h under aerobic conditions
(130 rpm). YEPD broth was used for pre-adaptation in order to reduce the lag-stage in the grape must,
which allows strains to grow immediately exponentially in the grape juice [22]. Once the pre-adaptation
process had finished, cells were counted with an improved Neubauer chamber.

2.3. Grapes and Vineyard Location

All experiments were carried out using Vitis vinifera L. cv. Malbec grapes harvested during the
2017 vintage from a vineyard located in Cañada Honda, San Juan, Argentina (31◦ 580 3400 S 68◦ 320 5200 W)
Fermentation 2019, 5, 65 3 of 15

at 610 m altitude. Grapes were manually destemmed and mixed to obtain a homogeneous solution.
The composition of the fresh juice was as follows: sugar (glucose and fructose), 238.2 g/L; pH, 3.8;
titratable acidity, 5.3 g/L; and yeast assimilable nitrogen (YAN), 175 mg/L. Then, 5-L vessels equipped
with a Muller valve were filled with juice (3 L) and supplemented with 50 ppm of free SO2 before
fermentation. The Muller valve was filled with a solution of 50% sulfuric acid and 50% sterile water
distilled. Vinifications were performed in triplicate.

2.4. Inoculation and Winemaking

Lab-scale fermentations were conducted under optimized factors previously determined by
Maturano et al. [22]. Treatment 1 (T1): H. uvarum BHu9 was inoculated (T0) at a concentration of
5 × 106 cells/mL, and 48 h later, 2 × 106 cells/mL of S. cerevisiae BSc114 were sequentially inoculated.
In parallel, a single culture of 2 × 106 cells/mL of S. cerevisiae yeasts was inoculated at T0 as control
treatment (TC). Both fermentations were performed at 25 ± 1 ◦ C under static conditions. Musts were
supplemented with nitrogen by adding 20 mg/L of (NH4 )2 HPO4 twice: after 48 h and in the middle of
the fermentation (when 5% weight loss was verified). Nitrogen supplement was established based
on nitrogen uptake previously determined with selected yeasts (data not shown). Punch down was
carried out every 24 h in order to keep acceptable dissolved oxygen levels throughout the process.
The fermentation progress was evaluated by the weight loss caused by CO2 production and vessels
were weighed every 24 h.
Samples were collected periodically and viable cell counts were determined by plating onto
Wallerstein Laboratory Nutrient (WLN) Agar medium (Oxoid, Hampshire, UK). Dilutions of 10−3 ,
10−4 and 10−5 were spread onto WLN agar medium and incubated for 7 days at 28 ◦ C. Green colonies
(H. uvarum BHu9) and creamy colonies (S. cerevisiae BSc114) were differentiated and counted [23].
After the sugar was completely consumed, 50 mg/L of free SO2 was added. The wines were
chemically stabilized, filtered, bottled, and conserved at 16 ± 1 ◦ C until sensorial analysis. Samples of
50 mL were stored at −20 ◦ C in order to carry out volatile composition analysis.

2.5. Chemical Analysis

Glycerol, residual sugars, total acidity and acetic, malic, lactic, and tartaric acid were measured
periodically using an ALPHA FT-IR Wine Analyzer (Bruker Optik Gmbh, Ettlingen, Germany). Ethanol
concentration was determined according to the OIV OENO 379-2009 ES official method. The pH was
measured with a multi-parameter Adwa (AD1030 PHM_MES_6362).

2.6. Sensorial Analysis

After 4 months of bottle stabilization, wines were evaluated by descriptive analysis according to
Lawless and Heymann [24]. A well-trained panel carried out the evaluation of 13 sensorial attributes:
three color/appearance descriptors (color intensity, red and brown color), five aroma descriptors
(mineral note, frutal, floral, chili pepper, and toasted) and five taste parameters (acidity, sweetness,
astringency, hotness, and bitterness). The intensity of each attribute was assessed using a structured
scale from 0 to 5, where 0 indicates that the descriptor was not perceived and values between 1 and 5
indicate that the intensity of the descriptors was very low to very high. The panel consisted of seven
individuals (five males and two females between 35 and 50 years old) from the Wine Sensorial Analysis
Department (Instituto Nacional de Vitivinicultura, Mendoza, Argentina). Vinifications were tasted
blindly and in duplicate from a constant volume of 30 mL at room temperature.

2.7. Free aromatic Analyses

2.7.1. Solid Phase Extraction (SPE)

The extraction of aroma compounds was performed by adsorption and the molecules were
separate elutions from an Isolute ENV+ cartridge (IST Ltd., Mid Glamorgan, UK) packed with 1 g of
Fermentation 2019, 5, 65 4 of 15

the highly cross-linked styrene divinylbenzene (SDVB) polymer according to Boido et al. [25] with
some modifications.

2.7.2. GC-MS Analyses

GC-MS analyses were conducted using a Shimadzu QP 2020 (Shimadzu Corporation, Kyoto,
Japan) mass spectrometer. A Carbowax 20 M capillary column (Agilent Technologies, Walt and Jennings
Scientific, Wilmington, DE, USA) (30m × 0.25mm × 0.25µm film thickness) was used. The experimental
conditions were as follows: The initial column temperature was 40 ◦ C (8 min), which was then
increased to 180 ◦ C (3 ◦ C/min) and then increased again to 250 ◦ C (20 min) at 20 ◦ C/min; injector
temperature, 250 ◦ C; injection mode, split; split ratio, 1:30; volume injected, 1.0 µL; carrier gas H2,
30 kPa; energy 70 eV. The wine aroma components were identified by comparison of their linear
retention indices (LRI) determined with a homologous series of n-alkanes (C9–C26), with those from
pure standards or reported in the literature according to their elution order with Carbowax 20 M [26–28].
Comparison of mass spectral fragmentation patterns with those stored in databases was also performed.
GC-MS instrumental procedures using 1-heptanol as an internal standard were applied for quantitative
purposes. GC-MS analyses were carried out with two samples of each wine.

2.8. Odor Activity Value (OAV) and Relative Odor Contributions (ROCs)
The contribution of each volatile compound was quantitatively evaluated using Odor Activity
Values (OAVs). The OAV was obtained by dividing the mean concentration of each volatile compound
by its odor threshold value in a hydroalcoholic solution [29]. The volatile compounds contribute to
wine aroma when its concentration in wine is above the perception threshold, therefore, the OAV value
is above 1. In this study, the threshold values were obtained from information available in the literature.
Moreover, the identified compounds were classified according to aromatic descriptors and grouped in
seven aromatic series which were classified according to the associated descriptor: 1, solvent; 2, sweet;
3, herbaceous; 4, floral; 5, fruity; 6, fatty; and 7, toasted.
From the volatile compounds that presented OAV > 1, the relative odor contribution (ROC)
was calculated. The relative odor contribution (ROC) represents the percentage of contribution of a
particular aroma compound and this was determined as the ratio between the OAV of the respective
P
compound and the total OAV of each wine ((individual OAV/ OAV) * 100) [30].

2.9. Statistical Analysis

Chemical data and population analysis were expressed as the means ± standard deviation from
three repetitions and aromatic analysis as the means of two repetitions. One-way ANOVA was used
to evaluate differences between treatments. Statistical analysis was performed using the InfoStat
professional version (Cordoba, Argentina, 2016).

3. Results
The current study assessed the contribution of H. uvarum BHu9 and S. cerevisiae BSc114 yeasts to
the ethanol content and sensorial and aromatic impact on wine.

3.1. Fermentative Kinetics and Population Dynamics

In the present study, fermentative kinetics are represented by sugar consumption and CO2 release
in both fermentations: BHu9/BSc114 (T1) and BSc114 (TC) (Figure 1). Both treatments completed
alcoholic fermentation after 8 days. During the first 24 h, both vinifications showed a similar sugar
consumption, but from day 2 until day 6, T1 exhibited a slower fermentation rate than TC (p < 0.05).
During day 7 and 8, sugar consumption was not significantly different (p > 0.05), and at the end of the
process, both treatments behaved similarly.
 Fermentation 2019, 5, x FOR PEER REVIEW 5 of 15

181 Total Fermentation

CO2 production
2019, 5, 65was 293.33 ± 17 g and 320 ± 10 g for the BHu9/BSc114 co-culture and S. 5 of 15
182 cerevisiae monoculture, respectively (Figure 1).

183 Figure 1. Release of CO2 (g) and sugar consumption in T1 (BHu9/BSc114) and TC (BSc114 control).
184 Figure 1. Release of CO2 (g) and sugar consumption in T1 (BHu9/BSc114) and TC (BSc114 control).
Like the sugar consumption, CO2 release showed a similar behavior for both treatments during
185 the first
The 24 h. From
population day 2 until
dynamics the(H.
of T1 end,uvarum
T1 demonstrated a lower rate
BHu9/S. cerevisiae than TC
BSc114) and(BSc114) < 0.05). Total
TC (S. (pcerevisiae
186 CO2 production was 293.33 ± 17 g and 320 ± 10 g for the BHu9/BSc114
BSc114) are shown in Figure 2. H. uvarum BHu9 population increased during the early stages reaching co-culture and S. cerevisiae
187 monoculture,
a maximum of 8.18respectively
× 107 cells/mL(Figure on1).day three. During the first 48 h, (previous S. cerevisiae
188 inoculation) BHu9 consumed 102.54 g/LT1of(H.
The population dynamics of uvarum
sugar withBHu9/S.
an ethanolcerevisiae BSc114)
production ofand
3.49% (S. cerevisiae
TCv/v. Therefore, BSc114)
189 are shown in Figure 2. H. uvarum BHu9 population increased during
when BSc114 was inoculated (after 48 h), the available sugar concentration was 135 g/L. In co- the early stages reaching a
7
× 10 cells/mL
190 maximum
inoculation of 8.18
trials, H. uvarum BHu9 on day three. During
maintained the first 48
its population uph,to day 4, S.after
(previous cerevisiae
whichinoculation)
the
191 BHu9 consumed 102.54 g/L of sugar with an ethanol production of 3.49%
concentrations were undetectable with the technique applied in this study. Hence, H. uvarum BHu9 v/v. Therefore, when BSc114
192 and S.was inoculated
cerevisiae (after
BSc114 48 h), theonly
coexisted available
during sugar concentration
2 days. During this wascoexistence
135 g/L. In period,
co-inoculation
the sugar trials, H.
193 uvarum BHu9
consumption maintained
was 51.74 its population
g/L, which means thatupthe to sugar
day 4, consumption
after which the byconcentrations
both strains was were undetectable
less than
194 with the technique
the consumption by BHu9 applied
beforeinS.
this study. Hence,
cerevisiae H. uvarum
inoculation, andBHu9 and S. cerevisiae
the ethanol production BSc114 coexisted
at this stage only
195 during
was 5.93% 2 days.
v/v. At theDuring this coexistence
final fermentation stageperiod,
(day 5 theto 8),sugar consumption
S. cerevisiae BSc114was 51.74 g/L,74.67
consumed which g/Lmeans
196 that the sugar consumption by both strains was less than the consumption
of sugar, and the average ethanol production was 3.22% v/v. The dynamic population of S. cerevisiae by BHu9 before S. cerevisiae
197 inoculation, and the ethanol production at this stage was
6 5.93% v/v.
in T1 presented an increase in the number of cells from 2 × 10 cells/mL to 1.82 × 10 cells/mL on day stage
At the final
8 fermentation
198 (day 5the
7, whereas to 8), S. cerevisiae
maximum BSc114 consumed
population achieved by 74.67 g/L of
BSc114 sugar,
(TC, and the
control) wasaverage
1.9 × 10ethanol
8 cells/mLproduction
on day was
199 6. 3.22% v/v. The dynamic population of S. cerevisiae in T1 presented an increase in the number of cells
6 8
from 2 × 10 cells/mL to 1.82 × 10 cells/mL on day 7, whereas the maximum population achieved by
BSc114 (TC, control) was 1.9 × 108 cells/mL on day 6.
 Fermentation 2019, 5, x FOR PEER REVIEW 6 of 15
Fermentation 2019, 5, 65 6 of 15

200
Figure 2. Dynamic populations of T1 (BHu9/BSc114) and TC (BSc114). * indicate the inoculation
201 moment
Figure of BSc114
2. Dynamic in T1 afterof48T1
populations h. (BHu9/BSc114) and TC (BSc114). * indicate the inoculation
202 moment of BSc114 in T1 after 48 h.
3.2. Enological Parameters
203 3.2. Enological Parameters
The analyses of the main chemical parameters at the end of the fermentations are summarized
204 in Table 1. Both
The analyses of treatments finished
the main chemical with sugar
parameters concentrations
at the below 1.8 are
end of the fermentations g/L,summarized
indicating that the
205 infermentations
Table 1. Both treatments finished with sugar concentrations below 1.8 g/L, indicating
had been successfully finished. Ethanol concentration in T1 was significantly that the lower
206 fermentations
(12.63 ± 0.05%had v/v)
beenthan
successfully
TC (13.15finished. Ethanol
± 0.28% v/v), concentration
representinginanT1average
was significantly
reductionlower
of 0.52% v/v.
207 (12.63 ± 0.05%
Likewise, pHv/v) thanwere
values TC (13.15
lowest ± 0.28% v/v),produced
in wines representing
withanco-cultures,
average reduction of 0.52%
but tartaric acidv/v.
was higher
208 Likewise, pH values were lowest in wines produced with co-cultures, but tartaric acid was
compared to control (TC) wines. No significant differences were observed for acetic, malic and lactic higher
209 compared to control (TC) wines. No significant differences were observed for acetic, malic and lactic
acid, or for glycerol and total acidity under the experimental conditions.
210 acid, or for glycerol and total acidity under the experimental conditions.
Table 1. Principal chemical parameters in wines obtained from BHu9/Bcs114 co-inoculation and
211 Table 1. Principal chemical parameters in wines obtained from BHu9/Bcs114 co-inoculation and
BSc114 control.
212 BSc114 control.
Chemicalcompounds
Chemical Compounds T1 T1 TC TC
Ethanol
Ethanol (%v/v)
(%v/v) 12.63 ± 0.05
12.6313.5±
± 0.050.28 (*) 13.5 ± 0.28 (*)
Acetic
Acetic acid
acid (g/L)
(g/L) 0.56 ± 0.020.56 ±0.49
0.02± 0.04 0.49 ± 0.04
Lactic
Lactic acid
acid (g/L)
(g/L) 0,5 ± 0.010.5 ±0,47
0.01± 0.02 0.47 ± 0.02
Malic acid (g/L)
Malic acid (g/L) 2,85 ± 0.212.85 ± 0.21± 0.07 2.85 ± 0.07
2,85
Tartaric acid (g/L) 1.31 ± 0.08 1 ± 0.01 (*)
Tartaric acid (g/L) 1,31 ± 0.08 1 ± 0.01 (*)
Glycerol (g/L) 10 ± 0.71 9.3 ± 0.57
Glycerol (g/L) 10 ± 0.71 9.3 ± 0.57
pH 3.43 ± 0.00 3.49 ± 0.01 (*)
Total pH
acidity (g/L) 3.43 ± 0.00 5.953.49 ± 0.01 (*) 5.80 ± 0.14
± 0.07
Total acidity
Residual (g/L)
sugar (g/L) 5.95 ± 0.07
1.55 ±5.80
0.64± 0.14 1.70 ± 0.28
Residual sugar (g/L) 1.55 ± 0.64 1.70 ± 0.28
REFERENCES: (*) indicate significant differences between treatments at p < 0.05.
213 REFERENCES: (*) indicate significant differences between treatments at p < 0.05.

214 3.3.Aromatic
3.3. Aromatic Composition
Composition
Volatile products of the fermented musts were quantified by SPE-GC-MS according to Boido et al.
(2003). Table 2 shows volatile compounds and their concentrations, odorant descriptors, perception
thresholds, odorant activity values (OAVs), and aromatic series found in the Malbec wines analyzed.
Fermentation 2019, 5, 65 7 of 15

A total of 38 volatile compounds were identified and quantified, and classified into four groups: esters
(ethyl and acetate esters), higher alcohols, fatty acids, and lactones.
Fermentation 2019, 5, 65 8 of 15

Table 2. Aromatic description of wines obtained (mean and SD of two measure).

Treatments Threshold Perception OAV

Compounds (µg/L) Descriptor Ref OAV TC
T1 TC p < 0.05 (µg/L) T1 Aromatic Serie
Ethyl esters
Ethyl hexanoate 600 ± 15 200 ± 83 * fruity, apple 14 1 42.85 14.28 5
Ethyl octanoate 872 ± 58 369 ± 187 * pineapple, pear 5 1 174.4 73.8 5
Ethyl-3-hydroxybutanoate 212 ± 53 174 ± 14 grape, caramel 67,000 2 0.003 0.002 2
Ethyl decanoate 1140 ± 40 590 ± 81 * floral 200 1 5.7 2.95 4
Ethyl dodecanoate 672 ± 52 nd leaf, fruity 1500 1 0.448 - 5
Ethyl tetradecanoate 31 ± 1 121 ± 22 * waxy 2000 1 0.015 0.060 6
Ethyl palmitate 345 ± 23 224 ± 14 * waxy 1500 1 0.23 0.149 6
Ethyl succinate 127 ± 0.011 145 ± 6 * ripe melon 1,000,000 1 0.0001 0.0001 5
Ethyl lactate 603 ± 21 409 ± 63 * strawberrry 14,000 1 0.043 0.029 5
P
Ethyl esters 4600 2232
Acetate esters
Isoamyl acetate 3210 ± 18 2892 ± 191 * banana 30 2 107 96.4 5
Hexyl acetate 24 ± 24 297 ± 11 * red fruit 1500 1 0.03 0.443 5
P
acetate esters 3234 3189
TOTAL ESTERS 7491 (1.77%) 5421 (1.03%)
Higher Alcohols
2-Methyl-1-propanol 18,480 ± 1620 12,990 ± 299 * solvent 7000 3 2.64 1.85 1
1-Butanol 444 ± 1 692 ± 72 * solvent 9000 3 0.049 0.076 1
3-Methyl-1-butanol 318,400 ± 12,300 371,000 ± 16,140 * burned, alcohol 30,000 3 10.61 12.36 1
1-Pentanol 40 ± 2 nd fruity, balsmic 4000 3 0.01 - 5
4-Methyl-1-pentanol 59 ± 8 60 ± 18 almond 50,000 3 0.001 0.001 2
3-Methyl-1-pentanol 214 ± 18 220 ± 10 herbaceous 50,000 3 0.004 0.044 3
1-Hexanol 1080 ± 142 852 ± 82 grass, green leaf 2500 4 0.432 0.340 3
trans-3-Hexenol 40 ± 0 nd herbaceous, land 400 1 0.1 - 3
3-Ethoxy-1-propanol 150 ± 11 65 ± 3 * ripe pear 100 1 1.5 0.65 5
cis-3-Hexenol 54 ± 8 57 ± 8 cutted grass 400 1 0.135 0.014 6
2-Ethyl hexanol 256 ± 12 255 ± 17 rose, citrus 8000 1 0.032 0.031 4
2,3-Butanediol 233 ± 15 340 ± 13 * butter 120,000 1 0.001 0.002 6
Furfurol 102 ± 19 190 ± 20 * floral 5000 4 0.02 0.038 4
3-(Methylthio)-1-propanol 819 ± 41 1450 ± 38 * cooked vegetal 1000 5 0.819 1.45 3
Benzyl alcohol 610 ± 50 11 ± 4 * caramelo, cítrico 10,000 4 0.061 0.0001 2
2-Phenylethyl alcohol 53,885 ± 3012 86,072 ± 731 * rose 14,000 4 3.848 6.148 2
Tyrosol 17,110 ± 895 23,071 ± 3245 * honey - - 2
Tryptophol 1910 ± 98 1780 ± 98 * - -
Fermentation 2019, 5, 65 9 of 15

Table 2. Cont.

Treatments Threshold Perception OAV

Compounds (µg/L) Descriptor Ref OAV TC
T1 TC p < 0.05 (µg/L) T1 Aromatic Serie
P 414,357 515,754
Higher alcohols
(98.14%) (98.45%)
Fatty acids
Acetic acid 39 ± 6 nd vinegar 200 1 0.195 - 6
Isobutanoic acid 181 ± 21 396 ± 9 * butter, cheese 8100 3 0.022 0.048 6
Butanoic acid 56 ± 3 295 ± 29 * fatty, rancid 1000 3 0.056 0.295 6
Hexanoic acid 176 ± 53 231 ± 42 * cheese, sudor 3000 3 0.058 0.077 6
Octanoic acid 235 ± 50 824 ± 9 * rancid butter 3000 3 0.078 0.276 6
Decanoic acid 94 ± 9 773 ± 310 * fatty, rancid 10,000 3 0.009 0.007 6
Dodecanoic acid 49 ± 1 43 ± 1 fatty, rancid 10,000 3 0.005 0.004 6
P
Acids 83 (0.019%) 2562 (0.49%)
Lactones
gamma-Valerolactone 39 ± 1 49 ± 3 * sweet, cocconut 10 7 3.9 4.9 2
gamma-Butyrolactone 236 ± 8 163 ± 26 * caramel 35 7 6.74 4.65 2
P
Lactones 275 (0.06%) 212 (0.04%)
P
compounds (µg/L) 422,206 523,858
References: (*) indicate significant differences between treatments. Grey cells indicate compounds with OAV > 1. References: [1] Tao and Zang (2010), [2] Moreno et al. (2005), [3] Rychlik
et al. (1996), [4] Leffingwell & Associates (2009), [5] Burdock (2016), [6] Culleré et al. (2004), [7] Lopez de Lerma and Peinado (2011).
Fermentation 2019, 5, 65 10 of 15

Alcohols formed the most abundant group of volatile compounds, followed by esters, fatty acids
and lactones. Higher alcohols represented 98.14 and 98.45% of the total aroma content in T1 and TC
wines, respectively, while esters and fatty acids constituted 1.77–1.03% and 0.019–0.49% in T1 and TC
wines, respectively (Table 2).
Overall, the total concentration of higher alcohols and fatty acids was higher in control treatment
TC, fermented with S. cerevisiae BSc114, than in wines produced by the sequential fermentation
of H. uvarum BHu9/S. cerevisiae BSc114 (T1). In contrast, esters and lactones (γ-butyrolactone and
γ-valerolactone) content was higher in T1 than in TC. These compounds represented 1.77 and 1.03 %
in esters, in T1 and TC respectively. The lactones proportions were 0.06 % and 0.04 % in T1 and TC
respectively. (Table 2).
Some compounds such as ethyl hexanoate, ethyl decanoate, 3-ethoxy-1-propanol, isoamyl acetate,
and γ-butyrolactone were detected at higher concentrations in T1 than in TC (p < 0.05). In contrast, S.
cerevisiae BSc114 fermentation showed higher concentrations of ethyl octanoate, 3-methyl-1-butanol,
3-(methyl thio)-1-propanol, 2-phenylethanol, and γ-valerolactone compared to wines obtained with
BHu9/BSc114 (p < 0.05) (Table 2).
Compounds such us ethyl hexanoate (fruity, apple), ethyl octanoate (pineapple, pear), isoamyl
acetate (banana), γ-butyrolactone (caramel, coconut), and γ-valerolactone (sweet, coconut) showed
OAVs > 1 in both treatments. Comparing pure with mixed, fermented 3-ethoxy-1-propanol (ripe pear)
exhibited an OAV > 1 only in T1, and 3-(methyl thio)-1-propanol (cooked vegetables) showed an OAV
> 1 in wine fermented by the S. cerevisiae Bc114 monoculture (Table 2).
Table 3 presents compounds with an OAV > 1 and their relative odor contribution (ROC). When
considering the ester contribution to the odorant composition, ethyl octanoate greatly contributed to
wines obtained with BHu9/BSc114 (49.35%), while isoamyl acetate was the main contributor to the
control treatment fermented with pure BSc114 control. The higher alcohols that demonstrated major
contributions to wines in both treatments were 3-methyl-1-butanol and 2-phenylethanol, but their
relative odor contributions were higher in TC wines.

Table 3. Compounds with an OAV > 1 and their relative odor contribution (ROC) in T1 and TC wines.

T1 TC
Compounds OAV ROC (%) OAV ROC (%) Aromatic Serie
Ethyl hexanoate 42.85 12.12 14.28 6.60 5 Fruity
Ethyl octanoate 174.40 49.35 73.8 34.09 5 Fruity
Ethyl decano ate 1.03 0.29 0 0.00 4 Floral
Isoamyl acetate 107.00 30.28 96.40 44.53 5 Fruity
2-Methyl-1-propanol 2.64 3.72 1.85 6.51 1 Solvent
3-Methyl-1-butanol 10.61 3.00 12.36 5.71 1 Solvent
3-Ethoxy-1-propanol 1.50 0.42 <1 - 5 Fruity
3-(Methylthio)-1-propanol <1 - 1.45 0.67 3 Herbaceous
2-Phenylethanol 5.38 1.52 8.61 3.98 4 Floral
gamma-Valerolactone 3.90 1.10 4.90 2.26 2 Sweet
gamma-Butirolactone 6.742 1.91 4.66 2.15 2 Sweet

Figure 3 shows the aromatic profile of the analyzed wines based on the sum of the components with
an OAV > 1 and ROC values according to each descriptor. Wines fermented with BSc114 were related
to the aromatic “floral”, “solvent”, “herbaceous” and “sweet” families, while co-culture fermented
wines were characterized by “frutal” descriptors.
 Fermentation 2019, 5, x FOR PEER REVIEW 2 of 15

Fermentation 2019,5,5,65x FOR PEER REVIEW

Fermentation2019, 112 of
of15
15

254

255254 Figure 3. Aromatic profile of wines produced by BHu9/BSc114 (T1) and BSc114 (TC control).
Figure 3. Aromatic profile of wines produced by BHu9/BSc114 (T1) and BSc114 (TC control).

256255 3.4.3.4. Figure

Sensorial 3. Aromatic profile of wines produced by BHu9/BSc114 (T1) and BSc114 (TC control).
Analysis
Sensorial Analysis
257256 Figure
3.4. 4 shows
Sensorial
Figure thethe
Analysis
4 shows sensorial analyses
sensorial of of
analyses thethe
wines
winesobtained. Wines
obtained. Winesfermented
fermented with
withH. H.
uvarum
uvarum
258 BHu9/S. cerevisiae BSc114 (T1) could be defined as fruity (p < 0.05). These results are in agreement
BHu9/S. cerevisiae BSc114 (T1) could be defined as fruity (p < 0.05). These results are in agreement with
259257 with
thethe
Figure 4 shows the sensorial analyses of the wines obtained. Wines fermented with H. uvarum
aromatic
aromatic profile
profile obtained
obtained withwith
ROCROC analysis.
analysis. Another
Another parameter
parameter that significantly
that significantly affected
affected reduced
260258 BHu9/S. cerevisiae BSc114 (T1) could be defined as fruity (p < 0.05). These results are in agreement
reduced ethanol wines (T1) was color intensity. Wines produced with S. cerevisiae
ethanol wines (T1) was color intensity. Wines produced with S. cerevisiae BSc114 (TC) were BSc114 (TC) were
more
261259 with the aromatic profile obtained with ROC analysis. Another parameter that significantly affected
more related to the floral descriptor, which is in agreement with the ROC results
related to the floral descriptor, which is in agreement with the ROC results due to 2-phenylethanol due to 2-
262260 reduced ethanol wines (T1) was color intensity. Wines produced with S. cerevisiae BSc114 (TC) were
phenylethanol
concentration; concentration;
moreover, thesemoreover, theseassociated
wines were wines were associated
with astringencywith
andastringency and hotness
hotness mouthfeel.
263261 more related to the floral descriptor, which is in agreement with the ROC results due to 2-
mouthfeel.
262 phenylethanol concentration; moreover, these wines were associated with astringency and hotness
263 mouthfeel.

264
265 Figure 4. Sensory
Figure analysis
4. Sensory of wines
analysis obtained
of wines from
obtained BHu9/BSc114
from (T1)
BHu9/BSc114 andand
(T1) BSc114 (TC).
BSc114 (*) (*)
(TC). Difference
Difference
266264 significant at 95% confidence level.
significant at 95% confidence level.
265 Figure 4. Sensory analysis of wines obtained from BHu9/BSc114 (T1) and BSc114 (TC). (*) Difference
266 significant at 95% confidence level.
Fermentation 2019, 5, 65 12 of 15

4. Discussion
The current wine market requires wines with lower ethanol concentrations and complex flavor
and color perception. Several microbiological strategies have been proposed in order to obtain these
characteristics. The present study intends to verify the behavior of selected yeasts regarding ethanol
production and the sensorial impact on the wine quality.
Several studies have reported reduced ethanol levels with sequential inoculations of
non-Saccharomyces and Saccharomyces yeasts and under different winemaking conditions [4,16,31].
It is well known that H. uvarum is the most representative yeast species found on grape surfaces
showing prevalence during early stages of spontaneous alcoholic fermentation [32]. This yeast has
several characteristics that could be used to reduce ethanol content in wines [21]. In the current
study, inoculation of H. uvarum BHu9 prior to inoculation of S. cerevisiae BSc114 demonstrated a sugar
consumption of 35.7 g/L for 1% of ethanol produced. In contrast, S. cerevisiae control (TC) used 17.5 g
/L of sugar for 1% of ethanol produced. It is reported that S. cerevisiae yeast uses 16.83 to 17 g/L on
average [1]. The decrease in ethanol production can sometimes be explained by an increase in glycerol
and acetic acid. However, in the present study, both glycerol and acetic acid did not show significant
differences. Sugars were probably partially consumed through the oxidative pathway to produce
biomass and other products.
It must be highlighted that when both strains remained together, sugar consumption was lower
than in the H. uvarum monoculture (prior to S. cerevisiae inoculation). There is evidence that presence
of non-Saccharomyces yeasts in the early stages of fermentation could affect the metabolic activity of S.
cerevisiae, probably encouraging a competition for nutrients [33–35]. For example, Bisson et al. [36]
demonstrated that K. apiculata consumed thiamine and other micronutrients, generating inefficiency in
the metabolic development of S. cerevisiae. Another study established that immobilized Starmerella
bombicola cells in a mixed fermentation affected decarboxylase and alcohol dehydrogenase levels of S.
cerevisiae [37]. Recently, the research of Petitgonnet et al. [38] demonstrated that sequential culture
between Lachaceae thermotholerans and S. cerevisiae provokes a negative interaction between the two
species to the detriment of S. cerevisiae, due to a cell–cell contact mechanism and essential nutrients
uptake. When H. uvarum and S. cerevisiae are mixed inoculated, the cultivability of H. uvarum is
significantly affected; however, the final ethanol concentrations are lower compared to the pure culture
of S. cerevisiae [39]. Nevertheless, to answer the results of this work, further studies should be carried
out to fully understand the interactions between H. uvarum and S. cerevisiae strains employed in the
present study.
With respect to the aromatic composition, many studies have shown that non-Saccharomyces yeasts
such as Candida, Debaryomyces, Pichia, Hansenula, and Hanseniaspora, that display oxidative metabolism
and/or are weakly fermentative produced higher ester levels than a single S. cerevisiae culture [40]. In
accordance, the total ester concentrations in wines produced by H. uvarum/S. cerevisiae co-cultures were
superior to that of wines produced by control treatment. The co-inoculation showed higher levels of
ethyl hexanoate, ethyl octanoate and ethyl decanoate which allowed the “fruity” aromatic profile of
the wines.
Fusel alcohol production was higher in wine fermented with a monoculture of S. cerevisiae.
Fusel alcohol production is related to amino acid production by yeasts, which varies according to
genera, species and strain [41]. S. cerevisiae yeasts have been reported to produce higher quantities of
these compounds compared with certain non-Saccharomyces yeasts [42]. The aromatic series that best
describes the TC profile is “floral”, which is associated with 2-phenylethanol levels. As was expected,
sensorial analysis of TC wine related it to floral descriptors (p < 0.05).
It is well known that ethanol significantly affects the sensorial perception of wines [43]; for
example, it decreases the perception of higher alcohols and aldehydes and shows a similar effect for
ethyl esters [44]. According to our results, wines obtained from a BSc114 (TC) monoculture could
be associated with attributes such us astringency, bitterness, hotness, and sweetness, which is in
agreement with the results by Tilloy et al. [45]. The authors found that high ethanol levels enhanced
Fermentation 2019, 5, 65 13 of 15

the perception of the abovementioned attributes. In contrast, wines with lower ethanol levels obtained
with H. uvarum/S. cerevisiae in the present study were related to red fruit by the sensorial panel (p < 0.05).
Although the control treatment presented elevated concentrations of chemical compounds which
are commonly related to fruity descriptors, it has been reported that high ethanol contents can mask
certain flavor-related volatile compounds like those related to fruity and floral profiles [46].
To our knowledge, this is the first time that a H. uvarum species, submitted to a previous selection
process, has been proposed to carry out sequential fermentations with S. cerevisiae under optimized
conditions to reduce ethanol in wines. The results obtained in the present study have demonstrated
the impact of this co-culture on the ethanol concentration and the chemical aromatic composition; and,
in addition, it has evidenced that ethanol levels affect sensorial perception. Therefore, the present
study could be considered an additional step to a successful change in the wine industry to face
current consumers’ demands. It is possible, however, that more research is necessary in order to fully
understand the impact of this co-culture on a major production scale.

Author Contributions: Investigation, M.V.M.; Methodology, M.V.M., Y.P.M., C.G. and E.D.; Project administration,
Y.P.M., M.E.T., F.V. and E.D.; Supervision, F.C.; Writing—original draft, M.V.M., F.V. and E.D.; Writing—review &
editing, M.V.M., Y.P.M., M.C., L.M., M.E.T. and F.C.
Funding: This research was funded by [UNSJ-SECITI] grant number [3635-2015-2017] and [UNSJ-CICITCA] grant
number [1531-2016].
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Zamora, F. Dealcoholised Wines and Low-Alcohol Wines. In Wine Safety, Consumer Preference, and Human
Health; Springer: Cham, Switzerland, 2016; pp. 163–182.
2. De Orduna, R.M. Climate change associated effects on grape and wine quality and production. Food Res. Int.
2010, 43, 1844–1855. [CrossRef]
3. Meillon, S.; Dugas, V.; Urbano, C.; Schlich, P. Preference and acceptability of partially dealcoholized white
and red wines by consumers and professionals. Am. J. Enol. Vitic. 2010, 61, 42–52.
4. Contreras, A.; Hidalgo, C.; Henschke, P.A.; Chambers, P.J.; Curtin, C.; Varela, C. Evaluation of
non-Saccharomyces yeasts for the reduction of alcohol content in wine. Appl. Environ. Microbiol. 2014, 80,
1670–1678. [CrossRef] [PubMed]
5. Fischer, U.; Noble, A.C. The effect of ethanol, catechin concentration, and pH on sourness and bitterness of
wine. Am. J. Enol. Vitic. 1994, 45, 6–10.
6. King, E.S.; Heymann, H. The effect of reduced alcohol on the sensory profiles and consumer preferences of
white wine. J. Sens. Stud. 2014, 29, 33–42. [CrossRef]
7. Kliewer, W.M.; Dokoozlian, N.K. Leaf area/crop weight ratios of grapevines: Influence on fruit composition
and wine quality. Am. J. Enol. Vitic. 2005, 56, 170–181.
8. Walker, R.R.; Blackmore, D.H.; Clingeleffer, P.R.; Tarr, C.R. Rootstock effects on salt tolerance of irrigated
field-grown grapevines (Vitis vinifera L. cv. Sultana). 3. Fresh fruit composition and dried grape quality.
Aust. J. Grape Wine Res. 2007, 13, 130–141. [CrossRef]
9. Chorti, E.; Guidoni, S.; Ferrandino, A.; Novello, V. Effect of different cluster sunlight exposure levels on
ripening and anthocyanin accumulation in Nebbiolo grapes. Am. J. Enol. Vitic. 2010, 61, 23–30.
10. Palliotti, A.; Panara, F.; Silvestroni, O.; Lanari, V.; Sabbatini, P.; Howell, G.S.; Gatti, M.; Poni, S. Influence of
mechanical postveraison leaf removal apical to the cluster zone on delay of fruit ripening in Sangiovese
(Vitis vinifera L.) grapevines. Aust. J. Grape Wine Res. 2013, 19, 369–377. [CrossRef]
11. Schmidtke, L.M.; Blackman, J.W.; Agboola, S.O. Production technologies for reduced alcoholic wines. J. Food
Sci. 2012, 77, R25–R41. [CrossRef]
12. Catarino, M.; Mendes, A. Dealcoholizing wine by membrane separation processes. Innov. Food Sci. Emerg.
2011, 12, 330–337. [CrossRef]
13. Belisario-Sanchez, Y.Y.; Taboada-Rodriguez, A.; Marin-Iniesta, F.; Lopez-Gomez, A. Dealcoholized wines
by spinning cone column distillation: Phenolic compounds and antioxidant activity measured by the 1,
1-diphenyl-2-picrylhydrazyl method. J. Agric. Food Chem. 2009, 57, 6770–6778. [CrossRef] [PubMed]
Fermentation 2019, 5, 65 14 of 15

14. Quirós, M.; Rojas, V.; Gonzalez, R.; Morales, P. Selection of non-Saccharomyces yeast strains for reducing
alcohol levels in wine by sugar respiration. Int. J. Food Microbiol. 2014, 181, 85–91. [CrossRef] [PubMed]
15. Varela, C.; Dry, P.R.; Kutyna, D.R.; Francis, I.L.; Henschke, P.A.; Curtin, C.D.; Chambers, P.J. Strategies for
reducing alcohol concentration in wine. Aust. J. Grape Wine Res. 2015, 21, 670–679. [CrossRef]
16. Englezos, V.; Rantsiou, K.; Cravero, F.; Torchio, F.; Ortiz-Julien, A.; Gerbi, V.; Rolle, L.; Cocolin, L. Starmerella
bacillaris and Saccharomyces cerevisiae mixed fermentations to reduce ethanol content in wine. Appl.
Microbiol. Biotechnol. 2016, 100, 5515–5526. [CrossRef] [PubMed]
17. Jolly, N.P.; Varela, C.; Pretorius, I.S. Not your ordinary yeast: Non-Saccharomyces yeasts in wine production
uncovered. FEMS Yeast Res. 2014, 14, 215–237. [CrossRef]
18. Varela, C.; Sengler, F.; Solomon, M.; Curtin, C. Volatile flavour profile of reduced alcohol wines fermented
with the non-conventional yeast species Metschnikowia pulcherrima and Saccharomyces uvarum. Food Chem.
2016, 209, 57–64. [CrossRef]
19. Gonzalez, R.; Quirós, M.; Morales, P. Yeast respiration of sugars by non-Saccharomyces yeast species: A
promising and barely explored approach to lowering alcohol content of wines. Trends Food Sci. Technol. 2013,
29, 55–61. [CrossRef]
20. Martin, V.; Valera, M.; Medina, K.; Boido, E.; Carrau, F. Oenological impact of the Hanseniaspora/Kloeckera
yeast genus on wines—A review. Fermentation 2018, 4, 76. [CrossRef]
21. Mestre Furlani, M.V.; Maturano, Y.P.; Combina, M.; Mercado, L.A.; Toro, M.E.; Vazquez, F. Selection of
non-Saccharomyces yeasts to be used in grape musts with high alcoholic potential: A strategy to obtain
wines with reduced ethanol content. FEMS Yeast Res. 2017, 17. [CrossRef]
22. Maturano, Y.P.; Mestre, M.V.; Kuchen, B.; Toro, M.E.; Mercado, L.A.; Vazquez, F.; Combina, M. Optimization
of fermentation-relevant factors: A strategy to reduce ethanol in red wine by sequential culture of native
yeasts. Int. J. Food Microbiol. 2019, 289, 40–48. [CrossRef] [PubMed]
23. Pallmann, C.L.; Brown, J.A.; Olineka, T.L.; Cocolin, L.; Mills, D.A.; Bisson, L.F. Use of WL medium to profile
native flora fermentations. Am. J. Enol. Vitic. 2001, 52, 198–203.
24. Lawless, H.T.; Heymann, H. Physiological and psychological foundations of sensory function. In Sensory
Evaluation of Food; Springer: New York, NY, USA, 2010; pp. 19–56.
25. Boido, E.; Lloret, A.; Medina, K.; Fariña, L.; Carrau, F.; Versini, G.; Dellacassa, E. Aroma composition of Vitis
vinifera cv. Tannat: The typical red wine from Uruguay. J. Agric. Food Chem. 2003, 51, 5408–5413. [CrossRef]
[PubMed]
26. Mondello, L. Mass Spectra of Flavors and Fragrances of Natural and Synthetic Compounds, 3rd ed.; MS Wil GmbH:
Zurich, Switzerland, 2015.
27. Adams, R.P. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry; Allured
publishing corporation: Carol Stream, IL, USA, 2007; Volume 456.
28. McLafferty, F.W.; Stauffer, D.B. The Wiley/NBS Registry of Mass Spectral Data; Wiley and Sons: New York, NY,
USA, 1991.
29. Hellin, P.; Manso, A.; Flores, P.; Fenoll, J. Evolution of aroma and phenolic compounds during ripening of
superior seedless grapes. J. Agric. Food Chem. 2010, 58, 6334–6340. [CrossRef] [PubMed]
30. Welke, J.E.; Zanus, M.; Lazzarotto, M.; Zini, C.A. Quantitative analysis of headspace volatile compounds using
comprehensive two-dimensional gas chromatography and their contribution to the aroma of Chardonnay
wine. Food Res. Int. 2014, 59, 85–99. [CrossRef]
31. Tristezza, M.; Tufariello, M.; Capozzi, V.; Spano, G.; Mita, G.; Grieco, F. The oenological potential of
Hanseniaspora uvarum in simultaneous and sequential co-fermentation with Saccharomyces cerevisiae for
industrial wine production. Front. Microbiol. 2016, 7, 670. [CrossRef] [PubMed]
32. Fleet, G.H.; Heard, G.M. Yeasts-growth during fermentation. In Wine Microbiology and Biotechnology;
Fleet, G.H., Harwood Academic, Eds.; CRC Press: Chur, Switzerland, 1993; pp. 27–54.
33. Mendoza, L.M.; de Nadra, M.C.M.; Farías, M.E. Kinetics and metabolic behavior of a composite culture of
Kloeckera apiculata and Saccharomyces cerevisiae wine related strains. Biotechnol. Lett. 2007, 29, 1057–1063.
[CrossRef] [PubMed]
34. Domizio, P.; Romani, C.; Lencioni, L.; Comitini, F.; Gobbi, M.; Mannazzu, I.; Ciani, M. Outlining a future
for non-Saccharomyces yeasts: Selection of putative spoilage wine strains to be used in association with
Saccharomyces cerevisiae for grape juice fermentation. Int. J. Food Microbiol. 2011, 147, 170–180. [CrossRef]
Fermentation 2019, 5, 65 15 of 15

35. Suzzi, G.; Arfelli, G.; Schirone, M.; Corsetti, A.; Perpetuini, G.; Tofalo, R. Effect of grape indigenous
Saccharomyces cerevisiae strains on Montepulciano d’Abruzzo red wine quality. Food Res. Int. 2012, 46,
22–29. [CrossRef]
36. Bisson, L.F. Stuck and sluggish fermentations. Am. J. Enol. Vitic. 1999, 50, 107–119.
37. Milanovic, V.; Ciani, M.; Oro, L.; Comitini, F. Starmerella bombicola influences the metabolism of
Saccharomyces cerevisiae at pyruvate decarboxylase and alcohol dehydrogenase level during mixed
wine fermentation. Microb. Cell Factories 2012, 11, 18. [CrossRef] [PubMed]
38. Petitgonnet, C.; Klein, G.L.; Roullier-Gall, C.; Schmitt-Kopplin, P.; Quintanilla-Casas, B.; Vichi, S.; Diane, J.D.;
Alexandre, H. Influence of cell-cell contact between L. thermotolerans and S. cerevisiae on yeast interactions
and the exo-metabolome. Food Microbiol. 2019, 83, 122–133. [CrossRef] [PubMed]
39. Wang, C.; Mas, A.; Esteve-Zarzoso, B. Interaction between Hanseniaspora uvarum and Saccharomyces
cerevisiae during alcoholic fermentation. Int. J. Food Microb. 2015, 206, 67–74. [CrossRef] [PubMed]
40. Erten, H.; Campbell, I. The production of low-alcohol wines by aerobic yeasts. J. Inst. Brew. 1953, 59, 207–215.
[CrossRef]
41. Giudici, P.; Romano, P.; Zambonelli, C. A biometric study of higher alcohol production in Saccharomyces
cerevisiae. Can. J. Microbiol. 1990, 36, 61–64. [CrossRef] [PubMed]
42. Mateos, J.R.; Pérez-Nevado, F.; Fernández, M.R. Influence of Saccharomyces cerevisiae yeast strain on the
major volatile compounds of wine. Enzym. Microb. Technol. 2006, 40, 151–157. [CrossRef]
43. Guth, H.; Sies, A. Flavour of wines: Towards an understanding by reconstitution experiments and an analysis
of ethanol’s effect on odour activity of key compounds. In Proceedings of the 11th Australian Wine Industry
Technical Conference, Glen Osmond, SA, Australia, 7–11 October 2002; pp. 128–139.
44. Conner, J.M.; Birkmyre, L.; Paterson, A.; Piggott, J.R. Headspace concentrations of ethyl esters at different
alcoholic strengths. J. Sci. Food Agric. 1998, 77, 121–126. [CrossRef]
45. Tilloy, V.; Ortiz-Julien, A.; Dequin, S. Reduction of ethanol yield and improvement of glycerol formation by
adaptive evolution of the wine yeast Saccharomyces cerevisiae under hyperosmotic conditions. Appl. Environ.
Microbiol. 2014, 80, 2623–2632. [CrossRef]
46. Escudero, A.; Gogorza, B.; Melus, M.A.; Ortin, N.; Cacho, J.; Ferreira, V. Characterization of the aroma of a
wine from Maccabeo. Key role played by compounds with low odor activity values. J. Agric. Food Chem.
2004, 52, 3516–3524. [CrossRef]

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