Identification and Characterization of Sulfur Heterocyclic Compounds That Contribute to the Acidic Odor of Aged Garlic Extract

Article abstract of DOI:10.1021/acs.jafc.0c06634

The aroma of aged garlic extract (AGE) has been recently characterized as a complexity of seasoning-like, metallic, fatty, and acidic notes; most of the important aroma compounds were identified in a previous study. Besides the 25 previously identified aromas of AGE, several of the odor compounds that contribute to the acidic notes were isolated and identified using various analytical techniques, including gas chromatography coupled with an olfactometry monitoring system (GC-O), accurate and high-performance preparative GC system, GC-MS analysis, and sensory evaluation. The identified aromas include: 2,4-dimethyl-1,3-dithiolane, 2,5-dimethyl-1,4-dithiane, and 2,6-dimethyl-1,4-dithiane. Interestingly, AGE contains all stereoscopic isomers of each of these components. An aroma recombinant composed of the newly identified acidic odors with other key odorants showed good agreement with the aroma of AGE.

Full text of DOI:10.1021/acs.jafc.0c06634

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Article
Identification and Characterization of Sulfur Heterocyclic
Compounds That Contribute to the Acidic Odor of Aged Garlic
Extract
Kazuki Abe, Takao Myoda, and Satoshi Nojima*
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ABSTRACT: The aroma of aged garlic extract (AGE) has been recently characterized as a complexity of seasoning-like, metallic,
fatty, and acidic notes; most of the important aroma compounds were identified in a previous study. Besides the 25 previously
identified aromas of AGE, several of the odor compounds that contribute to the acidic notes were isolated and identified using
various analytical techniques, including gas chromatography coupled with an olfactometry monitoring system (GC−O), accurate
and high-performance preparative GC system, GC−MS analysis, and sensory evaluation. The identified aromas include: 2,4-
dimethyl-1,3-dithiolane, 2,5-dimethyl-1,4-dithiane, and 2,6-dimethyl-1,4-dithiane. Interestingly, AGE contains all stereoscopic
isomers of each of these components. An aroma recombinant composed of the newly identified acidic odors with other key odorants
showed good agreement with the aroma of AGE.
KEYWORDS: aged garlic extract, acidic odor, organosulfur compounds, 2,4-dimethyl-1,3-dithiolane, 2,5-dimethyl-1,4-dithiane,
2,6-dimethyl-1,4-dithiane
1-propenylcysteine and S-allylcysteine,15 which exhibits
INTRODUCTION
■
beneficial pharmacological effects, such as antioxidant, anti-
Garlic (Allium sativum L.) is a vegetable that is used worldwide
as a seasoning and has been used traditionally as a medicinal
hypertensive, and immunomodulatory effects.16−18
plant.1 Various studies have been conducted to elucidate the
Because few studies have focused on the aroma of AGE, we
recently characterized the aroma components in AGE using
pharmacological effects of garlic; a number of active principle
compounds of garlic have been identified.2,3 Among the known
sensomics to elucidate the effect of the aging process on the
aroma of AGE.14 Twenty-five compounds, including sulfur-
active compounds, the sulfur-containing volatile compound
containing compounds, phenols, and esters, were identified as
key odorants, and these key aromas were mostly absent in fresh
garlic.19,20 The aroma components of AGE were also found to
be different from those of other garlic products, such as heated
garlic and black garlic.19−21 However, the key aroma
compounds contributing to the acidic note of AGE remain
unclear. Further investigation of the aroma of AGE using
various analytical techniques led to the isolation and
identification of additional important aroma compounds that
contribute to the acidic note; these results are reported herein.
diallylthiosulfinate, known as allicin, is an important contrib-
utor to the biological activities of garlic.4,5
Allicin is produced from alliin (S-allylcysteine sulfoxide) by
alliinase when fresh garlic is mechanically damaged, for
example, by slicing or grating.6 Allicin is gradually degraded
to ajoenes, allyl sulfides, and vinyl dithiins.7 These degradation
products have also been reported to exhibit pharmacological
effects, such as anticancer, antimicrobial, antioxidant effects,
and protect against cardiovascular disease.2,4,5 Although allicin
and some of its degradants have beneficial effects in humans,
allicin is also the principal compound associated with the
pungent odor of fresh garlic. Allicin is degraded to malodorous
volatiles (e.g., allyl methyl sulfide and allyl mercaptan) in the
mouth and gut, which results in an unpleasant breath odor.8−10
Because of these undesirable effects some people avoid fresh
garlic, despite its significant health benefits.
In an effort to reduce the pungent smell of fresh garlic,
various treatments and processes have been investigated;
however, most of these efforts also reduce the pharmacological
activities to some extent.11,12 Aged garlic extract (AGE) is
prepared by extracting sliced garlic in aqueous ethanol and
naturally aging for >10 months.13 During this process, allicin is
decomposed completely, and thus, the pungent odor of garlic
is substantially reduced.14 In addition, it was found that AGE
contains characteristic sulfur-containing amino acids, such as S-
MATERIALS AND METHODS
■
Materials. AGE was provided by Wakunaga Pharmaceutical Co.,
Ltd. AGE was prepared by slicing garlic cloves and soaking them in an
aqueous ethanol solution, followed by extraction and aging for >10
months at room temperature in an air tight container, and subsequent
filtering of the solution.13
Received: October 19, 2020
Revised: January 6, 2021
Accepted: January 6, 2021
Published: January 15, 2021
https://dx.doi.org/10.1021/acs.jafc.0c06634
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© 2021 American Chemical Society
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Chemicals. All chemicals used in this study were purchased from
Fujifilm Wako Pure Chemical Corporation (Osaka, Japan), Tokyo
Chemical Industry Co., Ltd. (Tokyo, Japan), Kishida Chemical
(Osaka, Japan), or Kanto Chemical Co., Ltd. (Tokyo, Japan). The
purities of the chemicals used for sensory analysis were >95% (GC-
FID).
4.64 (q, 1H J = 6.6 Hz); 13C NMR (100 MHz, CDCl3): δ 20.2, 24.1,
45.4, 48.8, 51.5; MS-EI m/z (intensity in relative %): 41 (28), 45
(33), 59 (53), 60 (19), 64 (20), 74 (25), 92 (41), 119 (81), 134
(100; M+). 1H NMR for (2S,4S)-1 and (2R,4R)-1 (400 MHz,
CDCl3): δ 1.41 (d, 3H, J = 6.8 Hz), 1.61 (d, 3H, J = 6.6 Hz), 2.92
(dd, 1H, J = 7.2 and 11.5 Hz), 3.35 (dd, 1H, J = 5.0 and 11.5 Hz),
3.86−3.95 (m, 1H), 4.67 (q, 1H, J = 6.6 Hz); 13C NMR (100 MHz,
CDCl3): δ 20.1, 25.5, 46.1, 47.9, 50.0; MS-EI m/z (intensity in
relative %): 41 (27), 45 (32), 59 (51), 60 (19), 64 (19), 74 (24), 92
(39), 119 (81), 134 (100; M+).
Synthesis of (2R,4S)- and (2S,4S)-2,4-Dimethyl-1,3-dithio-
lane (1). (R)-1,2-Dibromopropane (5). Powdery triphenylphosphine
(5.77 g, 22.0 mmol) was added to (S)-1,2-propanediol (4) (761 mg,
10.0 mmol) and tetrabromomethane (7.30 g, 22.0 mmol) in
dichloromethane (50 mL) at 0 °C. The mixture was stirred for 6 h
at room temperature. The reaction mixture was concentrated in vacuo
and extracted with n-pentane. The extracts were dried (Na2SO4) and
the solvent was removed in vacuo. The residue was purified on a short
silica gel column using n-pentane as an eluent to yield a mixture of
(R)-5, tetrabromomethane, and tribromomethane (6.60 g). The
mixture was used for the next step without further purification. (S)-5
was obtained from (R)-4 by the same manner as described above.
(S)-1,2-Propanedithiol, Diacetate (6). K2CO3 (1.52 g, 11.0 mmol)
was added to a solution of (R)-5 and thioacetic acid (1.67 g, 22.0
mmol) in dimethylformamide (DMF) (10 mL) at 0 °C. The mixture
was stirred for 2 h at room temperature. The reaction mixture was
then extracted with diethyl ether. The extracts were washed with
water and dried (Na2SO4), and then the solvent was removed in
vacuo. The residue was purified on a silica gel column using n-hexane/
AcOEt (9/1, v/v) to yield (S)-6 (810 mg, 4.21 mmol). (R)-6 was
Synthesis of 2,5-Dimethyl-1,4-dithiane (2). Compound 2 was
synthesized according to the method of Iranpoor and Owji.22 1H
NMR for (2S,5S)-2 and (2R,5R)-2 (400 MHz, CDCl3): δ 1.41 (d,
6H, J = 6.8 Hz), 2.74−2.82 (m, 2H), 2.94−3.03 (m, 4H); MS-EI m/z
(intensity in relative %): 41 (19), 45 (33), 59 (24), 60 (88), 74 (26),
1
75 (50), 88 (8), 106 (42), 133 (5), 148 (100; M+). H NMR for
meso-trans-2 (400 MHz, CDCl3): δ 1.21 (d, 6H, J = 6.8 Hz), 2.54−
2.70 (m, 4H), 3.12−3.22 (m, 2H); MS-EI m/z (intensity in relative
%): 41 (17), 45 (18), 59 (20), 60 (68), 74 (22), 75 (35), 106 (38),
133 (6), 148 (100; M+). 13C NMR analysis was not conducted
because of the limited amount of sample.
Synthesis of 2,6-Dimethyl-1,4-dithiane (3). A solution of
compound 7 (11 mg, 0.10 mmol) in DMF (20 mL) was added
dropwise for 30 min with stirring to a solution of compound 5 (20
mg, 0.10 mmol) and K2CO3 (14 mg, 0.20 mmol) in DMF (30 mL).
The mixture was stirred for 30 min in a N2 atmosphere, and then
dissolved in dichloromethane and washed with water. The extracts
were dried (Na2SO4), and the solvent was removed in vacuo. The
residue was purified on a silica gel column using n-pentane/Et2O (49/
1, v/v) followed by preparative GC. meso-trans-2 and meso-cis-3 were
obtained by the reaction of (S)-5 and (S)-7 under the same reaction
conditions used for the synthesis of racemic compound 3. meso-trans-
2 and meso-cis-3 were separated by preparative GC on a DB-5 column
(30 m × 0.53 mm i.d., 1 μm film thickness, J&W Scientific). (2R,5R)-
2 and (2R,6R)-3 were obtained by the reaction of (S)-5 and (R)-7
under the same reaction conditions. (2S,5S)-2 and (2S,6S)-3 were
obtained by the reaction of (R)-5 and (S)-7 by the same manner. cis-2
((2S,5S)-2 and (2S,5S)-2) and trans-3 ((2S,6S)-3 and (2R,5R)-3)
were purified by preparative GC on a DB-5 column (60 m × 0.25 mm
i.d., 0.25 μm film thickness, J&W Scientific). The enantiomeric
excesses of (2R,5R)-2, (2S,5S)-2, (2S,6S)-3, and (2R,6R)-3 were
determined to be >95% by GC analysis using a β-DEX 225 column
(30 m × 0.25 mm i.d., 0.25 μm film thickness). 1H NMR and MS of
1
obtained from (S)-5 by the same manner as described above. H
1
NMR of (S)-6 and (R)-6 were identical to that of the racemate. H
NMR for 6 (400 MHz, CDCl3): δ 1.32 (d, 3H, J = 7.2 Hz), 2.30 (s,
3H), 2.34 (s, 3H), 3.10−3.20 (m, 2H), 3.65−3.75 (m, 1H). 13C NMR
(100 MHz, CDCl3): δ 19.6, 30.5, 30.6, 35.0, 39.2, 194.9, 195.1.
(S)-1,2-Propanedithiol (7). (S)-6 (192 mg, 1.00 mmol) was
dissolved in anhydrous diethyl ether (5 mL) and added dropwise to a
suspension of lithium aluminum hydride (75.9 mg, 2.00 mmol) in
anhydrous diethyl ether (1 mL) at 0 °C. The mixture was stirred for 3
h at room temperature. Lithium aluminum hydride was quenched by
the slow addition of water and 15% NaOH aqueous solution at 0 °C.
The reaction mixture was filtered and neutralized with hydrochloric
acid. The solution was extracted with n-pentane and washed with
water. The extracts were dried (Na2SO4), and the solvent was
removed in vacuo. The residue was purified on a silica gel column
using n-pentane/Et2O (49/1, v/v) to yield (S)-7 (91.1 mg, 0.842
mmol). (R)-7 was obtained from (R)-6 by the same manner as
described above. 1H NMR and MS of (S)-7 and (R)-7 were identical
1
(2S,6S)-3 and (2R,5R)-3 were identical to those of the racemate. H
1
NMR for meso-cis-3 (400 MHz, CDCl3): δ 1.21 (d, 6H, J = 6.8 Hz),
2.70−2.91 (m, 4H), 2.95−3.07 (m, 2H); MS-EI m/z (intensity in
relative %): 41 (15), 45 (17), 59 (18), 60 (55), 74 (17), 75 (18), 88
(4), 106 (56), 133 (3), 148 (100; M+).1H NMR for (2S,6S)-3 and
(2R,5R)-3 (400 MHz, CDCl3): δ 1.40 (d, 6H, J = 6.8 Hz), 2.60 (dd,
2H, J = 7.2 and 13.6 Hz), 2.96 (dd, 2H, J = 2.4 and 13.6 Hz), 3.13−
3.22 (m, 2H); MS-EI m/z (intensity in relative %): 41 (18), 45 (19),
59 (24), 60 (63), 74 (18), 75 (23), 106 (54), 133 (3), 148 (100; M+).
13C NMR analysis was not conducted because of the limited amount
of sample.
Purification and Isolation of the Acidic Odorants of AGE.
AGE (500 mL) was diluted with 2.5 L of purified water, and the
solution was passed through a column packed with 12 g of Porapak Q
(GL Sciences, Tokyo, Japan), a ethylvinylbenzene−divinylbenzene
copolymer, to adsorb the organic compounds. After the column was
washed with water, the organic compounds adsorbed on the polymer
were eluted with dichloromethane (200 mL). The organic
compounds of AGE were collected from a total of 2000 mL of
AGE. The extracts in 800 mL of dichloromethane were fractionated
by solvent-assisted flavor evaporation (SAFE) without concentration
to collect relatively high volatile compounds from the extract matrix.23
The collected volatile compounds were dried (Na2SO4) and
concentrated to 1 mL using a Vigreux column (150 mm × 6 mm
i.d., Aldrich, Germany). The volatile compounds were then dissolved
in n-hexane and applied onto a silica gel column (8 g). The n-
pentane/Et2O solvent system, which exhibits a very low boiling point,
to those of the racemate. H NMR for 7 (400 MHz, CDCl3): δ 1.41
(d, 3H, J = 7.2 Hz), 1.63 (dd, 1H, J = 8.6 and 8.6 Hz), 1.82 (d, 1H, J
= 6.8 Hz), 2.69−2.74 (m, 2H), 2.99−3.10 (m, 1H); MS-EI m/z
(intensity in relative %): 39 (27), 41 (62), 45 (28), 47 (40), 59 (23),
60 (23), 61 (100), 74 (47), 75 (20), 108 (52; M+). 13C NMR (100
MHz, CDCl3): δ 23.3, 35.5, 38.4.
(2R,4S)- and (2S,4S)-2,4-Dimethyl-1,3-dithiolane (1). Acetalde-
hyde (22 mg, 0.50 mmol) was added to a solution of (S)-7 (55 mg,
0.50 mmol) and boron trifluoride diethyl ether complex (50 mg) in
dichloromethane (2 mL). The mixture was stirred for 0.5 h at room
temperature and then heated to 40 °C for 1 h. The reaction mixture
was cooled and subsequently dissolved in dichloromethane. The
extracts were washed with water and then dried (Na2SO4), and the
solvent was removed in vacuo. The residue was purified on a silica gel
column using n-pentane/Et2O (49/1, v/v) to yield compound 1 (35
mg, 0.26 mmol). (2R,4S)-1 and (2S,4S)-1 were purified and isolated
by preparative GC on an InertCap Pure WAX column (30 m × 0.53
mm i.d., 1 μm film thickness, GL Sciences, Tokyo, Japan). The
enantiomeric excesses of isolated (2R,4S)-1 and (2S,4S)-1 were
determined to be 75 and 74%, respectively, by GC analysis using a β-
DEX 225 column [30 m × 0.25 mm i.d., 0.25 μm film thickness,
(Merck, Darmstadt, Germany)]. 1H NMR, 13C NMR and MS of
1
(2R,4S)-1 and (2S,4R)-1 were identical to those of the racemate. H
NMR for (2R,4S)-1 and (2S,4R)-1 (400 MHz, CDCl3): δ 1.45 (d,
3H, J = 6.4 Hz), 1.64 (d, 3H, J = 6.6 Hz), 2.97 (dd, 1H, J = 7.8 and
11.5 Hz), 3.24 (dd, 1H, J = 4.8 and 11.5 Hz), 3.74−3.84 (m, 1H),
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was used as the eluent to prevent the loss of highly volatile
compounds. The column was eluted with 30 mL of n-pentane/Et2O
(97/3, v/v, Fr. 1), followed by 30 mL of n-pentane/Et2O (97/3, v/v,
Fr. 2), 30 mL of n-pentane/Et2O (97/3, v/v, Fr. 3), 50 mL of n-
pentane/Et2O (90/10, v/v, Fr. 4), 50 mL of n-pentane/Et2O (70/30,
v/v, Fr. 5), 50 mL of n-pentane/Et2O (50/50, v/v, Fr. 6), and 50 mL
of Et2O (Fr. 7). The fractions were subjected to GC−olfactometry
(GC−O) analysis. The acidity aromas were isolated by preparative
GC on an InertCap Pure WAX column (30 m × 0.53 mm i.d., 1 μm
film thickness).
Preparative GC. Preparative GC was performed using an Agilent
7890 A gas chromatograph (Agilent Technology, CA, USA) equipped
with collection traps (VPS 2800, GL Sciences). The column effluent
was split 5:95 to the flame ionization detector (FID, 230 °C) and the
collection trap (−15 °C).
For a fine separation and purification to prepare authentic
specimens at high purity for sensory analysis, another preparative
GC system equipped with a different collection system was used, as
described by Nojima et al.24 Short megabore capillary columns (DB-5,
20 cm, 1 μm film thickness) were used as traps for the collection
system. As it is not necessary to cool the traps during the collection
process, the same trap can be used for successive collections of the
same GC fraction during sequential injections.24 The traps were
extracted with 100 μL of dichloromethane to recover the volatile
compounds.
Preparative GC Condition for 2,4-Dimethyl-1,3-dithiolane.
An Inert cap Pure WAX column (30 m × 0.53 mm i.d., 1 μm film
thickness) was used. The injector temperature was 250 °C and the
samples (3.0 μL) were injected by the splitless mode. Helium was
used as the carrier gas at a flow rate of 6.0 mL/min. The oven
temperature was held at 40 °C for 2 min for the preparation of AGE
volatiles, then increased at a rate of 6°C/min to 230 °C, and further
held at 230 °C for 5 min. The oven temperature was held at 60 °C for
2 min for the preparation of compound 1, then increased at a rate of
1.5°C/min to 100 °C, further increased at a rate of 12°C/min to 230
°C, and then held at 230 °C for 5 min.
Preparative GC Condition for trans-2,5-Dimethyl-1,4-
dithiane and cis-2,6-Dimethyl-1,4-dithiane. A DB-5 column
(30 m × 0.53 mm i.d., 1 μm film thickness) was used. The injector
temperature was 250 °C, and the samples (3.0 μL) were injected by
the splitless mode. Helium was used as the carrier gas at a flow rate of
6.0 mL/min. The oven temperature was held at 60 °C for 2 min,
increased at a rate of 6°C/min to 150 °C, then further increased at a
rate of 12°C/min to 230 °C, and held at 230 °C for 5 min.
Preparative GC Condition for cis-2,5-Dimethyl-1,4-dithiane
and trans-2,6-Dimethyl-1,4-dithiane. A DB-5 column (60 m ×
0.25 mm i.d., 0.25 μm film thickness) was used. The injector
temperature was 250 °C, and the samples (1.0 μL) were injected by
the split mode (10:1). The oven temperature was held at 65 °C for 60
min, then increased at a rate of 12°C/min to 230 °C, and held at 230
°C for 5 min. Helium was used as the carrier gas at a flow rate of 1.6
mL/min.
GC−O Analysis. The odor qualities of each odorant were
evaluated using GC−O by at least 5 trained panelists at
concentrations near each threshold level. GC−O analysis was
performed using an Agilent 7890 A gas chromatograph (Agilent
Technology, CA, USA) equipped with an olfactory port (OP275, GL
Science). A β-DEX 225 column (30 m × 0.25 mm i.d.), 0.25 μm film
thickness, (Merck, Darmstadt, Germany) was used. The samples (1.0
μL) were injected by the split mode (10:1). The column temperature
was held at 85 °C for 27 min [except for (2R,4S)-1 and (2S,4R)-1],
then increased at a rate of 8 °C/min to 220 °C, and held at 220 °C for
5 min. For (2R,4S)-1 and (2S,4R)-1, the column temperature was
held at 75 °C for 35 min, then increased at a rate of 8 °C/min to 220
°C, and held at 220 °C for 5 min. Helium was used as the carrier gas
at a flow rate of 1.6 mL/min. The flow split ratio between the FID
and the olfactory port was 1:1. The FID and the sniffing port were
held at 250 °C.
energy for electron impact was 70 eV. The mass scan range was m/z
33−450. The oven temperature was held at 40 °C for 2 min, increased
at a rate of 6 °C/min to 230 °C, and then held at 230 °C for 5 min.
Selected ion monitoring (SIM) mode was used for quantitative
analysis of odorants. 1-Octanol was used an internal standard (IS).
The IS was spiked into 100 mL of AGE samples at a concentration of
0.106 mg/L and then subjected to purification steps. The
concentrations of the odorants were determined by calibration curves
of IS and each odorant. Preliminary purification of odorants was
performed using a Porapak Q column and SAFE. We monitored the
following ions by SIM (m/z): 1 (134, 119); 2 (148, 106); 3 (148,
106); and 1-octanol (IS, 84, 70). Linear retention indices (RIs) of
each odorant were calculated by GC−MS analysis by using n-alkanes
C6−C26 on a DB-WAX column (60 m × 0.25 mm i.d., 0.25 μm film
thickness) and C6−C18 on a DB-5 column (60 m × 0.25 mm i.d.,
0.25 μm film thickness).
1
Nuclear Magnetic Resonance (NMR) Spectroscopy. H, 13C
NMR and 1H−1H correlation spectroscopy (1H−1H COSY)
experiments were performed using an Agilent 400-MR (Agilent
Technologies, Tokyo, Japan). CDCl3 was used as the solvent. The
chemical shifts were referenced to tetramethylsilane.
Sensory Analysis. Sensory analysis was performed by 20 trained
panelists. The sensory attributes and aroma references were used to
define the following aromas: acid (acetic acid), cabbage (dimethyl
sulfide), cooked potato (methional), pungent (sliced fresh garlic),
seasoning (3-hydroxy-4,5-dimethyl-2(5H)-furanone), caramel (mal-
tol), metallic (uncured ham), and fatty (bacon). Panelists were
trained to share common sensory attributes and intensities using
aroma reference solutions before sensory analysis. The intensities
were scored from 0 (no odor) to 2.0 (strong odor) in increments of
0.5 (0, 0.5, 1.0, 1.5, and 2.0).
Odor thresholds in water were determined by the triangular test
according to Czerny et al.25 Two 45 mL glass containers containing
purified water (20 mL) were used as blank samples. One vessel
contained aqueous solution of odorants. The solution was diluted
stepwise 1:2 with water. The samples were tested by 20 trained
panelists. The detection odor threshold of each odorant was
calculated according to Czerny et al.25 Odor activity values (OAVs)
were calculated by dividing the concentrations by the odor thresholds
of the compounds in water.
Statistical Analysis. Sensory test results were analyzed by analysis
of variance (ANOVA) followed by Tukey−Kramer multiple
comparisons test using JMP 11 software (SAS Institute Inc., Cary,
NC, USA).
RESULTS AND DISCUSSION
■
Isolation and Specification of the Acidic Note of AGE.
Volatile compounds of AGE were collected and partially
purified by SAFE, and then separated into 7 fractions (Fr. 1−
7) by silica gel column chromatography (Figure 1). Significant
acidic and sulfury odors were found in Fr. 3 by the sensory
test; this fraction was then subjected to GC−O analysis. Three
acidic aromas and one strong sulfury aroma were found in the
fraction at RIs of 1422, 1542, 1599, and 1430 on a DB-WAX
column, respectively. One of these (RI = 1542) was specified
in our previous study as an important key aroma in AGE.14
The RIs of the odors are reported in Table 1. The mass spectra
of all 4 odorants clearly show sulfur isotopic patterns, but their
fragmentation patterns were not found in the databases (NIST
08 and Wiley W9N08). Their molecular ions were tentatively
determined as follows: RI 1422, m/z 134; RI 1430, m/z 134;
RI 1542, m/z 148; and RI 1599, m/z 148, respectively. The
compounds were further purified and isolated using prepara-
tive GC for NMR analysis.
Identification of 2,4-Dimethyl-1,3-dithiolane. The GC
peaks at RIs of 1422 and 1430 on a DB-WAX column were
collected in the same fraction by preparative GC (Fr. 3−1) and
GC−MS Analysis. An Agilent 7890 A gas chromatograph
equipped with a 5975 C mass spectrometer was used. The ion
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these isomers were completely separated on a β-DEX 225
column. The GC peaks with retention times of 28.3 and 29.0
min for cis-1 were determined to be (2S,4R)-1 and (2R,4S)-1,
respectively. For trans-1, retention times of 16.7 and 17.2 min
were determined to be (2S,4S)-1 and (2R,4R)-1, respectively.
AGE contains enantiomers of both diastereomers at a ratio of
about 50:50. To the best of our knowledge, this is the first time
these compounds were identified in food products.
The concentrations of cis-1 and trans-1 were found to be
1.27 and 0.78 mg/L in AGE, respectively (Table 1). These
odorants were not detected in fresh garlic (data not shown). In
our preliminary experiment, compound 1 was shown to be
generated by simply mixing and heating acetaldehyde,
hydrogen sulfide and allyl mercaptan in aqueous ethanol;
these starting materials are easily generated from ethanol and
allicin.27,28 Thus, it appears that these compounds are
generated by a similar process during aging.
Characterization of the Aroma of 2,4-Dimethyl-1,3-
dithiolane. The odor qualities of each stereoisomer were
evaluated by GC−O (Table 2). For cis-1, the odor quality of
(2R,4S)-1 was described as acid and malty, whereas that of
(2S,4R)-1 was described as garlic and gasoline. For trans-1,
(2S,4S)-1 was described as sulfur and sweet, whereas (2R,4R)-
1 was described as garlic and green. These results suggest that
the odor qualities depend on the stereochemistry of compound
1.
Odor thresholds of compound 1 were determined using
synthetic racemic mixtures because the threshold test needs a
relatively large amount and high purity of compound, which is
not easy to prepare. The thresholds of cis-1 and trans-1 were
determined as 14.7 and 5.7 μg/L, respectively (Table 1).
OAVs of cis-1 and trans-1 in AGE were 86 and 131,
respectively (Table 1). The OAV of trans-1 was the 9th
highest in AGE.
Identification of 2,5- and 2,6-Dimethyl-1,4-dithiane.
The GC peaks at RIs of 1542 and 1599 on a DB-WAX column
were collected by preparative GC (Fr. 3−2 and Fr. 3−3,
respectively; Figure 1) and subjected to further analysis by
GC−O and GC−MS using a different polarity column. Two
peaks were detected for each fraction (Fr. 3−2 and Fr. 3−3) by
GC analysis using a DB-5 column, indicating these were
coeluted on a DB-WAX column. All 4 peaks from the two
fractions exhibited strong odors by GC−O analysis using a
Figure 1. Isolation scheme for acidic odor components from aged
garlic extract (AGE). 1: 2,4-dimethyl-1,3-dithiolane, 2: 2,5-dimethyl-
1,4-dithiane, and 3: 2,6-dimethyl-1,4-dithiane.
subjected to further analysis by GC−O, GC−MS (using a
different polarity column), and NMR. Both peaks also
exhibited strong odors as confirmed by GC−O analysis using
a DB-5 column. The mass spectra showed similar patterns
(Figures S1 and S2), suggesting that the compounds were
related to each other. Fr. 3−1 was subjected to NMR analysis
without further purification (Figures S3 and S4). The 1H NMR
spectrum of Fr. 3−1, that is, a mixture of two compounds, was
easily assigned to each of the two individual compounds based
on the 1H−1H COSY results and integration results. Based on
the NMR and MS results of the mixture, these odorants were
tentatively identified as a mixture of diastereomers of
compound 1. Compound 1 was synthesized, and the structures
of the odorants eluted at RI 1422 and 1430 on a WAX column
were found to be cis- and trans-2,4-dimethyl-1,3-dithiolane,
respectively. The identities were subsequently confirmed by
1
matching their mass spectra, RIs, H NMR spectra, and odor
qualities with those of the synthesized authentic compounds.
The position of the cis−trans isomer was determined by
comparing the NMR data to that in previous report.26
Compound 1 exhibits two asymmetric centers, and thus, has
four stereoisomers. All four isomers were synthesized to
determine the absolute configuration of 1 (Figure 2). Each of
Table 1. RIs, Concentrations, Odor Thresholds, and OAVs of 2,4-Dimethyl-1,3-dithiolane, 2,5-Dimethyl-1,4-dithiane, and 2,6-
Dimethyl-1,4-dithiane and Their Respective cis−trans Isomers in AGE
RI
concentration
in AGE
(mg/L)
odor
threshold in
water (μg/L)
quantified
ion
(m/z)
calibration
range
(mg/L)
a
d
e
f
f
correlation
coefficient
LOD
LOQ
odorant
DB-5 DB-WAX
OAV
(μg/L) (μg/L)
b
cis-2,4-dimethyl-1,3-dithiolane
1034
1036
1163
1422
1430
1599
1.27 0.036 14.7
0.78 0.036 5.7
0.14 0.021 not
86
134
134
106
0.9998
0.9999
0.9970
0.28−2.27
0.32−2.57
0.024−0.24
2.1
2.5
4.6
6.4
7.5
b
trans-2,4-dimethyl-1,3-dithiolane
139
b
cis-2,5-dimethyl-1,4-dithiane
not
determined
647
13.7
c
c
c
c
determined
trans-2,5-dimethyl-1,4-dithiane
cis-2,6-dimethyl-1,4-dithiane
1138
1133
1162
1542
1542
1599
0.11 0.008 0.17
0.17 0.000 9.9
0.36 0.031 not
106
106
106
1.0000
0.9998
0.9996
0.027−0.27
0.063−0.63
0.060−0.60
6.0
4.7
4.4
18.0
14.0
13.3
17
b
trans-2,6-dimethyl-1,4-dithiane
not
determined
determined
a
b
Mean values of triplicate measurements with standard deviations. Concentration, odor threshold, and OAV were determined as a racemic
c
d
e
mixture. Preparation of high purity specimens for the odor threshold test was difficult. Ions selected for quantitative analysis. Calibration range of
f
the corresponding concentration in aged garlic extract. The limits of detection were estimated as the concentrations of a standard that produced a
signal-to-noise ratio of 3. The limits of quantitation were estimated as the concentrations of a standard that produced a signal-to-noise ratio of 10.33
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Figure 2. Synthesis of stereoisomers of 2,4-dimethyl-1,3-dithiolane.
Table 2. Odor Qualities of 2,4-Dimethyl-1,3-dithiolane, 2,5-
Dimethyl-1,4-dithiane, and 2,6-Dimethyl-1,4-dithiane and
Their Respective Stereoisomers by GC-O Analysis
odorant
odor quality
garlic, gasoline
acid, malty
garlic, sulfury, sweet
garlic, green
sulfury, leather, gassy
peppery, citrus
green, metallic, acid, mushroom
rubber, fruity, green, woody, acid
sulfury, rubber, garlic
woody, seasoning, sulfury, rubber
(2S,4R)-2,4-dimethyl-1,3-dithiolane
(2R,4S)-2,4-dimethyl-1,3-dithiolane
(2S,4S)-2,4-dimethyl-1,3-dithiolane
(2R,4R)-2,4-dimethyl-1,3-dithiolane
(2S,5S)-2,5-dimethyl-1,4-dithiane
(2R,5R)-2,5-dimethyl-1,4-dithiane
meso-trans-2,5-dimethyl-1,4-dithiane
meso-cis-2,6-dimethyl-1,4-dithiane
(2S,6S)-2,6-dimethyl-1,4-dithiane
(2R,6R)-2,6-dimethyl-1,4-dithiane
Figure 3. Synthesis of stereoisomers of 2,5-dimethyl-1,4-dithiane, and
2,6-dimethyl-1,4-dithiane.
DB-5 column. The mass spectra of these odorants showed
similar patterns (Figures S5−S8), suggesting that they are
related compounds that exhibit similar chromatographic
behaviors on a silica gel column, as well as both polar and
nonpolar GC columns. Fr. 3−2 and Fr. 3−3 were then
subjected to NMR analysis without further purification
meat flavors30 and thermal treatment of the meat broths in the
presence of alkyl-mercaptopropanol.31 To date, the aroma
properties of compounds 2 and 3 have not been well studied.
The concentrations of cis-2, trans-2, cis-3 and trans-3 in AGE
were determined to be 0.14, 0.11, 0.17, and 0.36 mg/L,
respectively (Table 1). These odorants were not detected in
fresh garlic (data not shown). Yu et al. proposed that
compound 2 is the cyclization product of two allyl mercaptan
molecules.28 Although it seems likely that a similar reaction
occurred during the aging process, this remains to be clarified.
To better understand the generation pathway of the various
component compounds and the corresponding change in the
aroma profile of AGE, a systematic investigation of the aroma
profiles of AGE during the aging process is necessary and
should be the subject of future research.
Characterization of the Aroma of 2,5- and 2,6-
Dimethyl-1,4-dithiane. Each stereoisomer of compounds 2
and 3 yielded different odor qualities (Table 2). In particular,
trans-2 exhibits a unique and strong odor (green, metallic,
garlic, and mushroom) compared with the other isomers. The
odorant reported as the key aroma compound was determined
to be trans-2 upon comparison of retention times and odor
quality. The odor detection thresholds of trans-2 and cis-3 in
water were determined to be 0.17 and 9.9 μg/L, respectively
(Table 2). The odor of trans-2 was just as strong as the well-
known flavoring methional (odor threshold: 0.43 μg/L) and
dimethyl sulfide (odor threshold: 0.84 μg/L).25 The quantities
of pure cis-2 and trans-3 were too small to determine odor
thresholds. The OAV of trans-2 was determined to be 647, the
fifth highest in AGE. Kubec et al. reported the aroma
characters of an isomeric pair of compound 2, noting that
one isomer had an unpleasant odor, while the other had a
mushroom-like odor with a considerably lower threshold
value.32 The aroma character reported in the literature for the
later isomer is very similar to our evaluation results for trans-2.
1
(Figures S9−S12). The H NMR spectrum of Fr. 3−2 was
easily assigned to each of the individual compounds based on
the 1H−1H COSY results and integration results. Based on the
NMR and MS results of the mixture, the odorants in Fr. 3−2
were tentatively identified as stereoisomers of compound 2 or
1
3. The H NMR spectrum of Fr. 3−3 was also easily assigned
to each of the individual compounds, and the odorants in Fr.
3−3 were also tentatively identified as stereoisomers of
compound 2 or 3.
All stereoisomers of compounds 2 and 3 were synthesized
and the structures of the odorants in Fr. 3−2 were identified as
(2R,6S)-2,6-dimethyl-1,4-dithiane (meso-cis-3) and (2R,5S)-
2,5-dimethyl-1,4-dithiane (meso-trans-2) by matching their
1
mass spectra, RIs, H NMR spectra, and odor qualities with
those of synthesized authentic compounds. The odorants in Fr.
3−3 were identified as cis-2 ((2S,5S)-2 or (2R,5R)-2), and
trans-3 ((2R,6R)-3 or (2S,6S)-3), without absolute config-
uration.
All isomers of cis-2 and trans-3 were synthesized to
determine their absolute configurations (Figure 3). The two
enantiomers of each of cis-2 and trans-3 were completely
separated on a β-DEX 225 column. For cis-2, the GC peaks
with retention times of 22.4 and 23.2 min were determined to
be (2S,5S)-2 and (2R,5R)-2, respectively. For trans-3, the GC
peaks with retention times of 21.8 and 22.3 min were
determined to be (2R,6R)-3 and (2S,6S)-3, respectively. AGE
contains enantiomers of both 2 and 3 at a ratio of about 50:50.
Yu et al. reported that compound 2 was tentatively identified in
baked blanched garlic, fried blanched garlic, and thermally
degraded alliin.28,29 Compound 3 was identified in simulated
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Journal of Agricultural and Food Chemistry
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Article
1H NMR, 1H−1H COSY and mass spectra of
compounds 1, 2, and 3 in AGE; mass spectrum of cis-
2,4-dimethyl-1,3-dithiolane in AGE; mass spectrum of
trans-2,4-dimethyl-1,3-dithiolane in AGE; 1H NMR
spectrum of the mixture of cis-2,4-dimethyl-1,3-dithio-
lane and trans-2,4-dimethyl-1,3-dithiolane in AGE;
1H−1H COSY spectrum of the isolated mixture of cis-
2,4-dimethyl-1,3-dithiolane and trans-2,4-dimethyl-1,3-
dithiolane in AGE; mass spectrum of cis-2,5-dimethyl-
1,4-dithiane in AGE; mass spectrum of trans-2,5-
dimethyl-1,4-dithiane in AGE; mass spectrum of cis-
2,6-dimethyl-1,4-dithiane in AGE; mass spectrum of
trans-2,6-dimethyl-1,4-dithiane in AGE; 1H NMR
spectrum of the mixture of trans-2,5-dimethyl-1,4-
dithiane and cis-2,6-dimethyl-1,4-dithiane in AGE;
1H−1H COSY spectrum of the isolated mixture of
trans-2,5-dimethyl-1,4-dithiane and cis-2,6-dimethyl-1,4-
While the configurations of compound 2 were not determined
in the previous report, we herein succeeded in determining the
absolute configurations.
Sensory Analysis of Aroma Recombinant. The aroma
profiles of recombinant samples were evaluated by sensory
analysis to determine the contributions of compounds 1, 2 and
3 to the acidic note of AGE. The following two aroma
recombinates were evaluated: (i) 25 compounds in aqueous
ethanol solution at their actual AGE concentrations reported in
a previous report14 and (ii) a blend of the 25 compounds and
compounds 1, 2, and 3 at their actual AGE concentrations.
The average scores of 20 panelists are shown in Figure 4.
1
dithiane in AGE; H NMR spectrum of the mixture of
cis-2,5-dimethyl-1,4-dithiane and trans-2,6-dimethyl-1,4-
1
dithiane in AGE; and H−1H COSY spectrum of the
isolated mixture of cis-2,5-dimethyl-1,4-dithiane and
trans-2,6-dimethyl-1,4-dithiane in AGE (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Satoshi Nojima − Laboratory of Aroma Chemistry,
Department of Food and Cosmetic Science, Faculty of
Bioindustry, Tokyo University of Agriculture, Abashiri City,
Hokkaido 099-2493, Japan; orcid.org/0000-0002-6055-
5994; Phone: +81-152-48-3820; Email: sn207003@
nodai.ac.jp
Figure 4. Aroma profiles of AGE and aroma recombinate of AGE. 1:
2,4-dimethyl-1,3-dithiolane, 2: 2,5-dimethyl-1,4-dithiane, and 3: 2,6-
dimethyl-1,4-dithiane. Each of the attributes denoted with different
letters indicate significant differences (ANOVA, Tukey’s post-hoc test,
p < 0.05).
Authors
ANOVA showed significant differences for the scores for “acid”
(p < 0.05) and no significant differences for that of other
sensory attributes. The score for “acid” of the aroma
recombinant without compounds 1, 2, and 3 (0.33 points)
was less intense (p < 0.05) compared with that of original AGE
(0.78 points). Addition of compounds 1, 2, and 3 resulted in
good agreement in the intensity of “acid” (0.65 points) with
that of original AGE. A blend of the 25 compounds and
compounds 1, 2, and 3 in aqueous ethanol was able to simulate
the overall aroma of AGE, including the other 7 descriptors.
In conclusion, we further investigated the aroma of AGE
using various analytical techniques, such as GC−O, accurate
and high-performance preparative GC system, GC−MS
analysis, and sensory evaluation, in an effort to better
understand the aroma of AGE. 2,4-Dimethyl-1,3-dithiolane
(1), 2,5-dimethyl-1,4-dithiane (2), and 2,6-dimethyl-1,4-
dithiane (3) and their respective stereoisomers were identified
as key aroma components of the acidic note of AGE. Our
studies revealed the chemical composition of the aroma of
AGE, and we hope this finding leads researchers to develop a
method and process to further reduce the pungent smell of
fresh garlic. In addition, we also hope these newly identified
sulfur heterocyclic odorants, which exhibit characteristic
flavors, lead to the development of new flavorings and
pharmacological compounds.
Kazuki Abe − Laboratory of Aroma Chemistry, Department of
Food and Cosmetic Science, Faculty of Bioindustry, Tokyo
University of Agriculture, Abashiri City, Hokkaido 099-2493,
Japan; Healthcare Research and Development Division,
Wakunaga Pharmaceutical Company Ltd., Akitakata,
Hiroshima 739-1195, Japan
Takao Myoda − Laboratory of Aroma Chemistry, Department
of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo
University of Agriculture, Abashiri City, Hokkaido 099-2493,
Japan
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.0c06634
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Yoji Hori for his helpful advice. We also thank
JAM Post (http://www.jamp.com/) for English language
editing.
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