Effects of different techniques of malolactic fermentation induction on diacetyl metabolism and biosynthesis of selected aromatic esters in cool- climate grape wines

Article abstract of DOI:10.3390/molecules23102549

Thr effects of different malolactic bacteria fermentation techniques on the bioconversion of aromatic compounds in cool-climate grape wines were examined. During three wine seasons, red and white grape wines were produced using various malolactic fermentation induction techniques: Coinoculation, sequential inoculation, and spontaneous process. Volatile compounds (diacetyl and the products of its metabolism, and selected ethyl fatty acid esters) were extracted by solid phase microextraction. Compounds were identified with a multidimensional gas chromatograph-GC × GC-ToFMS with ZOEX cryogenic (N2) modulator. Sensory evaluation of the wines was also performed. It was found that the fermentation-derived metabolites studied were affected by the malolactic bacteria inoculation regime. Quantitatively, ethyl lactate, diethyl succinate, and ethyl acetate dominated as esters with the largest increase in content. The total concentration of ethyl esters was highest for the coinoculation technique, while the highest concentration of diacetyl was noted for the spontaneous technique. Controlled malolactic fermentation, especially using the coinoculation technique, can be proposed as a safe and efficient enological practice for producing quality cool-climate grape wines enriched with fruity, fresh, and floral aromas.

Full text of DOI:10.3390/molecules23102549

Article
Effects of Different Techniques of Malolactic
Fermentation Induction on Diacetyl Metabolism and
Biosynthesis of Selected Aromatic Esters in Cool-
Climate Grape Wines
1,
2
1
Małgorzata Lasik-Kurdyś *, Małgorzata Majcher and Jacek Nowak
1
Department of Fermentation and Biosynthesis, Faculty of Food Science and Nutrition, Poznań University
of Life Sciences, Wojska Polskiego 31, 60-624 Poznań, Poland; jacek.nowaktz@up.poznan.pl
Department of Food Chemistry and Instrumental Analysis, Faculty of Food Science and Nutrition, Poznań
University of Life Sciences, Wojska Polskiego 31, 60-624 Poznań, Poland; majcherm@up.poznan.pl
Correspondence: malgorzata.lasik@up.poznan.pl; Tel.: +48-61-848-7288
2
*
Received: 17 August 2018; Accepted: 28 September 2018; Published: 6 October 2018
Abstract: The effects of different malolactic bacteria fermentation techniques on the bioconversion
of aromatic compounds in cool-climate grape wines were examined. During three wine seasons, red
and white grape wines were produced using various malolactic fermentation induction techniques:
Coinoculation, sequential inoculation, and spontaneous process. Volatile compounds (diacetyl and
the products of its metabolism, and selected ethyl fatty acid esters) were extracted by solid phase
microextraction. Compounds were identified with a multidimensional gas chromatograph—GC ×
GC-ToFMS with ZOEX cryogenic (N ) modulator. Sensory evaluation of the wines was also
2
performed. It was found that the fermentation-derived metabolites studied were affected by the
malolactic bacteria inoculation regime. Quantitatively, ethyl lactate, diethyl succinate, and ethyl
acetate dominated as esters with the largest increase in content. The total concentration of ethyl
esters was highest for the coinoculation technique, while the highest concentration of diacetyl was
noted for the spontaneous technique. Controlled malolactic fermentation, especially using the
coinoculation technique, can be proposed as a safe and efficient enological practice for producing
quality cool-climate grape wines enriched with fruity, fresh, and floral aromas.
Keywords: malolactic fermentation; coinoculation; diacetyl; esters; aromatic compounds; grape
wine
1
. Introduction
Malolactic fermentation (MLF) is a secondary fermentation that takes place after alcoholic
fermentation in the process of grape wine production. It is used in the majority of red wines, some
white wines, and generally for wines with enhanced acidity. The process is conducted by malolactic
bacteria (MLB), most often strains of Oenococcus oeni, and involves the decarboxylation of L-malic
acid into L-lactic acid. As a result of this bioconversion, a noticeable reduction in the total acidity of
the wine can be achieved. The MLB utilize not only malic acid, but also the residual sugars left by
yeast after the alcoholic fermentation. This reduces the potential carbon source for spoilage
microbiota, and increases microbial stabilization of the wine [1–5].
MLF is also a process known to modify the aroma profile of the wine through biosynthesis or
bioconversion of flavor-active compounds. Some authors suggest that MLF enhances fruity notes and
buttery aromas, while reduces vegetative green and grassy aromas [4,6]. Other researchers have
Molecules 2018, 23, 2549; doi:10.3390/molecules23102549
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2549
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postulated that MLF results in a creamier palate, less fruit intensity, and more nutty, vanilla, toasty
butter, and wet leather aromas [7].
A range of factors, including grape cultivar, the bacterial culture used, and the conditions of
vinification, have been described as having an effect on the character of wines treated by MLF [8,9].
However, little is known about the effect of different timings of malolactic bacteria inoculation on
wine aroma modification. To our knowledge, only a few research groups [9–12] have compared
different MLB inoculation timings and determined their effect on the differentiation of the quality
and quantity of the aroma compounds. However, none of these authors examined the spontaneous
variant of MLF, which involves a risk of producing undesirable aroma compounds in wines.
Our research focused on selected aroma compounds (diacetyl and its metabolic products, as well
as selected ethyl fatty acid esters) synthesized during malolactic fermentation in white and red grape
wines produced by three different methods of inoculation: (1) Coinoculation (COI), where the yeast
and bacteria were inoculated at the same time; (2) sequential inoculation (SEQI), where malolactic
fermentation was induced at the end of alcoholic fermentation; and (3) spontaneous malolactic
fermentation (SPONT), where MLB inoculation was not performed. This is the first study which
attempts to evaluate the effects of different timings of MLB inoculation (including the spontaneous
process which involves a significant risk of producing undesirable compounds) on the bioconversion
of aromatic compounds in red and white grape wines. The experiment was performed during three
wine seasons.
2
. Results and Discussion
2
.1. Chemical Characteristics of Wines and Dynamics of Malolactic Fermentation
The wines produced through different techniques of malolactic fermentation induction were
characterised three months after bottling (Table 1). Almost 90% of the sugars (glucose and fructose)
were utilised during the first week of the vinification process. Thus, for the sequential inoculation
variant in which MLF was induced at the end of alcoholic fermentation (AF), bacteria inoculation
was applied after one week. The final concentration of residual sugars ranged from 2.05 to 5.08 g/L,
therefore the obtained wines were classified as dry. No significant influence of MLF on the final
ethanol concentration was noted in all the wines. The volatile acidity did not exceed 0.85 g/L
(
expressed as acetic acid), which is in consonance with the standards of good-quality grape wines.
The course of malolactic fermentation was monitored for all the performed vinification
processes, as presented in our previous study [13]. The greatest dynamics of MLF were registered
during the first month of vinification. In the second and third months, the amount of malic acid still
decreased, but at a much slower rate. Beyond three months, a stabilization of the process was
observed, where malic acid reduction for the most part was not significantly important. Based on the
dynamics and effectiveness of malic acid reduction, the MLF induction techniques followed the
order: COI > SEQI > SPONT > AF [13]. The highest dynamics of malic acid reduction were observed
for the coinoculation variant, independent of the vine variety or season. Whereas, much lower
efficiency was noted for variants with no bacteria inoculation—alcoholic fermentation and
spontaneous MLF, as described in detail in our previous study [13]. No complete reduction of malic
acid was reached in any of the wines. The lowest final concentration of malic acid was noted for
coinoculation cases.

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Table 1. Chemical characteristics of wines produced by different methods of MLF induction; parameters were evaluated three months after bottling.
Residual Sugars (g/L)
Total Acidity
g/L) *
Volatile Acidity
(g/L) **
Ethanol
(% v/v)
pH
After
3 Months
after bottling
(
1
Week ***
Chardonnay
AF
a
c
7.15 ± 0.12 c
b
a
a
a
a
17.53 ± 0.51 b
5.08 ± 0.17 c
3.35 ± 0.02
3.65 ± 0.04
3.49 ± 0.06
3.38 ± 0.04
0.75 ± 0.03
0.69 ± 0.04
0.70 ± 0.01
0.85 ± 0.03
12.5 ± 0.12
12.4 ± 0.16
12.5 ± 0.19
12.4 ± 0.13
a
a
a
a
COI
SEQI
SPONT
Kerling
AF
COI
SEQI
SPONT
Pinot noir A
AF
COI
SEQI
SPONT
Pinot noir B
AF
COI
SEQI
SPONT
Rondo
AF
5.75 ± 0.09
6.34 ± 0.11
7.03 ± 0.14
11.12 ± 0.55
19.23 ± 0.42
18.92 ± 0.31 c
2.05 ± 0.53
2.52 ± 0.23
4.52 ± 0.41 b
b
a
b
a
c
a
c
c
White wines
3.26 ± 0.02a
7.35 ± 0.12c
0.66 ± 0.05 c
11.2 ± 0.11
11.1 ± 0.08
11.0 ± 0.06
11.2 ±0.13
a
a
a
16.71 ± 0.26 c
4.71 ± 0.09 d
d
a
a
b
a
3.58 ± 0.02
3.42 ± 0.05
3.37 ± 0.07
6.12 ± 0.09
7.17 ± 0.14
7.19 ± 0.11
0.41 ± 0.06
0.49 ± 0.03
14.39 ± 0.74
12.43 ± 0.41
2.09 ± 0.12
2.41 ± 0.11
c
b
b
a
b
b
b
0.77 ± 0.06 d
a
17.38 ± 0.55 d
4.05 ± 0.18 c
3.71 ± 0.05 a
5.69 ± 0.07
5.06 ± 0.09
5.77 ± 0.11
5.52 ± 0.09
b
0.44 ± 0.04
0.53 ± 0.07
0.39 ± 0.06
b
13.2 ± 0.12
13.1 ± 0.16
13.3 ± 0.22
13.3 ± 0.18
a
a
a
a
18.18 ± 0.42 d
4.78 ± 0.15 b
d
a
c
a
c
4.00 ± 0.09
3.90 ± 0.06
3.81 ± 0.03
12.53 ± 0.75
16.87 ± 0.39
2.14 ± 0.06
2.37 ± 0.08
c
c
a
b
a
b
b
0.69 ± 0.03 d
17.94 ± 0.46 c
3.94 ± 0.11 d
a
b
a
a
a
a
a
19.62 ± 0.38 d
5.08 ± 0.09 d
3.52 ± 0.04
3.81 ± 0.05
3.75 ± 0.02
3.58 ± 0.08
6.48 ± 0.16
5.81 ± 0.09
5.94 ± 0.07
6.44 ± 0.11
0.41 ± 0.04
0.42 ± 0.03
0.47 ± 0.09
0.66 ± 0.05
12.1 ± 0.11
12.1 ± 0.09
12.0 ± 0.13
12.0 ± 0.17
c
a
a
a
a
Red wines
13.93 ± 0.25
17.12 ± 0.74
3.12 ± 0.13
3.80 ± 0.11
b
a
a
b
b
a
b
b
18.27 ± 0.71 c
4.44 ± 0.16 c
3.30 ± 0.03 a
6.92 ± 0.12 c
0.49 ± 0.04 a
12.2 ± 0.14
12.2 ± 0.12
12.0 ± 0.06
12.1 ± 0.26
a
a
a
a
16.48 ± 0.52 d
4.45 ± 0.19 c
d
a
b
a
a
COI
SEQI
SPONT
3.66 ± 0.05
3.53 ± 0.03
3.47 ± 0.07
5.37 ± 0.13
6.52 ± 0.06
6.88 ± 0.12
0.59 ± 0.04
0.56 ± 0.09
0.69 ± 0.07
12.69 ± 0.84
14.36 ± 0.51
3.24 ± 0.12
4.00 ± 0.15
c
b
b
b
b
b
c
c
15.73 ± 0.73 c
5.03 ± 0.13 d
The data are the mean of triplicates ± SD; AF—only alcoholic fermentation; COI—coinoculation of yeast and bacteria; SEQI—sequential inoculation of yeast and bacteria;
SPONT—spontaneous malolactic fermentation; * as tartaric acid; ** as acetic acid; *** values of residual sugar concentration after 1 week are presented to explain why
a, b, c, d
bacteria inoculation in SEQI was performed after 7 days;
—denotes statistically significant differences (p < 0.05) between the different inoculation techniques.

Molecules 2018, 23, 2549
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2
.2. Ethyl Fatty Acid Esters
The synthesis and hydrolysis of esters during the malolactic fermentation of wines has been
described by many research groups, but there is a disagreement concerning the influence of this
secondary fermentation action on the final concentration of esters. Most authors indicate that there is
a significant enhancement in the ester content of wines that have undergone MLF [8,10–12,14–19],
but other researchers have presented a decreasing trend [20–22]. Malolactic bacteria strain selection
has also been described as an important factor that determines the final concentrations of esters
[
12,14,16,17].
Ethyl fatty acid esters are compounds considered to be of primary importance for the aroma of
wines. The compounds analyzed include ethyl lactate, diethyl succinate, ethyl propanoate, ethyl
hexanoate, ethyl octanoate, and acetate esters (ethyl acetate, isoamyl acetate, and 2-phenethyl
acetate).
Ethyl lactate, one of the most characteristic aromatic compounds produced during malolactic
fermentation, is synthetized in the course of the esterification of ethanol (produced by yeast during
alcoholic fermentation) and lactate (produced by malolactic bacteria during malolactic fermentation).
When the malolactic process takes place, the concentration of ethyl lactate progressively increases.
This is beneficial to the wine bouquet due to its fruity, buttery, and creamy aromas, and it also
contributes to the sensations of roundness in the mouth [16,23]. Some authors suggest that the
insensitivity of ethyl lactate biosynthesis depends on the strain of O. oeni used [9,15,17,24]. On the
other hand, some other authors did not register any dependence of ethyl lactate biosynthesis on the
bacteria strain [12].
In our study, the concentration of ethyl lactate after alcoholic fermentation falls in the 8.54–14.44
g/L range (Table 2), and this significantly increased as a result of malolactic fermentation. The highest
concentration of ethyl lactate was registered in the coinoculation technique (132.57–173.76 mg/L).
After sequential inoculation of MLB and spontaneous MLF, the concentrations of ethyl lactate were
significantly lower (41.64–115.63 mg/L). However, this value was several times higher than without
MLF (Table 2). The ethyl lactate biosynthesis was thus noted as strongly influenced by the method of
malolactic fermentation induction. For the coinoculation variant (with the highest dynamics and
effectiveness of malolactic bioconversion) [13], the highest ethyl lactate concentration in the final wine
was obtained (Table 2). Whereas, in the variants with lower MLF rate, a lower ethyl lactate synthesis
was noted.
Literature studies have reported a wide range of ethyl lactate concentrations in wines that have
undergone MLF. Values significantly higher than ours were described by Knoll et al. (up to 440 mg/L)
[
17], Pozo-Bayon et al. (up to 235 mg/L) [15], and Valade and Laurent (up to 190 mg/L) [25]. Our
results are similar to those of Lloret et al., who found ethyl lactate in wines after MLF at
concentrations ranging from 90 to 150 mg/L [26]. On the other hand, Fleet [27] and Maicas et al. [14]
described standard concentrations for ethyl lactate in red wines of up to 50 mg/L. The aroma
threshold for ethyl lactate has been determined to be 110 mg/L [26]. In line with this, ethyl lactate was
discernible in our experiment only in wines in which the yeast and MLB had been coinoculated.
Diethyl succinate is another volatile compound that contributes to wine aroma. Succinic acid (a
by-product of microbial α-ketoglutarate metabolism) is esterified to diethyl succinate, which brings
fruity melon notes. This compound occurs naturally in apples, grapes, and cocoa. Its odor threshold
has been set at 1.2 mg/L [28]. When only alcoholic fermentation was performed in our experiments,
the concentration of this compound was found to be under the threshold value (up to 0.68 mg/L). In
the wines that underwent MLF, the concentration of diethyl succinate was in the 0.49–2.63 mg/L
range (Table 2). Analyzing the influence of different malolactic bacteria inoculation techniques on
diethyl succinate concentration, a similar trend to ethyl lactate was noted. The highest concentration
of diethyl succinate was always observed in the case of coinoculated wines (1.14–2.63 mg/L) (Table
2
), in which the highest rate of MLF was observed [13]. Whereas, in the sequential and spontaneous
malolactic process, in which the deacidification rate were significantly lower [13], the concentration
of diethyl succinate was also respectively decreased. Similar observations have been described by

Molecules 2018, 23, 2549
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Knoll et al. [9], who noted that sequential inoculations resulted in lower concentrations of ethyl lactate
and diethyl succinate than in the case of coinoculation.
Table 2. Concentrations (mg/L) of esters in white and red grape wines produced by different methods
of MLF induction; parameters were evaluated three months after bottling
Ethyl
Ethyl
Diethyl
Ethyl Lactate
Ethyl Hexanoate
Propanoate
Octanoate
Succinate
Chardonnay
AF
8.54 ± 0.31 d
134.97 ± 3.93 a
115.63 ± 5.59 b
72.43 ± 2.64 c
0.31 ± 0.03 b
0.36 ± 0.05 a
0.42 ± 0.04 a
0.39 ± 0.05 a
0.83 ± 0.07 a
0.86 ± 0.04 a
0.79 ± 0.09 a
0.81 ± 0.05 a
1.17 ± 0.09 a
1.11 ± 0.09 a
1.14 ± 0.04 a
1.12 ± 0.06 a
0.68 ± 0.01 d
2.63 ± 0.02 a
1.42 ± 0.06 b
0.89 ± 0.03 c
COI
SEQ
White
wine
SPONT
Kerling
AF
8.62 ± 0.68 d
132.57 ± 4.04 a
111.66 ± 5.84 b
71.04 ± 3.61 c
0.14 ± 0.04 b
0.27 ± 0.07 a
0.23 ± 0.03 a
0.21 ± 0.04 a
0.53 ± 0.03 a
0.57 ± 0.05 a
0.53 ± 0.04 a
0.55 ± 0.04 a
0.88 ± 0.05 a
0.93 ± 0.04 a
0.89 ± 0.06 a
0.85 ± 0.08 a
0.32 ± 0.05 d
1.14 ± 0.03 a
0.55 ± 0.05 b
0.49 ± 0.05 c
COI
SEQI
SPONT
Pinot noir A
AF
14.44 ± 0.86 d
151.25 ± 4.23 a
97.32 ± 6.59 b
64.97 ± 4.44 c
0.51 ± 0.03 b
0.73 ± 0.05 a
0.77 ± 0.03 a
0.74 ± 0.03 a
0.74 ± 0.06 a
0.77 ± 0.07 a
0.74 ± 0.05 a
0.71 ± 0.05 a
1.23 ± 0.04 b
1.31 ± 0.03 a
1.26 ± 0.02 b
1.22 ± 0.05 b
0.54 ± 0.03 d
2.07 ± 0.09 a
1.32 ± 0.07 b
0.76 ± 0.06 c
COI
SEQI
SPONT
Pinot noir B
AF
11.43 ±0.41 d
173.76 ± 5.72 a
91.26 ± 3.66 b
65.33 ± 3.43 c
0.39 ± 0.03 b
0.62 ± 0.03 a
0.60 ± 0.04 a
0.61 ± 0.03 a
0.66 ± 0.08 a
0.71 ± 0.04 a
0.68 ± 0.05 a
0.65 ± 0.06 a
1.08 ± 0.06 a
1.14 ± 0.08 a
1.09 ± 0.05 a
1.06 ± 0.09 a
0.47 ± 0.03 d
1.96 ± 0.07 a
0.88 ± 0.05 b
0.61 ± 0.07 c
Red
COI
wine
SEQI
SPONT
Rondo
AF
9.01 ± 0.56 d
137.41 ± 6.92 a
84.78 ± 4.47 b
41.64 ± 4.33 c
0.27 ± 0.03 b
0.34 ± 0.04 a
0.38 ± 0.02 a
0.35 ± 0.03 a
0.64 ± 0.03 a
0.69 ± 0.04 a
0.66 ± 0.03 a
0.62 ± 0.07 a
0.94 ± 0.05 a
0.99 ± 0.04 a
0.93 ± 0.07 a
0.93 ± 0.08 a
0.51 ± 0.06 d
1.88 ± 0.11 a
1.12 ± 0.08 b
0.74 ± 0.06 c
COI
SEQI
SPONT
The data are the mean of triplicates ± SD; AF—only alcoholic fermentation; COI—coinoculation of
yeast and bacteria; SEQI—sequential inoculation: bacteria at the end of AF; SPONT—spontaneous
a, b, c, d
MLF;
—denotes statistically significant differences (p < 0.05) between the different inoculation
techniques.
With regard to other esters, no effects of the malolactic process on the biosynthesis of ethyl
hexanoate (fruity, strawberry, and green apple aromas) or ethyl octanoate (fruity, sweet, banana, and
pear aromas) were observed. All wines that underwent malolactic fermentation showed a
significantly enhanced concentration of ethyl propanoate (pineapple aroma) (Table 2). Different
observations have been described by Knoll et al. [9], in which a decrease in the concentrations of ethyl
hexanoate and ethyl octanoate were reported. Similar to our findings, there was an increase in ethyl
propanoate concentration after MLF. Summarizing fluctuations in ethyl fatty acid esters, ethyl lactate
and diethyl succinate quantitatively dominated the process, and were the esters showing the greatest
increase in concentration. Similar to our results, a significant increase in the concentration of ethyl

Molecules 2018, 23, 2549
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lactate, ethyl propanoate, and diethyl succinate was also observed after MLF during vinification of
Riesling wine [9,17,29], Aglianico wine [16], and Tempranillo and Merlot wine [12,15].
A second important group of wine esters is the acetate esters group. For this study, we selected:
Ethyl acetate, isoamyl acetate, and 2-phenethyl acetate.
Ethyl acetate is synthesized from ethanol and acetic acid, which are key metabolites in the
vinification process. When its concentration does not exceed 100 mg/L, a desirable and fruity aroma
enriches the wine. Its presence in higher concentrations leads to solvent, nail, varnish, and chemical
aromas [4,14,30]. Isoamyl acetate introduces pleasant fruity notes (mostly banana) to wine aroma
profiles. This ester is formed from isoamyl alcohol and acetic acid, intermediate metabolites of
alcoholic and malolactic fermentation. The highest concentrations of both ethyl acetate (88.13–119.33
mg/L) and isoamyl acetate (0.76–0.94 mg/L) were always noted with spontaneous MLF (Table 3). This
may be due to the notably higher concentrations of volatile acidity (as acetic acid) for this variant of
vinification, as described in our previous research [13]. The different inoculation methods
significantly affected the final concentration of ethyl acetate and isoamyl acetate in the white and red
wines, but the concentrations did not exceed 86.87 and 0.66 mg/L, respectively (Table 3). Maicas et al.
[
14] found additionally that the production of these compounds was dependent on the MLB strain.
In their study, the concentrations after MLF varied from 36.94 to 216.04 mg/L of ethyl acetate, and
from 0.25 to 0.68 mg/L of isoamyl acetate.
Table 3. Concentrations (mg/L) of esters in white and red grape wines produced by different methods
of MLF induction; parameters were evaluated three months after bottling
Sum of Esters
Isoamyl
Acetate
2-Phenethyl
Acetate
Total/Without
Ethyl Lactate and
Ethyl Acetate
Ethyl Acetate
Chardonnay
AF
63.43 ± 1.27 c
51.67 ± 2.03 d
68.44 ± 3.16 b
88.41 ± 2.87 a
0.56 ± 0.07 b
0.42 ± 0.04 c
0.63 ± 0.08 b
0.89 ± 0.03 a
0.23 ± 0.01 b
0.69 ± 0.03 a
0.62 ± 0.08 a
0.73 ± 0.05 a
75.75 / 3.78
192.71 / 6.07
189.09 / 5.02
165.67 / 4.83
COI
SEQI
White
wine
SPONT
Kerling
AF
72.66 ± 3.016 b
58.12 ± 5.37 d
63.27 ± 5.82 c
110.26 ± 4.93 a
0.66 ± 0.02 b
0.49 ± 0.04 c
0.51 ± 0.03 c
0.94 ± 0.03 a
0.33 ± 0.02 b
0.49 ± 0.07 a
0.52 ± 0.03 a
0.55 ± 0.04 a
84.14 / 2.86
194.58 / 3.89
178.16 / 3.23
184.89 / 3.59
COI
SEQI
SPONT
Pinot noir A
AF
77.42 ± 3.28 c
71.33 ± 6.71 c
81.58 ± 4.55 b
94.11 ± 5.19 a
0.33 ± 0.05 c
0.28 ± 0.06 c
0.44 ± 0.02 b
0.76 ± 0.03 a
0.45 ± 0.03 b
0.92 ± 0.03 a
0.88 ± 0.04 a
0.94 ± 0.06 a
95.66 / 3.8
228.66 / 6.08
184.31 / 5.41
164.21 / 5.13
COI
SEQI
SPONT
Pinot noir B
AF
Red
wine
69.31 ± 6.07 c
66.93 ± 4.31 c
75.21 ± 4.89 b
88.13 ± 5.26 a
0.66 ± 0.09 b
0.47 ± 0.03 c
0.58 ± 0.09 b
0.83 ± 0.04 a
0.37 ± 0.03 b
0.84 ± 0.07 a
0.79 ± 0.09 a
0.87 ± 0.07 a
84.37 / 3.63
246.43 / 5.74
171.09 / 4.62
158.09 / 4.63
COI
SEQI
SPONT
Rondo
AF
79.17 ± 4.11 c
0.31 ± 0.02 c
0.41 ± 0.02 b
91.26 / 3.08

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COI
SEQI
78.33 ± 4.85 c
86.87 ± 3.17 b
119.33 ± 4.69 a
0.44 ± 0.07b
0.38 ± 0.06 b
0.79 ± 0.03 a
0.83 ± 0.05 a
0.81 ± 0.09 a
0.88 ± 0.05 a
220.91 / 5.17
175.93 / 4.28
165.28 / 4.31
SPONT
The data are the mean of triplicates ± SD; AF—only alcoholic fermentation; COI—coinoculation of
yeast and bacteria; SEQI—sequential inoculation: bacteria at the end of AF; SPONT—spontaneous
a, b, c, d
MLF;
—denotes statistically significant differences (p < 0.05) between the different inoculation
techniques.
2
-phenethyl acetate is a volatile metabolite which gives wine floral, honey, and raspberry
aromas. In all the examined wines, malolactic bioconversion significantly increased (by almost a
factor of two) its concentration over that of the control process (only alcoholic fermentation).
However, no effect of inoculation variant on the biosynthesis of 2-phenethyl acetate was noted. Knoll
et al. [9] found that wines with sequential MLF had lower concentrations of acetate esters and ethyl
esters than coinoculated wines. Ester concentrations were also affected by the bacteria strain used.
2
.3. Diacetyl and Its Metabolic Products
Diacetyl (2,3-butanedione) is produced by yeast during alcoholic fermentation by a pathway
linked to the amino acid metabolism. At the end of alcoholic fermentation, diacetyl is reduced by
diacetyl reductase to acetoin (3-hydroxy-2-butanone) and 2,3-butanediol. At this stage of the
winemaking process, the concentration of diacetyl thus has no olfactive effect. Significantly higher
amounts of diacetyl can be produced during malolactic fermentation as an intermediate product in
citric acid metabolism [4,8,14,30,31]. In the course of the MLB carbohydrate metabolism pathway,
pyruvate is reduced to lactate. However, when the concentration of residual sugars is too low, citric
acid begins to be utilized as a carbon source, and additional pyruvate is synthesized. This pyruvate
is then a precursor in the process of diacetyl production. The utilization of citric acid starts
simultaneously with malic acid degradation, but it is a very slow process. It is particularly observed
in states of sugar deficiency [31]. Subsequently, because of its low chemical stability, diacetyl can be
easily transformed into acetoin and 2,3-butanediol [4,7,32].
Malolactic fermentation is thus highly recommended to allow diacetyl to enter into the wine
aroma profile. During the secondary fermentation, diacetyl, acetoin, and 2,3-butanediol appear in
different concentrations, which has a direct effect on wine aroma. Each of these compounds has a
different odor detection threshold. Acetoin and 2,3-butanediol have significantly higher threshold
values of perceptibility than diacetyl, at an average of 150 and 600 mg/L, respectively [7,33]. The
aroma detection threshold value for diacetyl depends on the type and style of wine. In general, for
good quality young red wines it ranges from about 0.2 to about 1.84 mg/L, and for aged red wines
from 1.25 to 3.39 mg/L [33].
The lowest concentration of diacetyl noted in our study occurred in the case of alcoholic
fermentation without MLF. The concentration of diacetyl synthesized by the yeasts then ranged from
0
.94 to 1.72 mg/L (Table 4), being too low to have an impact on the wine aroma (Figure 1). A
significantly higher concentration of diacetyl was noted with malolactic fermentation. In the
coinoculation variant this ranged from 2.19 to 4.06 mg/L, and in the sequential inoculation it varied
from 3.42 to 5.91 mg/L. For these wines, light, pleasant buttery and nutty aromas were perceptible
(
Figure 1). The highest concentration of diacetyl was observed for the spontaneous malolactic
fermentation (7.44 to 9.22 mg/L); this was characterized as an intensive and unacceptable buttery
aroma.

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Table 4. Concentrations (mg/L) of diacetyl, acetoin, and 2,3-butanediol in white and red grape wines
produced by different methods of MLF induction; parameters were evaluated three months after bottling.
2
,3-Butanedione
Diacetyl)
3-Hydroxy-2-Butanone
(Acetoin)
2,3-Butanediol
(
Chardonnay
AF
COI
SEQI
SPONT
Kerling
AF
COI
SEQI
SPONT
Pinot noir A
AF
COI
SEQI
SPONT
Pinot noir B
AF
COI
SEQI
SPONT
Rondo
AF
COI
SEQI
SPONT
d
d
d
1.13 ± 0.22
0.86 ± 0.06
254.32 ± 11.34
331.56 ± 15.33
479.93 ± 9.14
2.19 ± 0.15 c
3.11 ± 0.39 c
c
b
b
b
3.42 ± 0.19
5.95 ± 0.26
White
wines
7.44 ± 0.08 a
8.79 ± 0.42 a
712.42 ± 23.65 a
d
d
d
0.94 ± 0.24
0.73 ± 0.04
165.36 ± 12.75
349.62 ± 16.83
513.88 ± 8.31
3.02 ± 0.16 c
4.31 ± 0.22 c
c
b
b
b
4.09 ± 0.08
6.18 ± 0.42
8.33 ± 0.22 a
11.05 ± 0.98 a
783.63 ± 10.63 a
d
d
d
1.71 ± 0.15
1.62 ± 0.17
288.97 ± 12.06
3.80 ± 0.19 c
5.11 ± 0.36 c
361.35 ± 9.72 c
b
b
b
5.24 ± 0.16
6.94 ± 0.45
647.55 ± 17.83
9.22 ± 0.23 a
12.39 ± 0.41 a
806.37 ± 14.97 a
d
1.04 ± 0.12 c
6.48 ± 0.48 b
d
1.31 ± 0.14
267.42 ± 8.53
Red
wines
4.06 ± 0.09 c
493.52 ± 11.84 c
b
b
b
5.91 ± 0.11
7.17 ± 0.56
631.67 ± 17.59
8.72 ± 0.15 a
12.52 ± 0.46 a
718.13 ± 13.77 a
d
d
d
1.72 ± 0.05
1.53 ± 0.14
264.94 ± 6.48
3.81 ± 0.09 c
4.97 ± 0.32 c
355.13 ± 15.83 c
b
b
b
5.50 ± 0.13
7.03 ± 0.26
584.42 ± 18.28
8.80 ± 0.07 a
11.43 ± 0.31 a
652.36 ± 15.75 a
The data are the mean of triplicates ± SD; AF—only alcoholic fermentation; COI—coinoculation of
yeast and bacteria; SEQI—sequential inoculation: bacteria at the end of AF; SPONT—spontaneous
a, b, c, d
MLF;
—denotes statistically significant differences (p < 0.05) between the different inoculation
techniques.
According to Lerm et al. [34], the accumulation of diacetyl and acetoin depends on the dynamics
of malolactic fermentation. The higher the MLF rate, the lower the concentration of diacetyl and
acetoin. In our study, the dynamics of MLF followed the order: COI < SEQI < SPONT [13]. This
explains the reason for a lesser concentration of diacetyl in the case of coinoculation, and higher
concentration for the SEQI and SPONT variants, in which the dynamics of the MLF process were
significantly lower. The presence of oxygen during MLF can also affect the diacetyl content in wine.
This is directly associated with the oxidation of α-acetolactate to diacetyl [30,34]. In our study, micro-
oxygenation was performed to support the initiation of spontaneous MLF (Figure 2) [13]. The
additional amount of oxygen present during the vinification process could thus also have led to the
significantly higher concentration of diacetyl in this variant (Table 4).
The products of diacetyl degradation—acetoin (3-hydroxy-2-butanone) and 2,3-butanediol—
were also evaluated. In all the wines, the lowest levels of both compounds were noted when only
alcoholic fermentation was performed; this concentration ranged from 0.73 to 1.62 mg/L of acetoin,
and from 165 to 288 mg/L of 2,3-butanediol (Table 4). Similarly, as in the case of diacetyl, MLF
significantly increased the concentration of both metabolites. The highest concentrations were noted
for the spontaneous process, in which the biosynthesis yield was as high as 8.79–12.52 mg/L of
acetoin, and 652–806 mg/L of 2,3-butanediol. Coinoculation resulted in significantly lower values of
both metabolites than sequential inoculation. According to Francis and Newton [33], and Bartowsky
and Henschke [7], acetoin levels remained under the sensory threshold, but 2,3-butanediol reached a
3
concentration of sensory significance for wine (over 600 mg/dm ), though only in the spontaneous
variants. This was reflected in the sensory evaluation of the produced wines (Figure 1). Generally,
2
,3-butanediol is not expected to affect the sensory qualities of wine appreciably [35], but we did note
a bitter taste in these variants. Some authors have also described very low or undetectable levels of

Molecules 2018, 23, 2549
6 of 15
diacetyl in wines that had undergone MLF [36]. They suggest that this may be a result of the
enzymatic reduction of diacetyl to 2,3-butanediol. Acetoin, the other intermediate metabolite of
diacetyl involved in the same metabolic pathway, is also reduced to 2,3-butanediol. This may explain
the high levels of 2,3-butanediol found in our wines.
2
.4. Sensory Evaluation
AF
AF
fruity
fruity
(
white wines)
5
4
3
2
1
0
(red wines)
5
4
3
2
1
0
earthy
fresh
earthy
fresh
vegetal
bitter
floral
vegetal
floral
buttery
bitter
buttery
nutty
fruity
nutty
COI
white wines)
COI
(red wines)
fruity
(
5
4
3
2
1
0
5
4
3
2
1
0
earthy
fresh
earthy
vegetal
bitter
fresh
vegetal
bitter
floral
floral
buttery
buttery
nutty
nutty
SEQI
white wines)
SEQI
fruity
5
4
3
2
1
0
(
(red wines)
fruity
earthy
fresh
5
4
3
2
1
0
earthy
fresh
vegetal
bitter
floral
vegetal
bitter
floral
buttery
buttery
nutty
nutty
SPONT
white wines)
SPONT
(red wines)
(
fruity
fruity
5
4
3
2
1
0
5
4
3
2
1
0
earthy
vegetal
bitter
fresh
earthy
fresh
floral
vegetal
floral
buttery
bitter
buttery
nutty
nutty
Figure 1. Descriptive sensory evaluation of white and red wines produced by different methods of
MLF induction; evaluation was performed three months after bottling. Scale 0–5: 0—lack, 1—very
light, 2—light, 3—noticeably, 4—intensive, 5—very intensive sensibility (value 5 is the most desirable
for fruity, fresh, and floral aromas, but undesirable and not acceptable for buttery, nutty, bitter,
vegetal, and earthy aromas).

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The sensory evaluation indicated that the timing and method of MLB inoculation significantly
affected the taste and aroma of the wines. In general, malolactic fermentation diversified the wine
aroma profile (Figure 1). The coinoculated wines were noted to be higher in fruity, fresh, and floral
sensations than the wines which had used sequential MLF. The spontaneous process was perceived
as producing wines with more buttery and bitter notes. Pleasant balanced buttery and nutty aromas
were also found in the coinoculated wines. The wines after sequential and spontaneous processes
had no nutty aromas, but strong buttery aromas were noted instead.
3
. Materials and Methods
3
.1. Microorganisms
Saccharomyces cerevisiae yeast (Lalvin EC-118, Lallemand, Petaluma, CA, USA) and Oenococcus
oeni bacteria (Lalvin VP41, Lallemand, Petaluma, CA, USA) were used in the experiments.
Rehydration protocols were followed as per the producer’s instructions.
3
.2. Grape Variety
During three wine seasons, two white varieties, Chardonnay 2009 and Kerling 2010, and two
red varieties, Pinot noir 2009 and 2012, and Rondo 2012, were used to produce the experimental
wines. The grapes were obtained from Mierzecin Vineyard in Poland. Typically for grapes from cool-
climate countries, the musts were characterized with enhanced total acidity (9.38–12.14 g/L, as tartaric
acid) and low pH (3.19–3.64) (Table 5) [13], which significantly singularize them from others studied
and described in the literature [9–12,37,38].
Table 5. Characteristic of grape must used for the vinification process [13].
Pinot Noir
Chardonnay
Kerling
Pinot Noir B
Rondo
A
°
Bx
22.5 ± 0.1
193.12 ± 2.11
3.31 ± 0.03
11.64 ± 0.12
8.14 ± 0.09
0.33 ± 0.02
20.5 ± 0.1
171.33 ± 2.54
3.19 ± 0.03
24.0 ± 0.2
22.0 ± 0.1
22.0 ± 0.2
Sugars: glucose + fructose (g/L)
pH
203.54 ± 3.05 185.47 ± 3.17 181.62 ± 2.84
3.64 ± 0.05
9.38 ± 0.09
6.54 ± 0.07
0.28 ± 0.02
3.41 ± 0.06
10.95 ± 0.23
8.11 ± 0.14
0.35 ± 0.02
3.26 ± 0.07
11.52 ± 0.26
7.83 ± 0.21
0.31 ± 0.02
Total acidity (g/L) *
Malic acid (g/L)
Citric acid (g/L)
12.14 ± 0.14
9.5 ± 0.05
0.41 ± 0.02
*
as tartaric acid.
3
.3. Parameters of the Vinification Process.
The wines were produced on a laboratory scale in 15-liter glass containers. Four different
variants of the vinification process were performed: (1) Alcoholic fermentation only, as a control (AF);
2) coinoculation (COI), where the yeast and bacteria were inoculated at the same time; (3) sequential
(
inoculation (SEQI), where malolactic fermentation was induced at the end of alcoholic fermentation;
and (4) spontaneous malolactic fermentation (SPONT), where we did not perform MLB inoculation.
The consecutive steps of the vinification process are presented in Figure 2 as a flowchart, and follow
those of our previous study [13].

Molecules 2018, 23, 2549
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Figure 2. Flowchart of white and red wine production [13].
The process commenced with the inoculation of yeast in the first day for all variants. The timing
of malolactic bacteria inoculation was dependent on the variant. This was done on the first day
(
together with yeasts) in COI samples, and on the seventh day of winemaking for SEQI samples. To
avoid spontaneous MLF in the AF variant, an additional sulfitation process was performed after one
month of vinification (10 g/hL of K ). In the SPONT samples, MLF was induced by micro-
2
S
2O5
oxygenation, supplementation with bacteria nutrients, and lower sulfitation. No malolactic bacteria
starter culture was added in this case. The winemaking process lasted six months. Next, after bottling,
the wines were subjected to maturation (temp. 7–10 °C) for three more months. Finally, after nine
months, the obtained wines were submitted for chemical and sensory analysis. The samples were
stored at 7–10 °C, without freezing.
3
.4. Analysis of Volatile Compounds
The chemical analysis of volatile compounds was performed for all the wines of the three
seasons at the same time (3 months after bottling). Before analysis, samples were only filtered with
.45-µm Millipore filters.
0

Molecules 2018, 23, 2549
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The volatiles were extracted by solid phase microextraction using a 2 cm DVB/CAR/PDMS fiber
(
Supelco) with a CTC Combipal autosampler (Agilent Technologies). For each analysis, 10 mL
2
samples of wine were placed into 20 mL vials, spiked with an internal standard ([ H
8]-naphthalene),
sealed with PTFE/silicon septa caps, and incubated for 2 min at 50 °C prior to extraction. Compounds
were extracted from the headspace at 50 °C for 35 min. Compounds were identified using
multidimensional gas GCxGC-ToFMS chromatography with a ZOEX cryogenic (N
2
) modulator
(
Pegasus IV, LECO, St. Joseph, MI, USA). The GC was equipped with a DB-5 column (30 m × 0.25
mm × 0.25 μm), and had a Supelcowax 10 (1 m × 0.1 mm × 0.1 μm) as a second column, with a helium
flow rate of 0.8 mL/min. For the two-dimensional analysis, the modulation time was optimized and
set at 3 seconds, and the mass spectra were collected at a rate of 150 scans/s. The transfer line was
heated to 280 °C, and the ion source was heated to 220 °C. The injector temperature was set to 280 °C
for the DVB/CAR/PDMS fiber. During the injection, the fiber was maintained for 5 min in the splitless
mode, and then for 1 min in the split mode (20:1). Identification of volatiles was performed by
comparison of the retention indices and mass spectra of eluting compounds to those of the NIST 05
library match. The calculation was done using Chroma TOF software (version 4.23) upgraded with
additional post data processing Statistical Compare software (LECO, St. Joseph, MI, USA) for
calculation of the Fisher ratio. Semiquantification of the volatile compounds was performed using
the internal standard; thus, they do not represent the absolute amount of the compound present in
the wine samples, but were instead calculated and used to observe the differences between the wine
samples. Each measurement was repeated three times.
3
.5. Chemical Analysis of Must and Wine
The chemical analysis of the wines was performed for all the wines of the three seasons at the
same time (3 months after bottling). Before analysis, samples were only filtered with 0.45-µm
Millipore filters. The soluble solids (°Bx) were evaluated by refractometer. To control the chemical
parameters, total acidity was measured according to OIV-MA-AS313-01, pH according to OIV-MA-
AS313-15, and volatile acidity according to OIV-MA-AS-313-02 [39]. The concentration of glucose,
fructose, malic acid, citric acid, and ethanol was measured using HPLC (Waters Alliance 2695
combined with BioRad Aminex HPX-87H column, 300 × 7.8 mm and RI detector; measurement
conditions: 1.5 mM H
2
SO as eluent, flow rate 0.4 mL/min, column temp. 50 °C, and detector temp.
4
4
5 °C).
3
.6. Sensory Evaluation
The sensory evaluation was performed for all the wines of the three seasons at the same time (3
months after bottling). Sensory analysis was undertaken in order to evaluate the differences between
the wines obtained with different inoculation techniques. The sensory panel members included 120
people between 24 and 55 years old. They evaluated the wine samples using a 0–5 point scale (0 =
very low discernible aroma; 5 = very intensive discernible aroma). The applied serving temperature
was kept at 13–15 °C for white wines, and at 17–18 °C for red wines.
3
.7 Statistical Analysis
All data are presented as the mean value of at least three repetitions ± standard deviation.
Statistical data analysis was performed using analysis of variance (ANOVA) in Statistica V.8 (Statsoft
Inc., Tulsa, OK, USA). Tukey’s test was used for significantly different samples (p < 0.05).
4
. Conclusions
The fermentation-derived metabolites examined in this study were affected by the malolactic
bacteria inoculation technique. The total concentration of the analyzed ethyl esters was highest for
the coinoculation case. Quantitatively, ethyl lactate, diethyl succinate, and ethyl acetate dominated
as the esters that showed the greatest increase in concentration. An excess of diacetyl, perceived as a
serious danger of MLF which can negatively affect the quality of wine, was noted only for the

Molecules 2018, 23, 2549
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spontaneous processes. Whereas, coinoculation was a treatment with unbeatably balanced nutty
notes.
The present investigations highlighted that controlled malolactic fermentation, and especially
the coinoculation technique, can be proposed as a safe and efficient enological practice for producing
quality grape wines. Our results clearly indicate that simultaneous inoculation of yeast and bacteria
offer not only dynamic deacidification of low pH grape wines [13], but also modify qualitatively and
quantitatively the profile of volatile compounds, enriching the cool-climate and low aromatic wines
with fruity, fresh, and floral aromas.
Author Contributions: M.L.-K. (leader of the project) conceived, designed and performed the experiments;
analyzed the data; and wrote the manuscript. M.M. analyzed the volatiles in the wine samples. J.N. designed the
experiment. All authors read and approved the final manuscript.
Acknowledgments: This research was realized with the financial support of a research grant obtained from the
National Science Centre in Poland (project No NN312 206036).
Conflict of Interest: The authors declare that they have no conflict of interest.
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