Biosci. Biotechnol. Biochem., 71 (12), 3105–3109, 2007
Note
Ethyl Esterification of Long-Chain Unsaturated Fatty Acids Derived
from Grape Must by Yeast during Alcoholic Fermentation
Keita Y UNOKI,1 Shuji H IROSE,2 and Masao O HNISHI1; y
1
Department of Agricultural and Life Science, Obihiro University of Agriculture and Veterinary Medicine,
Obihiro, Hokkaido 080-8555, Japan
2
Tokachi-Ikeda Research Institute for Viticulture and Enology, Ikeda, Hokkaido 083-0002, Japan
Received July 12, 2007; Accepted August 28, 2007; Online Publication, December 7, 2007
[doi:10.1271/bbb.70444]
The composition of total fatty acid ethyl ester (FAEE)
in yeast cells and the liquid phase separated from grape
must during alcoholic fermentation at different temperatures was investigated by using the solid-phase extraction method. Thirteen FAEE from butyric to linolenic
acids were detected during fermentation. Significant
amounts of long-chain unsaturated FAEE, including
linoleic and linolenic acids derived from grape material,
had already accumulated in the yeast cells by day 3
during fermentation.
Key words:
red wine; fatty acid ethyl ester; linoleic acid;
alcoholic fermentation; yeast
Grapevines can adapt to various climate and soil
conditions, and can be grown in cold regions such as
Hokkaido. We have already determined the relationship
between the lipid components in grapes and their
cryotolerance, and found that the membrane lipid
composition of a grapevine was related to its cryotolerance.1) Moreover, we reported that lipid components
of the must from grapes grown in cold regions had
characteristically high levels of linoleic and linolenic
acids, of which linoleic acid may inhibit the formation
of FAEE by yeast.2) Wines have various components
derived from grape materials and microorganism activities which can contribute to multiple variations in their
color and flavor.3,4) It was therefore assumed that the
lipid components, including long-chain unsaturated fatty
acids, derived from grape materials could greatly
influence wine quality, although there has been little
research on the lipid components in wine-making,2,5,6)
compared to Japanese sake-making.7) On the other hand,
particular investigations have been made of lower
FAEE, including ethyl esters of caproic and caprylic
acids, with direct relation to wine flavor.8,9) Thus, in the
present study, a comprehensive and quantitative determination of total FAEE from lower and long-chain
unsaturated types, existing in both intra- and extracellular yeast during the alcoholic fermentation of
y
grapes with many long-chain unsaturated fatty acids,
was carried out by using the solid-phase extraction
method. Additionally, the effect of fermentation temperatures on total FAEE formation was also investigated,
since it is known that low-temperature fermentation can
change the FAEE composition in wine.10,11)
The grape sample (Kiyomi) was that used for wine
production at Tokachi-Ikeda Research Institute for
Viticulture and Enology, Ikeda, Hokkaido, Japan. Must
was obtained from the free run of destemmed and
crushed grapes prior to alcoholic fermentation. Alcoholic fermentation was carried out by using a commercial yeast (Lalvin EC1118, Lallemand Inc., Canada), at
25 C and 15 C. Samples were taken on days 3, 5 and 7
during alcoholic fermentation.
The lipophilic constituents were extracted by using
chloroform and methanol by the method of Bligh and
Dyer.6) Total lipids were methanolyzated with 5%
methanolic HCl. Fatty acid methyl esters were analyzed
by using GC as previously reported.6)
The fatty acid ethyl esters (FAEE) were determined
according to a previous method with slight modification.2) A fermented must sample (50 ml) was centrifuged
at 1,500 g for 15 min. After transferring the supernatant
and washing the pellet, the combined supernatant was
used for solid-phase extraction. To further determine
FAEE in yeast cells in the pellet, the pellet was twice
subjected to sonication in ethanol (UD-200, Tomy,
Japan) for 15 min and then shaken. After centrifugation,
the combined supernatant was adjusted to 12% ethanol,
and used for solid-phase extraction. Briefly, a cartridge
(200 mg/3 ml, LiChrolut EN, Merck) was successively
rinsed with 4 ml of dichloromethane, 4 ml of methanol
and finally 4 ml of 12% ethanol. The solutions were
loaded into the preconditioned cartridge. The sorbent
was then dried by letting air pass through it via an
aspirator for 10 min and eluted with 1.3 ml of hexanediethyl ether (9:1, v/v). An ethyl tridecylate solution in
hexane as an internal standard and an appropriate
amount of anhydro-Na2 SO4 were added to the eluted
To whom correspondence should be addressed. Fax: +81-155-49-5549; E-mail: mohnishi@obihiro.ac.jp
3106
K. YUNOKI et al.
A
TIC
8:0 10:0
6:0
4:0
10
12:0
13:0 (IS)
16:0
m/z 88
20
30
40
Retention time (min)
B
18:2
18:3
18:1
16:1
TIC
13:0
8:0
10:0 12:0
14:0
16:0
18:0
5
m/z 88
10
30
20
40
Retention time (min)
Fig. 1. Total Ion and Mass Chromatograms of FAEE Separated by the Solid-Phase Extraction Method.
A, FAEE from the supernatant after centrifugation of the must on day 7 at 25 C; B, FAEE from the precipitate after centrifugation of the must.
FAEE were monitored at m=z 88, the characteristic ion of FAEE.
sample. FAEE in these two extracts were analyzed by
GC-MS2) and GC-FID,2) using an ULBON HR-1 column
(50 m 0:25 mm I.D., Df = 0.25 mm; Shinwa Chemical
Industries, Japan). The column temperature was initially
held at 40 C for 5 min, then programmed from 40 C
to 70 C at 4 C/min, and at 8 C/min to 230 C, and
finally held for 15 min. When the reproducibility of the
method had been determined by using standard ethyl
butyrate, ethyl capriate, ethyl palmitate, ethyl oleate and
ethyl linoleate in a model wine solution, the recovery of
the five compounds ranged from 92% to 97%.2)
All data for the lipid analyses are averages from at
least three experiments, and the mean SD is presented
for each. Statistical significance was evaluated by an
analysis of variance (ANOVA) and tested by a Scheffe
analysis.
In the lipophilic constituents of must on day 0 of
fermentation, fourteen fatty acids with carbon lengths
from 12 to 26, including five unsaturated fatty acids,
were detected. The proportion of linoleic acid (18:2) was
highest at 47.0%, followed by palmitic acid (16:0) at
27.8% and linolenic acid (18:3) at 12.1% as major
components, with these three acids accounting for
86.9% of the total acids. The palmitoleic acid (16:1)
content was only 0.2%. Moreover, the lauric (12:0) and
myristic acid (14:0) contents were 0.1% and 0.2%,
respectively, both negligible amounts. These compositions almost corresponded to those of the previous
report.2)
The supernatant (extracellular) and precipitate (intracellular) of must fermenting at 25 C on day 7 were
analyzed by a combination of solid-phase extraction and
GC-MS (Fig. 1A and B, respectively). When the fragment ion of m=z 88, characteristic of FAEE, was
monitored, 6 saturated-type FAEE from butyric acid
(4:0) to 16:0, except for tridecanoic acid (13:0, used as
the internal standard), were detected in the extracellular
fraction (Fig. 1A). Moreover, 6 saturated-type FAEE
from caprylic (8:0) to stearic acids (18:0) and 4
unsaturated-type FAEE from 16:1 to 18:3 were detected
in the intracellular fraction (Fig. 1B). In addition, the
large peaks with retention times of 7.2, 12.8, 13.3 and
20.7 min were isoamyl alcohol, hexanol, isoamyl acetate
and 2-phenyl ethanol, respectively. The FAEE components were significantly different between the intra- and
extra-cellular fractions, the former consisting of volatile
Esterification of Grape Unsaturated Fatty Acids by Yeast
3107
Table 1. Changes in the Fatty Acid Ethyl Ester Composition (%) of Must during Alcoholic Fermentation at 25 C
Day 3
FAEE
Day 5
Day 7
Intracellular
Extracellular
Intracellular
Extracellular
Intracellular
Extracellular
4:0
6:0
8:0
10:0
10:1
12:0
14:0
16:0
16:19
18:0
18:19
18:29;12
18:39;12;15
—
—
3:3 0:1
9:0 0:1
—
7:7 0:9
4:5 0:7
25:8 2:0
10:2 0:1
5:7 0:1
3:2 0:5
22:0 2:6
8:6 0:6
4:6 1:1
11:3 0:6
36:9 1:4
27:0 0:8
2:3 0:2
15:9 1:8
—
2:0 0:5
—
—
—
—
—
—
—
2:0 0:1
4:8 0:1
—
10:5 0:5
5:5 0:3
27:4 0:3
9:3 0:2
3:6 0:3
1:9 0:1
23:4 0:9
11:6 0:6
6:8 0:5
40:4 2:5
36:8 0:3
9:2 1:1
0:3 0:1
5:2 0:6
—
1:3 0:5
—
—
—
—
—
—
—
2:6 0:1
6:1 0:2
—
4:9 0:4
4:4 0:2
41:9 0:4
5:4 0:1
6:9 0:6
2:0 0:2
18:3 0:5
7:5 0:4
12:0 1:1
33:3 2:2
33:5 0:6
13:2 1:3
—
5:9 0:5
—
2:1 1:0
—
—
—
—
—
Total acylsa
1;930 150
150 15
5;590 90
1;410 210
2;680 190
1;020 150
Alcohol (%)
0.8
9.7
11.1
a
nmol/100 ml of fermented must
Significantly different with at least 95% confidence on days 5 vs. 3 and on days 7 vs. 5.
Each value is expressed as the mean SD.
lower FAEE and the latter consisting of unsaturated
FAEE, including 18:2, as the major components. Thus,
this method was very effective for determining the
various FAEE moieties produced by yeast, because the
headspace method, which is generally used to detect
volatile flavor compounds, cannot detect non-volatile
long-chain FAEE.
The amount and composition of FAEE during
alcoholic fermentation at 25 C is shown in Table 1.
The total amount of extracellular FAEE on day 3 was at
a low level, 150 (nmol/100 ml of fermented must),
whereas a significant amount of intracellular FAEE was
present at 1,930 (nmol/100 ml of fermented must),
although the alcoholic concentration was only 0.8%.
Extracellular FAEE on day 5 had increased by more
than nine times that (1,410 nmol/100 ml) on day 3;
however, it decreased on day 7. Six saturated-type
FAEE from 4:0 to 16:0 were detected as extracellular
FAEE. Slight amounts of decenoic acid (10:1) were also
detected on days 3 and 5. On the other hand, ten FAEE
from 8:0 to 18:3 were detected throughout fermentation
as intracellular FAEE. 18:2 and 18:3 are the fatty acids
derived from grapes which the yeast, Saccharomyces
bayanus cannot synthesize.12,13) In this way, the extracellular fraction mainly consisted of lower FAEE, which
were synthesized by yeast, while the intracellular
fraction mainly consisted of long-chain unsaturated
FAEE which were mainly derived from the grape
materials. Our previous study has indicated that ethyl
esters of 18:2 and 18:3 were also present in wine after
alcoholic fermentation,2) suggesting that they were
released from yeast cells by autolysis.
Fatty acids with a short carbon length increased with
the development of fermentation in the extracellular
fraction, although 8:0, 10:0 and 12:0 were predominantly present on day 3. Significant amounts of 16:0,
18:2 and 18:3 were detected in the intracellular fraction
on day 3, showing that the fatty acids derived from
grapes were present as major FAEE in yeast cells at the
early stage of fermentation. On the other hand, 16:1,
which was present at only 0.2% in the lipophilic
components of must, accounted for 10.2% on day 3. It
is assumed that 16:0 synthesized de novo by yeast was
desaturated to 16:1 and then esterified with ethanol,
since this acid is known to be a major component of the
Saccharomyces yeast species.13) The major FAEE
composition had hardly changed by day 5; however,
on day 7, unsaturated fatty acids, including 16:1, 18:2
and 18:3, had significantly decreased, the proportion of
16:0 being particularly high. These results indicate that
FAEE up to C6 were released from yeast cells and that
FAEE longer than C14 (except for a trace amount of
16:0) were accumulated in the yeast cells.
In conclusion, it was found that some polyunsaturated
fatty acids, such as 18:2, abundantly expressed in grape
materials were immediately incorporated into yeast cells
and accumulated as ethyl esters. This high availability of
exogenous unsaturated fatty acids by yeast could
suppress yeast fatty acid de novo synthesis or the
esterification reaction.12) As a result, 18:2 in the must
probably inhibited FAEE formation by the yeast during
alcoholic fermentation, inducing the small amount of
FAEE in wine after fermentation, as we have previously
reported.2)
In order to increase the lower FAEE during fermentation, low-temperature fermentation at 15 C was
carried out and the FAEE composition was investigated
(Table 2). Although it is known that low-temperature
fermentation can increase lower FAEE in wine,10,11) the
present study focused on the total FAEE components in
the intra- and extra-cellular fractions, including unsaturated fatty acids. The alcoholic concentration on day 7
3108
K. YUNOKI et al.
Table 2. Changes in the Fatty Acid Ethyl Ester Composition (%) of Must during Alcoholic Fermentation at 15 C
Day 3
FAEE
Day 5
Day 7
Intracellular
Extracellular
Intracellular
Extracellular
Intracellular
Extracellular
4:0
6:0
8:0
10:0
10:1
12:0
14:0
16:0
16:19
18:0
18:19
18:29;12
18:39;12;15
—
—
6:6 0:1
7:7 0:2
—
11:4 3:2
6:0 0:2
19:0 0:3
18:8 2:2
1:3 0:1
3:4 0:4
19:1 3:0
6:7 0:4
1:8 0:9
12:4 1:0
33:3 1:1
32:3 1:5
4:5 0:3
15:0 1:5
—
0:7 0:3
—
—
—
—
—
—
—
12:2 0:2
13:0 0:1
—
11:9 0:2
5:7 0:1
19:2 0:3
9:9 0:7
1:9 0:1
1:8 0:1
16:9 0:5
7:5 0:6
3:1 0:2
31:9 2:4
43:2 2:0
12:7 0:8
0:6 0:1
8:1 0:7
—
0:4 0:1
—
—
—
—
—
—
—
2:0 0:1
5:9 0:2
—
11:3 0:7
5:2 0:1
35:0 1:1
12:8 0:4
3:6 0:1
2:8 0:1
15:3 0:1
6:1 0:2
7:1 0:9
39:1 1:1
39:6 1:1
8:9 0:6
0:9 0:1
3:8 0:3
—
0:6 0:1
—
—
—
—
—
Total acylsa
810 100
120 4
4;700 160
1;610 200
6;580 200
2;750 260
Alcohol (%)
0.7
5.2
9.5
a
nmol/100 ml of fermented must
Significantly different with at least 95% confidence on days 5 vs. 3 and on days 7 vs. 5.
Each value is expressed as the mean SD.
at 15 C was similar to that on day 5 at 25 C due to
lowering the fermentation temperature. The amount of
extracellular FAEE (2,750 nmol/100 ml) on day 7, in
spite of having a low alcoholic concentration, was
approximately twice that (1,410 nmol/100 ml) on day 5
at 25 C, which peaked at 25 C, although these values
on days 3 and 5 were not significantly different between
15 C and 25 C. Moreover, the amount of intracellular
FAEE on days 3 and 5 was less than that at 25 C,
whereas this value on day 7 (6,580 nmol/100 ml) was
slightly more than that on day 5 at 25 C (5,590 nmol/
100 ml), which peaked at 25 C. The proportions of 18:2
and 18:3 in intracellular FAEE were low compared to
those at 25 C, and those of medium-chain fatty acids
and 16:1 were high. This suggests that yeast de novo
fatty acid synthesis was increased due to the decreased
incorporation of exogenous unsaturated fatty acids into
the yeast cells, which would inhibit yeast desaturation.
In addition, it was assumed that the high proportion of
16:1 had increased due to the promotion of delta-9
desaturation by low-temperature exposure.14)
Taken together, even for alcoholic fermentation by
using grape must with many unsaturated fatty acids from
grapes grown in Hokkaido, low-temperature fermentation has the potential to increase lower FAEE, which can
increase the fruit profile of wine.
2)
3)
4)
5)
6)
7)
Acknowledgments
8)
This work was supported by a grant-aid from Oil and
Fat Industry Kaikan, 2006.
9)
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