Characterization of the aroma-active compounds in pink guava (Psidium guajava, L.) by application of the aroma extract dilution analysis

Article abstract of DOI:10.1021/jf8005245

The volatiles present in fresh, pink-fleshed Colombian guavas (Psidium guajava, L.), variety regional rojo, were carefully isolated by solvent extraction followed by solvent-assisted flavor evaporation, and the aroma-active areas in the gas chromatogram were screened by application of the aroma extract dilution analysis. The results of the identification experiments in combination with the FD factors revealed 4-methoxy-2,5-dimethyl-3(2H)-furanone, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, 3-sulfanylhexyl acetate, and 3-sulfanyl-1-hexanol followed by 3-hydroxy-4,5-dimethyl-2(5H)-furanone, (Z)-3-hexenal, trans-4,5-epoxy-(E)-2-decenal, cinnamyl alcohol, ethyl butanoate, hexanal, methional, and cinnamyl acetate as important aroma contributors. Enantioselective gas chromatography revealed an enantiomeric distribution close to the racemate in 3-sulfanylhexyl acetate as well as in 3-sulfanyl-1-hexanol. In addition, two fruity smelling diastereomeric methyl 2-hydroxy-3- methylpentanoates were identified as the (R,S)- and the (S,S)-isomers, whereas the (S,R)- and (R,R)-isomers were absent. Seven odorants were identified for the first time in guavas, among them 3-sulfanylhexyl acetate, 3-sulfanyl-1-hexanol, 3-hydroxy-4,5-dimethyl-2(5H)-furanone, frans-4,5-epoxy-(E)-2-decenal, and methional were the most odor-active.

Full text of DOI:10.1021/jf8005245

4120 J. Agric. Food Chem. 2008, 56, 4120–4127
Characterization of the Aroma-Active Compounds in
Pink Guava (Psidium guajava, L.) by Application of
the Aroma Extract Dilution Analysis
†
‡
†
‡
MARTIN STEINHAUS, DIANA SINUCO, JOHANNES POLSTER, CORALIA OSORIO,
,
†
AND PETER SCHIEBERLE*
Deutsche Forschungsanstalt für Lebensmittelchemie, Lichtenbergstrasse 4,
D-85748 Garching, Germany, and Universidad Nacional de Colombia, Bogota, Colombia
The volatiles present in fresh, pink-fleshed Colombian guavas (Psidium guajava, L.), variety regional
rojo, were carefully isolated by solvent extraction followed by solvent-assisted flavor evaporation,
and the aroma-active areas in the gas chromatogram were screened by application of the aroma
extract dilution analysis. The results of the identification experiments in combination with the FD
factors revealed 4-methoxy-2,5-dimethyl-3(2H)-furanone, 4-hydroxy-2,5-dimethyl-3(2H)-furanone,
3-sulfanylhexyl acetate, and 3-sulfanyl-1-hexanol followed by 3-hydroxy-4,5-dimethyl-2(5H)-furanone,
(Z)-3-hexenal, trans-4,5-epoxy-(E)-2-decenal, cinnamyl alcohol, ethyl butanoate, hexanal, methional,
and cinnamyl acetate as important aroma contributors. Enantioselective gas chromatography revealed
an enantiomeric distribution close to the racemate in 3-sulfanylhexyl acetate as well as in 3-sulfanyl-
1-hexanol. In addition, two fruity smelling diastereomeric methyl 2-hydroxy-3-methylpentanoates were
identified as the (R,S)- and the (S,S)-isomers, whereas the (S,R)- and (R,R)-isomers were absent.
Seven odorants were identified for the first time in guavas, among them 3-sulfanylhexyl acetate,
3-sulfanyl-1-hexanol, 3-hydroxy-4,5-dimethyl-2(5H)-furanone, trans-4,5-epoxy-(E)-2-decenal, and
methional were the most odor-active.
KEYWORDS: Extract dilution analysis; 3-sulfanyl-1-hexanol; 3-sulfanylhexyl acetate; methyl (R,S)- and
(
S,S)-2-hydroxy-3-methylpentanoate
INTRODUCTION
green, grassy note with fruity and sweet attributes and a distinct
grapefruit-like aroma contributes to the characteristic tropical
fruit character. First attempts to identify the odorous principle
of guavas date back to the 1960s (1, 2). Since then numerous
studies on guava volatiles (3–21) have been performed, which
have led to the identification of ∼400 volatile compounds until
today (22). However, little attention has been paid so far to the
contribution of individual compounds to the typical aroma.
Because it is well-documented in the recent literature (23) that
only a limited share of the volatiles present in a food actually
contribute to a given aroma, closing this gap would be helpful,
for example, to assess aroma changes caused by industrial fruit
processing. Whether or not an individual volatile actually plays
a role in generating the overall food aroma depends on its
individual odor threshold and its concentration in the food. Since
the odor thresholds of food volatiles often cover several orders
of magnitude, minor compounds may be important odor
contributors, while major volatiles might be negligible.
The fruits of Psidium guajaVa, L., a tree native to tropical
America and a member of the myrtle family Myrtaceae, are
widely cultivated in different tropical and subtropical countries
with increasing popularity all over the world. Guava fruits are
spherical in shape, about 4 to 10 cm in diameter, and covered
by a leathery, green to yellowish skin (exocarp). Depending on
the variety, either a white or a pink fruit flesh (meso- and
endocarp) houses numerous small seeds.
The fruits are rich in pectin, carotenoids, vitamins, in
particular vitamin C, and dietary fiber and are consumed as
whole fruits, either fresh or cooked. In addition, a major part
of the harvest is processed into jam, sherbet, pastes, or puree,
which is a common ingredient in multifruit juices popular in
many Northern countries. The estimated guava production in
Colombia amounts to about 100 000 tons/year, a major part of
which is processed to a candy bar named “bocadillo”.
Guava fruits elicit a pleasant aroma in combination with a
sour-sweet taste. The characteristic aroma profile combines a
Only two investigations were aimed at clarifying the aroma
activity of individual guava constituents. McLeod and de
Troconis (6) applied GC-Olfactometry on an extract isolated
from Venezuelan guavas and detected 37 odor-active regions.
Chemical structures were tentatively assigned for 28 areas based
on GC-MS measurements. However, identifications were not
*
Corresponding author. Phone: +49 89 289 13265. Fax: +49 89
2
89 14183. E-mail: peter.schieberle@ch.tum.de.
†
Deutsche Forschungsanstalt für Lebensmittelchemie.
Universidad Nacional de Colombia.
‡
1
0.1021/jf8005245 CCC: $40.75  2008 American Chemical Society
Published on Web 05/14/2008

Key Aroma Compounds in Guava
J. Agric. Food Chem., Vol. 56, No. 11, 2008 4121
confirmed by using reference compounds and thus, unidentified
minor volatiles with high odor potencies may have coeluted
with major peaks in the chromatogram, thereby erroneous
correlations between odor perception and the mass spectrum
recorded may occur. Attempts to overcome this problem, e.g.,
by fractionation of the extract, were not performed, and also
no efforts were made to rank the detected odorants according
to their individual odor contribution. By applying GC-O on a
pink guava puree, Jord a´ n et al. (19) recently detected 46 odor-
active compounds, 40 of which could be characterized. These
authors tried to rank the importance of a single odorant on the
basis of the number of assessors detecting an odor during GC-
O. According to this approach, 23 volatiles were suggested to
be of major importance, because these were detected by all
assessors in all runs.
or D-allo-isoleucine, respectively, according to the procedure described
recently for the respective ethyl 2-hydroxy-3-methylpentanoates (27).
The following data were obtained:
Methyl (2R,3S)-2-Hydroxy-3-methylpentanoate. Yield: 52 mg )
4
5
1
0%. MS-EI: m/z (intensity in %): 90 (100), 87 (71), 45 (70), 41 (47),
7 (38), 69 (25), 43 (14), 58 (10), 39 (9). MS-CI: m/z (intensity in %)
+
47 (100; M + H ).
Methyl (2S,3S)-2-Hydroxy-3-methylpentanoate. Yield: 29 mg )
2%. MS-EI: m/z (intensity in %) 90 (100), 87 (62), 45 (55), 41 (44),
2
57 (41), 69 (23), 39 (8). MS-CI: m/z (intensity in %) 147 (100; M +
+
H ).
Methyl (2R,3R)-2-Hydroxy-3-methylpentanoate. Yield: 57 mg )
4
5
1
3%. MS-EI: m/z (intensity in %) 90 (100), 45 (62), 87 (59), 41 (44),
7 (36), 69 (20), 43 (14), 39 (9), 58 (9). MS-CI: m/z (intensity in %)
+
47 (100; M + H ).
Methyl (2S,3R)-2-Hydroxy-3-methylpentanoate. Yield: 41 mg )
1%. MS-EI: m/z (intensity in %): 90 (100), 87 (65), 45 (51), 57 (35),
3
4
To obtain more knowledge on the aroma contribution of
volatiles present in fresh guava, the aim of the present study
was to screen the odor-active compounds by application of the
aroma extract dilution analysis (AEDA) on an extract obtained
by careful solvent extraction and SAFE-distillation from fresh,
ripe Colombian pink-fleshed guavas, variety regional roja.
1 (34), 69 (21), 85 (8), 58 (7). MS-CI: m/z (intensity in %) 147 (100;
+
M + H ).
(R/S)-3-Sulfanylhexyl Acetate. The compound was prepared fol-
lowing closely a method previously described (28). To a solution of
racemic 3-sulfanyl-1-hexanol (20 mmol; 2.69 g) in dichloromethane
(
15 mL) was added acetyl chloride (50 mmol; 3.93 g) dissolved in
dichlormethane (15 mL) at 0 °C under argon atmosphere, and the
mixture was stirred for 2 h at room temperature. The solvent and the
excess of acetyl chloride were removed in vacuo, and the (R/S)-3-
sulfanylhexyl acetate formed was purified by distillation in vacuo.
Yield: 2.64 g ) 75%. MS-EI: m/z (intensity in %) 43 (100), 55
MATERIALS AND METHODS
Fruits. Fresh pink-fleshed guavas (Psidium guajaVa, L.), variety
regional roja, grown in Puente Nacional (Santander, Colombia) were
purchased at a local market in the full ripe state (skin greenish yellow)
and transported overnight to Germany by air freight.
Reference Odorants. The following compounds were purchased
from the commercial sources given in parentheses: numbers 1, 2, 4, 7,
(
(
49), 88 (45), 116 (45), 83 (38), 73 (33), 87 (25), 67 (22), 41 (21), 82
+
14). MS-CI: m/z (intensity in %) 117 (100; M + H - CH
3
COOH).
To separate both isomers in a preparative scale, the racemic mixture
was reacted with enantiomerically pure (1S,4R)-camphanic acid chloride
to yield two diastereoisomers (28). Separation was done by the
following procedure:
1
0-15, 17, 19, 20, 22, 23, 25-31 (Aldrich, Sigma-Aldrich Chemie,
Taufkirchen, Germany) and 5 and 18 (Alfa Aesar, Karlsruhe,
Germany).
The diastereomers of 3-{[(1S,4R)-camphanoyl]sulfanyl}hexyl ac-
etates (5.6 mmol; 2 g) were applied onto a water-cooled (12 °C) glass
column (60 × 3.5 cm i.d.) packed with a slurry of purified silica gel in
pentane. Elution was performed with pentane/diethyl ether (80:20; by
volume; 4 L). The eluate was collected in portions of 50 mL and
monitored for the analytes by TLC (silica gel 60 F254, 5 × 10 cm,
Merck, Darmstadt, Germany), using the solvent mixture pentane/diethyl
ether, 80:20, v/v. The target compounds were detected in fractions
Syntheses. Compounds 21 and 24 were synthesized by reacting either
(
E,E)-2,4-decadienal (Aldrich) or (E,E)-2,4-dodecadienal (Alfa Aesar)
with 3-chloroperbenzoic acid (Aldrich) as described in ref 24.
E)-3-Hexenal (3). To a solution of 1,1,1-tris(acetyloxy)-1,1-dihydro-
,2-benz-iodoxol-3-(1H)-one (Dess-Martin-periodinane; 1.3 mmol; 550
mg) in dichloromethane (5 mL) was added (25) (E)-3-hexenol (1 mmol;
(
1
1
1
00 mg) in dichloromethane (5 mL), then the mixture was stirred for
h at room temperature. After addition of diethyl ether (80 mL), the
3
9-72 (1900-3600 mL). These fractions were submitted to HPLC
solution was washed with an aqueous sodium thiosulfate solution (1
mol/L; 100 mL; saturated with sodium hydrogen carbonate), followed
by aqueous sodium hydrogencarbonate (0.5 mol/L; 100 mL), and finally
water (50 mL). The solution was dried over anhydrous sodium sulfate,
filtered, and, after addition of hexane (2 mL), concentrated to 2 mL
with use of a Vigreux column (50 × 1 cm i.d.). To isolate the target
compound, the concentrate was applied onto a water-cooled (12 °C)
glass column (25 × 1 cm i.d.) filled with a slurry of purified silica gel
analysis to determine the isomeric distribution. Fractions 45 ((R)-
derivative) and 69 ((S)-derivative), respectively, showed the highest
purities and were used to prepare the enantiopure thiols for the sensory
experiments.
Fractions 39-44 and 46-48 ((3R)-derivative) as well as 65-68 and
7
0-72 ((3S)-derivative) were combined and used for the preparation
of the {[(2S)-2-phenylpropanoyl]sulfanyl}hexyl acetates needed in the
NMR experiments.
(
9 g; silica 60, 0.040-0.063 mm, Merck, Darmstadt, Germany) in
(R)-3-Sulfanyl-1-hexanol and (S)-3-Sulfanyl-1-hexanol. The enan-
pentane (23). The aldehyde was eluted with pentane/diethyl ether (90:
0, by volume, 50 mL), and its concentration was determined by GC-
tiopure 3-{[(1S,4R)-camphanoyl]sulfanyl}hexyl acetates were treated
with lithium aluminum hydride (28) to obtain the free thiols. The
concentrations of the target compounds were determined by GC-FID,
using methyl octanoate as the internal standard. To take into account
the different detector responses, a correction factor was determined from
a mixture of known amounts of racemic 3-sulfanyl-1-hexanol and
methyl octanoate.
Yield: 7.46 mg of (3R)-3-sulfanyl-1-hexanol with an enantiomeric
purity of 99.8% and 6.78 mg of (3S)-3-sulfanyl-1-hexanol with an
enantiomeric purity of 99.7%. MS-EI: m/z (relative intensity in %) 55
1
FID, using (E)-2-hexenal as the internal standard.
Yield: 44 mg of (E)-3-hexenal ) 44%. MS-EI: m/z (intensity in %)
4
8
+
1 (100), 69 (44), 39 (27), 55 (24), 42 (15), 83 (12), 98 (12), 70 (12),
0 (11), 53 (10). MS-CI (isobutane): m/z (intensity in %) 99 (100; M
+
H ).
(
Z)-1,5-Octadien-3-one (6). (Z)-3-Hexenal (3 mmol; 294 mg) was
reacted with vinyl magnesium bromide (10 mmol) (26) and the resulting
Z)-1,5-octadien-3-ol was subsequently oxidized into the target com-
(
pound with use of Dess-Martin-periodinane (2.2 mmol, 933 mg) as
described above. The concentration of the target compound was
determined by using 1-octen-3-one as the internal standard.
(
(
(
100), 100 (64), 57 (57), 41 (49), 61 (43), 67 (35), 82 (34), 83 (28), 47
19), 73 (16), 88 (15), 39 (15), 43 (14), 134 (14, M ), 56 (13), 71
13), 69 (13), 45 (12); 42 (11), 59 (11).
+
Yield: 87 mg ) 35%. MS-EI: m/z (intensity in %) 55 (100), 41
(R)-3-Sulfanylhexyl Acetate and (S)-3-Sulfanylhexyl Acetate.
(
(
23), 39 (9), 95 (8), 109 (7), 67 (6), 69 (5). MS-CI (isobutane): m/z
Enantiopure 3-sulfanylhexyl acetates were prepared from (R)-3-sulfanyl-
1-hexanol (0.028 mmol; 3.73 mg) or (S)-3-sulfanyl-1-hexanol (0.025
mmol; 3.39 mg), respectively, as described above for the racemic
compound. The crude products were purified by column chromatog-
raphy, using a water-cooled (12 °C) glass column (15 × 2 cm i.d.)
+
intensity in %) 125 (100; M + H ).
Methyl 2-Hydroxy-3-methylpentanoates. The synthesis of the four
stereoisomers of methyl 2-hydroxy-3-methylpentanoate started from
the four chiral amino acids L-isoleucine, D-isoleucine, L-allo-isoleucine,

4122 J. Agric. Food Chem., Vol. 56, No. 11, 2008
Steinhaus et al.
filled with a slurry of purified silica gel in pentane. Elution was
performed with pentane/diethylether (90:10, by volume, 300 mL). The
eluate was collected in fractions of 50 mL, and the target compound
was obtained in fractions 3-4 (100-200 mL). The concentration was
determined by GC-FID, using methyl octanoate as the internal standard.
To take into account the different detector responses, a correction factor
was determined from a mixture of known amounts of racemic
Fractionation of Volatiles by Column Chromatography. Fraction
NBV, isolated as detailed above, was concentrated to 1 mL with use
of a Vigreux column (50 × 1 cm) and applied at 12 °C onto a water-
cooled glass column (25 × 1 cm i.d.) filled with a slurry of purified
silica gel (9 g) in pentane. Elution was performed with pentane (50
mL) followed by pentane/diethyl ether (99:1; v/v; 50 mL), pentane/
diethyl ether (90:10; v/v; 50 mL), pentane/diethyl ether (70:30; v/v;
50 mL), and finally diethyl ether (50 mL). The eluate was collected in
10 mL fractions. The odorants detected during AEDA were localized
in the fractions by GC-O, and mass spectra were recorded by GC-MS,
using capillaries DB-5 and FFAP, respectively.
Selective Enrichment of Thiols. Fraction NBV was concentrated
to 5 mL, and the thiols were isolated by affinity chromatography on
mercurated agarose gel as recently reported (31).
Gas Chromatography-Mass Spectrometry (GC-MS). Mass spec-
tra were recorded on a gas chromatograph 5890 series II (Hewlett-
Packard, Waldbronn, Germany) connected to a sector field mass
spectrometer MAT 95 S (Finnigan, Bremen, Germany). Mass spectra
in the electron ionization mode (MS-EI) were recorded at 70 eV
ionization energy, and mass spectra in the chemical ionization mode
3
-sulfanylhexyl acetate and methyl octanoate.
Yield: 2.30 mg of (3R)-3-sulfanylhexyl acetate ) 41% with an
enantiomeric purity of 100% and 2.71 mg of (3S)-3-sulfanylhexyl
acetate ) 53% with an enantiomeric purity of 99.5%.
1
The absolute configuration of the thiol group was derived from H
NMR data of the hydratropic acid thiol esters prepared by a reaction
with (R)-2-phenylpropanoyl chloride as described in ref 28. The chloride
was generated by a treatment of (R)-2-phenylpropanoic acid with oxalyl
chloride.
Isolation of the Volatiles. Whole fruits (100 g) were blended using
a commercial stainless steel blender. After 5 min, dichloromethane (300
mL) was added to the puree, and the mixture was cooled in an ice
bath. With continuous stirring and cooling, sodium sulfate (250 g) was
added in small portions. The mixture was filtered through defatted cotton
wool and the sodium sulfate/guava powder obtained was washed with
another portion of dichloromethane (200 mL). The combined organic
phases, exhibiting the characteristic aroma of guava fruits, were
submitted to SAFE distillation (29). The distillate obtained was extracted
three times with an aqueous sodium carbonate solution (0.5 mol/L;
total volume: 400 mL) to remove the acidic volatiles. The organic phase,
containing the neutral and basic volatiles (NBV), was dried over
anhydrous sodium sulfate and concentrated to 1 mL by distilling off
the solvent by means of a Vigreux column (50 × 1 cm). The combined
aqueous phases were washed with dichloromethane (50 mL), acidified
(
MS-CI) at 115 eV with isobutane as the reactant gas.
Ratio of 2-Methylbutanoic Acid to 3-Methylbutanoic Acid. The
ratio of the amounts of 2- and 3-methylbutanoic acid was determined
by GC-MS calculating the intensities of the fragments m/z 60
(
3-methylbutanoic acid) and m/z 74 (2-methylbutanoic acid). Seven
defined mixtures of 2- and 3-methylbutanoic acid (100:0; 90:10; 70:
0; 50:50; 30:70; 10:90; 0:100) were analyzed under the same
3
conditions and a calibration line was drawn plotting the intensity ratio
of m/z 60 over the sum of m/z 60 + m/z 74 against the percentage of
3
-methylbutanoic acid in the mixture.
Two-Dimensional Gas Chromatography-Mass Spectrometry
TD-GC-MS). The system consisted of a Combi PAL autosampler
(
(
pH 2) with hydrochloric acid (16%), and extracted with dichlo-
(
CTC Analytics, Zwingen, Switzerland), a Trace GC 2000 series,
romethane (total volume: 300 mL) to yield the fraction of acidic
volatiles (AV), which was also concentrated to 1 mL.
equipped with a cold on column injector and a moving column stream
switching system (Thermo Finnigan, Egelsbach, Germany), a heated
Gas Chromatography-FID and Gas Chromatography-Olfacto-
metry (GC-O). GC-FID and GC-O analyses were performed with a
gas chromatograph 8160 (Fisons Instruments, Mainz, Germany) with
helium as the carrier gas. Samples were applied by the cold-on-column
injection technique. Capillaries used were DB-5, DB-1701, and DB-
FFAP (each 30 m × 0.32 mm i.d., 0.25 µm film thickness, 70 kPa
head pressure) (J&W Scientific, Chromatographie-Handel M u¨ ller,
Fridolfing, Germany). Columns BGB-175 and BGB-176 (each 30 m
(
250 °C) transfer line to the cold trap (SGE, Darmstadt, Germany)
localized in the second GC (CP 3800, Varian, Darmstadt, Germany),
and an ion trap mass spectrometer (Saturn 2000, Varian). Spectra were
recorded either in the EI or in the CI mode with methanol as the
reactant. For the determination of the enantiomeric distribution of 16
and 18, the first oven was equipped with an FFAP column and the
second with a BGB-175 column. The determination of the enantiomeric
distribution in the methyl 2-hydroxy-3-methylpentanoates was per-
formed on a DB-5 column in the first and a BGB-176 column in the
second dimension. The start temperature was 40 °C in all cases.
Temperature gradients were 6 deg/min in the first and 2 deg/min in
the second dimension.
×
0.25 mm i.d., 0.25 µm film thickness, 140 kPa head pressure) were
obtained from BGB Analytik (Schlossb o¨ ckelheim, Germany).
For GC-O applications, the end of the capillaries were connected to
a deactivated Y-shaped glass splitter (Chromatographie Handel Mueller,
Fridolfing, Germany) dividing the effluent into two equal parts, which
were transferred via two deactivated fused silica capillaries (50 cm ×
Determination of Odor Thresholds. These were determined by
aroma extract dilution analysis of a mixture containing known amounts
of the target odorant and (E)-2-decenal as internal standard. Thresholds
were calculated from the FD factors determined by using the method
of Ullrich and Grosch (32) and a threshold of 2.7 ng/L for (E)-2-decenal
0
.25 mm) to a sniffing port and an FID, respectively. The sniffing port
consisted of a cylindrically shaped aluminum device (80 mm × 25
mm i.d.) with a beveled top and a central drill hole (2 mm) housing
the capillary. It was mounted on a detector base of the GC and heated
to 180 °C. The FID was operated at 250 °C with hydrogen (20 mL/
min) and air (200 mL/min). Nitrogen (30 mL/min) was used as the
makeup gas. The injection volume was 0.5 µL. During a GC-O run,
the nose of the panelist was placed closely above the top of the sniffing
port and the odor of the effluent was evaluated. If an odor was
recognized, the retention time was marked in the chromatogram, and
the odor quality was assigned. The panelists were trained on a “flavor
language” in several sessions, in which pure reference odorants were
evaluated. The GC-O analyses were performed by at least three
panelists.
(
33).
High-Performance Liquid Chromatography (HPLC). To deter-
mine the isomeric purity of (S)- and (R)-3-{[(1S,4R)-
camphanoyl]sulfanyl}hexyl acetate, an HPLC-System consisting of a
Jasco PU-1580 HPLC pump (Jasco, Grossumstadt, Germany), a
Nucleosil-100-5 C18 column (Macherey-Nagel, D u¨ ren, Germany), and
a Jasco UV-1575 UV/vis detector was used. The acetates were isolated
with hexane/ethyl acetate (90:10; by volume; 1 mL/min) as the eluent,
which was monitored at 254 nm.
Aroma Extract Dilution Analysis (AEDA). Fractions NBV and
AV were stepwise diluted to obtain dilutions of 1:1, 1:2, 1:4, 1:8, 1:16,
RESULTS AND DISCUSSION
Aroma-Active Compounds in Guavas. Guava fruits were
blended and mixed with anhydrous sodium sulfate to obtain a
dry powder, and the homogenate was extracted with dichlo-
romethane by vigorous stirring. The addition of sodium sulfate
was necessary to achieve a homogeneous mixture because, due
to the high pectin content (34), the fruit mash otherwise formed
an unextractable, gum-like matter.
..., 1:4096 of the original solutions (23, 30). Each dilution was submitted
to GC-O, using capillary FFAP. The oven temperature was programmed
as follows: 2 min at 40 °C, then at 6 deg/min to 190 °C and finally at
1
2 deg/min to 240 °C. The odor-active compounds were located in the
chromatograms, and each odorant detected was assigned an FD factor
representing the highest dilution in which the odorant was detectable.
The FD factors obtained by three panelists were averaged.

Key Aroma Compounds in Guava
J. Agric. Food Chem., Vol. 56, No. 11, 2008 4123
Table 1. Aroma-Active Compounds (FD g 8) Detected in a Solvent Extract Obtained from Fresh, Pink Guavas
earlier reported
as volatile
a
b
c
d
RI (FFAP)
d
RI (DB-5)
e
no.
odorant
odor quality
fruity
grassy
grassy
grassy
mushrooms
FD factor
constituent, ref no.
1
2
3
4
5
6
7
8
9
ethyl butanoate
hexanal
1028
1075
1130
1135
1289
1364
1443
1473
1493
1540
1591
1610
1615
1652
1658
1708
1726
1831
1906
1943
2006
2021
2023
2094
2138
2144
2188
2200
2284
2383
2555
802
802
800
796
978
987
903
992
256
256
8
512
8
8
256
32
16
8
2048
64
8
32
8
2048
32
2048
8
8
512
32
2048
32
8
128
64
4
3
7
7
7
(E)-3-hexenal
(Z)-3-hexenal
1-octen-3-one
f
f
(Z)-1,5-octadien-3-one
methional
geranium leaves
cooked potato
fruity
fruity
flowery, citrus
caramel
violet, floral
rancid, cheese
violet, floral
rancid, cheese
black currant
rancid, cheese
grapefruit
methyl (2R,3S)-2-hydroxy-3-methylpentanoate
methyl (2S,3S)-2-hydroxy-3-methylpentanoate
994
f
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
linalool
1103
1041
1096
817
1170
885
10
17
3
7
7
4-methoxy-2,5-dimethyl-3(2H)-furanone
methyl benzoate
butanoic acid
ethyl benzoate
2- and 3-methylbutanoic acid
g
9
3-sulfanylhexyl acetate
pentanoic acid
3-sulfanyl-1-hexanol
γ-octalactone
heptanoic acid
1249
918
9
1418
1257
1112
1383
1370
1081
1486
1468
1453
1495
1136
1335
1678
1261
coconut
10
7
rancid, cheese
metallic
coconut
f
trans-4,5-epoxy-(E)-2-decenal
f
γ-nonalactone
4-hydroxy-2,5-dimethyl-3(2H)-furanone
trans-4,5-epoxy-(E)-2-undecenal
γ-decalactone
cinnamyl acetate
δ-decalactone
3-hydroxy-4,5-dimethyl-2(5H)-furanone
cinnamyl alcohol
10
7
caramel
metallic
peach
floral, honey
coconut
f
7
3
11
f
seasoning-like
floral, honey
peach
1024
512
64
7
11
9
f
γ-dodecalactone
phenylacetic acid
f
honey
8
a
b
Odorants were consecutively numbered according to their retention time on the FFAP column. Odorants were identified by comparing their retention indices on the
c
FFAP and DB-5 column, their mass spectra obtained by GC-MS, and their odor characteristics with respective data of reference compounds. Odor quality as perceived
d
e
f
at the sniffing port during GC-O. RI ) retention index. FD factor ) flavor dilution factor. Unequivocal mass spectra could not be obtained in the guava extract,
identification was based on the resting criteria detailed in footnote b. 2- and 3-methylbutanoic acid were not separated on the GC-column used; mass spectral data
g
showed a mixture of both isomers and their ratio was determined from the intensities of characteristic mass fragments as 78% 2- and 22% 3-methylbutanoic acid.
To avoid odorant degradation and/or artifact formation, high
temperatures were avoided throughout the entire workup
procedure. In particular, addition of sodium sulfate to the
homogenate was done in small portions under ice-cooling to
compensate for the heat liberated by hydration. Extraction with
dichloromethane was performed at ambient temperature and
during SAFE distillation the temperature was always kept below
were further fractionated by column chromatography on silica
gel. In a parallel experiment, thiols were selectively enriched
by affinity chromatography on mercurated agarose gel (35).
In the FD factor range of 8 to 2048 a total of 31 aroma-
active compounds were identified. The highest FD factors (range
512-2048), were found for eight compounds, the structures of
which are displayed in Figure 1. The two compounds eliciting
a caramel-like odor note at the highest FD factor of 2048 were
identified as 4-methoxy-2,5-dimethyl-3(2H)-furanone (11) and
4-hydroxy-2,5-dimethyl-3(2H)-furanone (23). In addition, the
3-sulfanylhexyl acetate (16) with a black-currant-like aroma and
the grapefruit-like smelling 3-sulfanyl-1-hexanol (18) were
identified with high FD factors. Also the seasoning-like smelling
3-hydroxy-4,5-dimethyl-2(5H)-furanone (28), the green, grassy
(Z)-3-hexenal (4), the metallic trans-4,5-epoxy-(E)-2-decenal
(21), and the floral cinnamyl alcohol (28), followed by ethyl
butanoate (1), hexanal (2), methional (7), and cinnamyl acetate
(26) showed high odor activity. Among these compounds,
3-sulfanylhexyl acetate, 3-sulfanyl-1-hexanol, 3-hydroxy-4,5-
dimethyl-2(5H)-furanone, trans-4,5-epoxy-(E)-2-decenal, and
methional were identified for the first time in guavas.
4
0 °C. The distillate obtained represented the typical guava
flavor with its green, sweet, tropical-fruit and also grapefruit-
like notes.
The volatiles were separated into a fraction containing the
neutral and basic volatiles (NBV) and a fraction containing the
acidic volatiles (AV) by treatment with aqueous sodium
carbonate. Both fractions were concentrated at 40 °C with use
of a Vigreux column, and the aroma-active compounds in the
concentrates were screened by application of the aroma extract
dilution analysis. A total of 31 odor-active regions were detected
in both fractions, among which two areas with caramel-like
aromas (11 and 23), a grapefruit-like odor (18), and a black-
currant aroma quality (16) showed the highest flavor dilution
(
FD)-factors (Table 1). Structural assignments of the odorants
detected were performed by comparing retention indices, odor
qualities, and odor intensities with data available in an in-house
database consisting of nearly 1000 compounds previously
identified as odorants in foods. On the basis of the suggested
structure, mass spectra of the analyte and the corresponding
reference compound were recorded. To avoid coelutions during
mass spectrometric analysis, the volatiles present in the NBV
Structural Assignment of 16 and 18. Several substituted
cyclodextrine derivatives have already been applied to separate
either 3-sulfanylhexyl acetate (16) or 3-sulfanyl-1-hexanol (18)
into its enantiomers (36–38). Good separations were found in
our study with 2,3-diacetyl-6-tert-butyldimethylsilyl-ꢀ-cyclo-
dextrine dissolved in 1701 polysiloxane as the stationary
phase.

4124 J. Agric. Food Chem., Vol. 56, No. 11, 2008
Steinhaus et al.
Figure 1. Aroma-active compounds showing the highest FD factors in pink guava fruits (Psidium guajava L.).
Table 2. Chromatographic Parameters and Sensory Properties of the
Enantiomers of 3-Sulfanyl-1-hexanol and 3-Sulfanylhexyl Acetate and Their
Distribution in Guavas.
enantiomeric
odor
distribution
in guavas
[%]
a
c
RI
odor
threshold
b
compd
R)-3-sulfanyl-1-hexanol
S)-3-sulfanyl-1-hexanol
R)-3-sulfanylhexyl acetate
(BGB-175)
quality
[ng/L in air]
(
(
(
1497
1490
1461
1466
grapefruit
grapefruit
black-currant
black-currant
0.08
0.07
0.10
0.03
43
57
42
58
(S)-3-sulfanylhexyl acetate
a
b
RI ) retention index. Odor quality as perceived at the sniffing port during
GC-O. Odor threshold in air as determined according to ref 32.
c
Table 3. Analytical and Sensory Data of the Four Isomeric Methyl
2-Hydroxy-3-methylpentanoates
odor
threshold
[ng/L]
a
a
a
a
c
RI
RI
RI
RI
odor
b
isomer
(DB-5) (1701) (FFAP) (BGB-176) quality
(
(
(
2S,3S)-
2R,3R)-
2S,3R)-
991
991
990
990
1101
1101
1095
1095
1492
1492
1471
1471
1114
1100
1086
1092
fruity
10
>200
110
11
Figure 2. Synthetic pathway used in the preparation of enantiopure (R)-
and (S)-3-sulfanyl-1-hexanol and the respective O-acetates.
fruity
fruity
(2R,3S)-
a
b
RI ) retention index. Odor quality perceived at the sniffing port during GC-
O. Odor threshold in air as determined according to ref 26.
To assign the correct elution order, enantiopure samples of
both isomers were prepared. This was achieved by a separation
of the diastereomeric compounds obtained by forming a
derivative of 3-sulfanylhexyl acetate with the enantiomerically
pure (1S,4R)-camphanoyl chloride (28). The diastereomers were
separated by column chromatography on silica gel and, after a
reductive cleavage of the esters, the enantiopure (R)- and (S)-
c
As to be expected, mass spectra of all isomers were quite
similar and were, thus, inappropriate for differentiation. Also
no reliable differentiation was possible on the basis of their
retention indices on nonpolar GC columns like DB-1 or DB-5
(Table 3). However, the diastereomers showed good separation
on polar GC columns, and on a chiral ꢀ-cyclodextrine column
(BGB-176) all four isomers were well separated. Interestingly,
the odor thresholds of the (2S,3S)- and (2R,3S)-isomer were
much lower as compared to the respective (3R)-isomers (Table
3). A comparison of the retention indices of 8 and 9 with those
of the reference compounds (Figure 3) showed the presence of
the fruity smelling methyl (R,S)-2-hydroxy-3-methylpentanoate
(8) and methyl (S,S)-2-hydroxy-3-methylpentanoate (9) in a ratio
of 60% to 40%, while the sensorially less active (S,R)- and
(R,R)-isomers where absent.
As a result of the identification experiments, 31 key odorants
of pink guavas could be identified (Table 1), among which nine
aroma compounds, namely (Z)-1,5-octadien-3-one, methional,
methyl (2R,3S)-2-hydroxy-3-methylpentanoate, methyl (2S,3S)-
2-hydroxy-3-methylpentanoate, 3-sulfanylhexyl acetate, 3-sul-
fanyl-1-hexanol, trans-4,5-epoxy-(E)-2-decenal, trans-4,5-epoxy-
(E)-2-undecenal, and 3-hydroxy-4,5-dimethyl-2(5H)-furanone
were identified for the first time in guavas.
3
-sulfanyl-1-hexanols were obtained in an enantiomeric purity
of nearly 100%. The entire procedure applied to prepare the
enantiopure compounds is schematically shown in Figure 2.
The assignment of the correct stereochemistry was performed
1
by H NMR of the O-acetyl-S-[(2S)-2-phenylpropanoyl] deriva-
tives (28).
The results revealed that 3-sulfanyl-1-hexanol occurred almost
in a racemic ratio (Table 2) with the (R)- and the (S)-isomer
eliciting the same grapefruit-like odor at the extremely low odor
threshold of 70-80 pg per liter in air. The corresponding
acetates also occurred as a nearly racemic mixture with both
enantiomers eliciting a black-currant like aroma. The odor
thresholds were in the same order of magnitude as those of the
corresponding thiols (Table 2).
Structural Assignment of Diastereomeric Methyl 2-Hy-
droxy-3-methylpentanoates. The four possible stereoisomers
were separately synthesized from the respective chiral amino
acid. All four isomers were characterized by their retention
indices on different GC columns, their mass spectra, and their
odor properties (Table 3).
The results of the AEDA suggested that the most important
aroma compounds of guavas are the following: (Z)-3-hexenal,

Key Aroma Compounds in Guava
J. Agric. Food Chem., Vol. 56, No. 11, 2008 4125
Our results demonstrate that the enantiomeric distribution in
both 16 and 18 was virtually the same and was close to the
racemate (Table 2). But in passion fruits, the (S)-isomers
prevailed with 58-90% for the alcohol (36–38) and 91-96%
for the acetate (36, 38). In wine, also the (S)-isomer predomi-
nated with 68-73% in the alcohol, whereas the ester showed
an R/S-ratio of 41/59 to 57/43, except for wine made from
botrytized grapes, in which the (S)-isomer predominated (46).
The low odor thresholds in air determined for 18 and 16
(
Table 2) corresponded well with the low thresholds in water
previously published by Bouchiloux et al. (18: 12-15 ng/L;
6: 2-3 ng/L) (47). The two enantiomers of 18 exhibited nearly
1
the same odor thresholds, which was in agreement with results
of Tominaga et al. (46). However, these authors found some
differences in the odor quality of the enantiomers. They
described the (R)-isomer as grapefruit, citrus-peel-like, while
the (S)-isomer was found to be more passion fruit-like.
For the two enantiomers of 3-sufanylhexyl acetate (16), the
thresholds in air differed by a factor of 3-4 with the (S)-isomer
being more odor-active than the (R)-isomer. This result also
confirmed recent data found in a hydro-alcoholic wine model
solution (46). However, while in our study, both enantiomers
were described as black-currant-like during GC-O, Tominaga
et al. (46) and Weber et al. (36) described the odor of the (R)-
isomer as more passion fruit-like and that of the (S)-isomer as
more herbaceous.
Figure 3. GC-separation of the synthesized isomers of methyl 2-hydroxy-
3-methylpentanoate (A) and their occurrence in the distillate obtained from
pink guava (B)
In passion fruits (48), grapes (41, 49), and also in axilliary
sweat (44, 45), 3-sulfanyl-1-hexanol was shown to be derived
from the cleavage of a nonvolatile precursor, (S)-(1-hydroxy-
3
-hexyl)cysteine,mostprobablybytheactionofaꢀ-lyase(45,50).
hexanal, 3-sulfanyl-1-hexanol, 3-sulfanylhexyl acetate,
In grapes, this precursor is located predominantly in the
skin (49, 51) and liberates the thiol during fermentation (42).
The gasoline-like smelling 2-pentanethiol, earlier held re-
sponsible for the tropical odor note in white-fleshed guavas by
K o¨ nig et al. (52), was not detected in our study.
4
-hydroxy-2,5-dimethyl-3(2H)-furanone, and 4-methoxy-2,5-
dimethyl-3(2H)-furanone, with (Z)-3-hexenal and hexanal being
responsible for the green aroma note, and 3-sulfanyl-1-hexanol
and 3-sulfanylhexyl acetate for the tropical and grapefruit-like
note. 4-Hydroxy-2,5-dimethyl-3(2H)-furanone, together with
some other compounds, such as ethyl butanoate, methyl and
ethyl benzoate, cinnamyl alcohol, and cinnamyl acetate, might
account for the sweet and fruity character.
4
-Hydroxy-2,5-dimethyl-3(2H)-furanone (23) was earlier
found by Idstein and Schreier (7) and also by Jordan et al. (19)
in guavas, while its methoxy derivative 11 was only reported
by Pino et al. (17) so far. Methyl and ethyl benzoate have
been reported as guava constituents in numerous studies
(Z)-3-Hexenal and hexanal have frequently been reported in
Psidium guajaVa (3, 9–15, 18, 19). Together with (E)-2-hexenal
and the corresponding odorless alcohols these compounds have
previously been reported as major volatiles in guavas (11) and
are suggested to be formed by an enzymatic degradation of
unsaturated fatty acids upon disruption of the plant tissue (39).
Several thiols other than the 3-sulfanyl-1-hexanol and 3-sul-
fanylhexyl acetate have been reported in Psidium guajaVa
before (11, 16). Nishimura et al. (11) identified 2-methylpro-
panethiol and 6-sulfanyl-1-hexanol, while Bassols and Demole
(3,7,10–12,15,17,19,34)aswellascinnamylalcohol(12,13,19)
and cinnamyl acetate (7, 14, 15, 34).
Although the chiral methyl 2-hydroxy-3-methylpentanoates
have already been found in other fruits, both esters were found
for the first time as aroma compounds of guavas in this study.
The enantiomeric distribution in the methyl 2-hydroxy-3-
methylpentanoates (Figure 2) in guavas suggests their biogen-
eration from L-isoleucine via the corresponding ꢀ-keto-acid
followed by a nonstereospecific reduction to the alcohol.
The aroma compounds of different varieties may also be
different. Therefore, quantitative studies are under way to
confirm the contribution of the odor-active compounds to the
overall aroma of pink and also white guavas.
(
16) detected 2-pentanethiol, a compound earlier found by
Idstein and Schreier (7), but was assigned as 3-pentanethiol by
these authors. However, neither 3-sulfanyl-1-hexanol (18) nor
3-sulfanylhexyl acetate (16) have been reported as a constituent
of pink guavas up to now. The crucial problem with these
compounds might be their instability during workup. In earlier
studies on guava volatiles often high temperatures were applied,
either to isolate the volatiles, e.g., by steam-distillation (6), or
by using split injection in GC analysis (19).
ACKNOWLEDGMENT
The authors thank Anja Mialki and Magdalena Uzunova for
their skillful technical assistance.
3
-Sulfanyl-1-hexanol was first identified in yellow passion
fruit (Passiflora edulis f. flaVicarpa) by Engel et al. (40). Later
it was also found in wine (41), grapefruit juice (42), and beer
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