Analysis of the volatile aroma constituents of parental and hybrid clones of pepino (Solanum muricatum)

Article abstract of DOI:10.1021/jf040107w

The volatile constituents of 10 clones (4 parents with different flavors and 6 hybrids from selected crossings among these parents) of pepino fruit (Solanum muricatum) were isolated by simultaneous distillation-extraction and analyzed by gas chromatography-mass spectrometry (GC-MS). Odor-contributing volatiles (OCVs) were detected by GC-olfactometry-MS analyses and included 24 esters (acetates, 3-methylbutanoates, and 3-methylbut-2-enoates), 7 aldehydes (especially hexenals and nonenals), 6 ketones, 9 alcohols, 3 lactones, 2 terpenes, β-damascenone, and mesifurane. Among these compounds, 17, of which 5 had not been reported previously in pepino, were found to contribute significantly to pepino aroma. OCVs can be assigned to three groups according to their odor quality: fruity fresh (acetates and prenol), green vegetable (C6 and C9 aidehydes), and exotic (lactones, mesifuran, and β-damascenone). Quantitative and qualitative differences between clones for these compounds are clearly related to differences in their overall flavor impression. The positive value found for the hybrid-midparent regression coefficient for volatile composition indicates that an important fraction of the variation observed is inheritable, which has important implications in breeding for improving aroma. Significant and positive correlations were found between OCVs having common precursors or related pathways.

Full text of DOI:10.1021/jf040107w

J. Agric. Food Chem. 2004, 52, 5663−5669 5663
Analysis of the Volatile Aroma Constituents of Parental and
Hybrid Clones of Pepino (Solanum muricatum)
ADRIAÄ N RODRIÄGUEZ-BURRUEZO,† HUBERT KOLLMANNSBERGER,§ JAIME PROHENS,†
SIEGFRIED NITZ,§ AND FERNANDO NUEZ*,†
Centro de Conservacio´n y Mejora de la Agrodiversidad Valenciana, Universidad Polite´cnica de
Valencia, Camino de Vera 14, CP 46022, Valencia, Spain, and Lehrstuhl fu¨r Chemisch-Technische
Analyse und Chemische Lebensmitteltechnologie, Technische Universita¨t Mu¨nchen,
Weihenstephaner Steig 23, D-85350 Freising-Weihenstephan, Germany
The volatile constituents of 10 clones (4 parents with different flavors and 6 hybrids from selected
crossings among these parents) of pepino fruit (Solanum muricatum) were isolated by simultaneous
distillation-extraction and analyzed by gas chromatography-mass spectrometry (GC-MS). Odor-
contributing volatiles (OCVs) were detected by GC-olfactometry-MS analyses and included 24 esters
(acetates, 3-methylbutanoates, and 3-methylbut-2-enoates), 7 aldehydes (especially hexenals and
nonenals), 6 ketones, 9 alcohols, 3 lactones, 2 terpenes, â-damascenone, and mesifurane. Among
these compounds, 17, of which 5 had not been reported previously in pepino, were found to contribute
significantly to pepino aroma. OCVs can be assigned to three groups according to their odor quality:
fruity fresh (acetates and prenol), green vegetable (C6 and C9 aldehydes), and exotic (lactones,
mesifuran, and â-damascenone). Quantitative and qualitative differences between clones for these
compounds are clearly related to differences in their overall flavor impression. The positive value
found for the hybrid-midparent regression coefficient for volatile composition indicates that an
important fraction of the variation observed is inheritable, which has important implications in breeding
for improving aroma. Significant and positive correlations were found between OCVs having common
precursors or related pathways.
KEYWORDS: Solanum muricatum; pepino; volatile compounds; flavor; parental clones; hybrid clones;
GC-MS
INTRODUCTION
more, qualitative and quantitative differences between cultivars
have been detected in many species (3, 4). Therefore, the use
of a representative degree of variation for aroma is required if
one is interested in determining the compounds contributing to
the aroma of pepino fruits and responsible for the differences
in aroma between cultivars.
The success of the establishment of pepino (Solanum muri-
catum), an Andean domesticate (1), as a new crop in Europe
depends on the development of new cultivars with improved
fruit quality. Aroma is an important component of fruit quality
in pepino and is a key factor in determining the type of use of
the fruits. Cultivars yielding berries with fruity and exotic
aromas are preferentially used as a fresh dessert fruit. On the
other hand, cultivars with fruits releasing herbaceous/vegetable-
like aromas are preferred for being used in vegetable salads in
the same way as cucumber (2).
Because aroma is important for pepino fruit quality, it must
be considered as a main trait in breeding programs aimed to
improve quality. As in other fruits and vegetables (3), pepino
aroma is the result of a complex, and specific, combination of
individual volatile compounds. Thus, the isolation, identification,
and quantification of the pepino fruit volatiles is important in
studying the differences in aroma between cultivars. Further-
Despite the wide spectrum for aroma that can be found
between cultivars, studies about this topic in S. muricatum are
scarce. Shiota et al. (5) conducted a pioneer study of the volatile
fraction of pepino, in which they identified up to 30 compounds
from three cultivars of pepino. The main constituents of the
volatile fraction in pepino were 3-methylbut-2-en-1-ol,
3-methylbut-3-en-1-ol, (Z)-non-6-en-1-ol, 3-methylbut-2-en-1-
yl acetate, 3-methylbut-3-en-1-yl acetate, butyl acetate, and
hexyl acetate (all of them related to fruity aromas) and a series
of linear C9 aldehydes (related to green-herbaceous odors).
Recently, it was confirmed that 3-methylbut-2-en-1-ol, 3-meth-
ylbut-3-en-1-ol, and their corresponding acetates were also main
compounds in the volatile fraction of two cultivars of pepino
(6).
* Author to whom correspondence should be addressed (telephone +3496
387 7421; fax +3496 387 9422; e-mail fnuez@btc.upv.es).
† Universidad Polite´cnica de Valencia.
Because of the limited genotypic diversity utilized in these
experiments, compounds of the volatile fraction of other pepino
§ Technische Universita¨t Mu¨nchen.
10.1021/jf040107w CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/04/2004

5664 J. Agric. Food Chem., Vol. 52, No. 18, 2004
Rodr´ıguez-Burruezo et al.
Preliminary experiments with fruits from Sm-23 and Sm-29 (rep-
resentatives of the two extreme aromas in pepino) were conducted to
determine an adequate protocol for the preparation of the samples.
Different sample weights (100, 250, and 500 g of fruit flesh), aqueous
media (distilled water or a 10% NaCl solution), and preparations of
the sample (pieces dipped into the liquid or as a puree obtained after
homogenization with the aqueous medium in a commercial blender)
were tested. Preparation of samples as a puree of 250 g of fruit flesh
in distilled water (1:1) was chosen because it was found to represent
the optimum combination. In addition, the analysis of the peel revealed
that this part of the fruit was very poor in volatiles compared to the
flesh, which was in agreement with the findings of Ruiz-Bevia´ et al.
(6). Consequently, our work was focused on the flesh. SDE volatile
extracts were concentrated to 1 mL on a Vigreux column (40 °C).
Methyl decanoate (2.23 mg kg-1 of fruit) was used as internal standard.
The use of enzyme-inactivating agents in the preparation of samples
was found to be unnecessary. A preliminary gas chromatography-
mass spectrometry (GC-MS) analysis, comparing SDE extractions,
following the protocol described above, with solvent extraction (diethyl
ether) of homogenates inhibited with methanol [preparation as puree
of 250 g of fruit flesh in methanol (1:1)] revealed that the first method
gives the best and reproducible yields of all volatiles.
Table 1. Agronomic and Morphological Traits of Interest of Parents
and Hybrids of Pepino Fruits Evaluated
shape
(length/
width)
soluble
solids
(°Brix)
yield
(t ha-1
fruit
wt (g)
genotype
)
parental clones
Sm-4
Sm-23
Sm-26
Sm-29
0a
40−65
0−20
150−250
250−400
100−200
100−250
0.90−1.10
3.00−3.50
1.20−1.40
2.30−3.00
7.0−9.0
5.0−6.5
8.0−9.0
8.0−9.0
20−30
hybrid clones
Sm-4 × Sm-23 (c4)b
Sm-4 × Sm-23 (c5)
Sm-4 × Sm-23 (c16)
Sm-4 × Sm-29 (c30)
Sm-23 × Sm-26 (c25)
Sm-29 × Sm-26 (c27)
30−50
30−60
50−75
30−40
30−50
25−45
150−250
100−200
200−350
200−350
100−200
150−250
1.50
1.70−1.80
1.30
1.80−1.90
2.20−2.70
2.10−2.20
8.0−8.5
8.0
7.0−8.0
8.0
8.0−8.5
9.0−10.0
a Unviable pollen, nil yield under natural self-pollination conditions. It needs
hand pollination in order to set fruits. b c4 means clone 4 of this segregating hybrid
generation.
Analysis of Volatiles. Volatiles were identified by GC-MS. A
Siemens SiChromat II gas chromatograph directly coupled through a
Live-T effluent splitter (1:1) to a Finnigan-MAT 8222 mass spectrom-
eter and a sniffing-port and a SPB-5 fused silica capillary column (30
m length, 0.53 mm i.d., 1.5 µm film thickness; Supelco, Bellefonte,
PA) were used. Injector and sniffing-port temperature was held at 250
°C. The column was programmed from 100 °C at a rate of 5 °C min-1
to 250 °C. Helium was used as carrier gas at a flow of 3 mL min-1
and split ratio of 1:5. The volume of the samples injected was 2 µL.
Identification of volatiles was done by comparing mass spectra
(ionization energy ) 70 eV) and linear retention indices (13) with
commercially available (Merck, Fluka, Sigma-Aldrich, Oril) or syn-
thesized (see ref 14 and below) reference substances.
Quantitative analysis was performed with a Siemens SiChromat 1-4
gas chromatograph, equipped with the same type of column, carrier
gas, and temperature conditions. The flame ionization detector tem-
perature was 260 °C. Quantitative data were obtained by comparison
to the peak area of the internal standard without considering response
factors.
cultivars might remain unidentified. Furthermore, the identifica-
tion of odor-contributing volatiles (OCVs) of different S.
muricatum cultivars from the total volatiles (7) is of great
importance for investigating the differences in the aroma
between cultivars.
Also, information on the inheritance of the aroma and its
constituents will provide valuable information for plant breeders.
No data exist for pepino regarding this subject, but differences
exist in the mode of inheritance for several individual volatiles
in tomato (8), a species phylogenetically close to pepino (9).
In this work, we analyzed the volatiles of fruits from a
collection of pepino clones, including parents differing in aroma,
and hybrids between them by means of GC-olfactometry-
MS. This will provide information not only on the compounds
relevant for the different patterns of aroma in pepino but also
on their inheritance.
Clones having the highest concentration of the volatiles in the
quantitative analysis [the four parents and hybrid Sm-29 × Sm-26 (c27)]
were chosen to identify OCVs. Samples from these clones were
analyzed by three trained panelists in a qualitative test using GC-
olfactometry-MS. Compounds detected by the panelists in at least one
of the clones evaluated were considered as OCVs in pepino.
Synthesis of Reference Compounds. (Z)-Non-6-en-1-yl acetate was
prepared from (Z)-non-6-en-1-ol (Oril SA, Paris, France) and ethyl
acetate by the addition of KOH according to the method of ref 14.
Methyl, butyl, 3-methylbutyl, 3-methylbut-2-enyl, and 3-methylbut-3-
enyl 3-methylbut-2-enoates were prepared by the addition of ∼10 mg
of KOH and 20 mg of the corresponding alcohol (each obtained from
Fluka Chemie GmbH) to 20 mg of senecioic acid (3-methylbut-2-enoic
acid and 3,3-dimethylacrylic acid, Fluka). After 30 min, 50 µL of a
HCl solution (10%) was added and the flask incubated at 100 °C for
15 min. The reaction products were identical by mass spectra and
retention time compared to the corresponding compounds of pepino
fruit [butyl 3-methylbut-2-enoate, m/e (%) 83 (100), 100 (74), 55 (38),
39 (24), 101 (24), 82 (21), 41 (20), 156 (18); 3-methylbutyl 3-methylbut-
2-enoate, m/e (%) 83(100), 100 (43), 55(42), 43 (36), 70 (29), 101
(27), 71 (23), 170 (10)].
MATERIALS AND METHODS
Plant Material. Fruits from 10 clones of pepino (4 parents and 6
hybrids from selected crossings among these parents) were analyzed
to detect the volatiles present in S. muricatum (Table 1). The population
of parents represents a comprehensive spectrum of the different patterns
of aromas in this species: from the intense green-herbaceous aroma
of Sm-23 (used in salads as a vegetable) to the fruity and exotic aroma
of Sm-29 (dessert fruit), including clones with intermediate odor quality
(Sm-26 and Sm-4, intense green-fruity and light green-fruity, respec-
tively). The six clonal hybrids are the result of an advanced program
for the improvement of yield and soluble solids concentration (SSC),
and they were selected from segregating hybrid offsprings obtained
after the four parental clones had been crossed (10). Three of the six
clonal hybrids studied were selected from the same segregating hybrid
population [Sm-4 × Sm-23 (c4), Sm-4 × Sm-23 (c5), and Sm-4 ×
Sm-23 (c16)]. Plants were grown in a greenhouse in Valencia (Spain)
under autumn-winter growing season conditions (11, 12). Fruits were
harvested when commercially ripe and analyzed 2 days later.
Experimental Design. Two samples per clone were analyzed, except
for Sm-4 and Sm-4 × Sm-29 (c30), for which only one sample each
could be analyzed. Each sample was prepared from three fruits, each
of which came from a different plant of the same clone. Thus, a total
of six plants were sampled per clone [three in the case of Sm-4 and
Sm-4 × Sm-29 (c30)].
Parental Effect and Correlations. An estimate of the parental effect
for the total volatiles and total OCVs was studied by mid-parent (MP)-
hybrid regression analysis. Phenotypic correlations between OCVs were
also calculated.
Preparation of Samples and Extraction of Volatiles. Extraction
of volatiles was conducted as described by Shiota (5) according to the
Lickens-Nickerson simultaneous extraction-distillation (SDE) for 2
h, utilizing diethyl ether as solvent.
RESULTS AND DISCUSSION
Constituents of Pepino Volatiles. A total of 53 volatiles,
including esters, alcohols, aldehydes, ketones, and other com-

Volatiles of Pepino Clones
J. Agric. Food Chem., Vol. 52, No. 18, 2004 5665
Table 2. Volatiles Identified in the Population of 10 Clones of Pepino Fruit
not reported
previouslya
sensory
testb
retention
idexc
compound
identificationd
esters
ethyl acetate
ND
ND
(3)
(3)
(1)
ND
ND
ND
ND
(5)
608
696
797
1
1
1
1
1
1
1
1, 3
1
propyl acetate
butyl acetate
pentyl acetate
hexyl acetate
octyl acetate
(Z)-hex-3-en-1-yl acetate
(Z)-non-6-en-1-yl acetate
2-methylpropyl acetate
3-methylbutyl acetate
904
1006
1208
999
1308
753
*
*
862
1, 4
1, 2, 3, 4
1, 2, 3
1, 2
1, 2
1, 2
1, 2, 3, 4
1, 2, 3, 4
1, 2
1, 3
1, 5
1, 4, 5
1, 3, 4
1, 3, 4
1, 2
3-methylbut-3-en-1-yl acetate
3-methylbut-2-en-1-yl acetate
2-methylene-butan-1,4-diyl diacetate
(Z)-2-methylbut-2-en-1,4-diyl diacetate
(E)-2-methylbut-2-en-1,4-diyl diacetate
3-methylbut-3-en-1-yl 3-methylbutanoate
3-methylbut-2-en-1-yl 3-methylbutanoate
3-methylbut-2-en-1-yl 2-methylbutanoate
methyl 3-methylbut-2-enoate
butyl 3-methylbut-2-enoate
3-methybutyl 3-methylbut-2-enoate
3-methylbut-3-en-1-yl 3-methylbut-2-enoate
3-methylbut-2-en-1-yl 3-methylbut-2-enoate
3-methylbut-2-en-1-yl 2-methylbut-2-enoate
aldehydes and ketones
hexanal
(Z)-hex-3-enal
(E)-hex-2-enal
(Z)-non-6-enal
(E,Z)-nona-2,6-dienal
(E)-non-2-enal
(E,E)-deca-2,4-dienal
acetoin (2-hydroxy-3-butanone)
nonan-2-one
undecan-2-one
tridecan-2-one
pentadecan-2-one
heptadecan-2-one
alcohols
butan-1-ol
(E)-hex-2-en-1-ol
(Z)-non-6-en-1-ol
(E,Z)-nona-2,6-dienol
2-methyl-3-buten-2-ol
3-methyl-3-buten-1-ol
3-methyl-2-buten-1-ol
pentadecan-2-ol
heptadecan-2-ol
miscellaneous
â-damascenone
2,5-dimethyl-4-methoxy-3(2H)-furanone (mesifuran)
linalool
(5)
(5)
872
914
*
*
*
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
1255
1278
1301
1115
1146
1138
833
1121
1184
1195
1237
1245
*
*
*
*
(5)
ND
(2)
(5)
(5)
789
782
846
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1, 4
1
1
1
1
1
1
1
1
1
1
1
1107
1160
1165
1331
700
1094
1297
1499
1703
1902
(5)
*
*
ND
ND
ND
ND
ND
ND
ND
*
*
*
*
ND
ND
ND
ND
ND
ND
(5)
651
856
1172
1170
611
716
756
1710
1909
*
*
ND
ND
*
*
*
*
*
*
*
(5)
(2)
ND
ND
(2)
(2)
(2)
1404
1064
1104
1574
1387
1494
1508
nerolidol
4-nonanolide (γ- nonalactone)
4-decanolide (γ-decalactone)
5-dec-2-enolide (massoia lactone)
a According to ref 3. 2-Methyl-3-buten-2-ol was identified by ref 5, but is not listed in ref 3. b ND ) not detected at the sensory test. In parentheses is the number of
clones (of a total of 5) in which the compound was detected at the sensory test. c Calculated according to ref 13. d 1, by comparison of mass spectra and retention indices
with authentic reference substances; 2, by mass spectra and retention index according to ref 14; 3, by mass spectra according to ref 5; 4, by mass spectra according to
ref 15; 5, by mass spectra (see Materials and Methods).
pounds, were identified (Table 2). The analyses of clones having
a wide spectrum of volatiles allowed the identification of 24
compounds not previously reported in pepino: 8 esters, 5
ketones, 2 aldehydes, 2 alcohols, 3 lactones, 3 isoprenoids, and
a furanone (Table 2).
The most abundant components in pepino flesh are esters.
Among these esters 3-methylbutyl 3-methylbutanoate and
3-methylbut-2-en-1-yl 3-methylbut-2-enoate (Table 2) were
identified in the venom of the European hornet (Vespa crabro)
by Wheeler et al. (15). The 2-methylbutanoate and 2-methylbut-
2-enoate (tiglinate) of 3-methylbut-2-en-1-ol (prenol), as well
as the diacetates of the isomeric 2-methylbut-2-ene-1,4-diols,
were described recently as new natural constituents of Helenium
aromaticum by Kollmannsberger et al. (14), whereas to our
knowledge the diacetate of 2-methylenebutane-1,4-diol was not
found in nature up to now. Ketones and aldehydes (mainly C6
and C9 linear aldehydes) ranked second in number of com-
pounds identified (Table 2). Nine alcohols were also identified,

5666 J. Agric. Food Chem., Vol. 52, No. 18, 2004
Rodr´ıguez-Burruezo et al.
Table 3. Odor Description of Each Odor-Contributing Volatile (OCV)
Perceived at the Sniffing Port
Table 4. Total Volatiles (TVs) and Total Odor-Contributing Volatiles
(TOCVs) (Micrograms per Kilogram) with Respective Midparent−Hybrid
(MP−H) Correlations and Ratio of TOCVs to TVs (Percent) of the
Clones Evaluated
OCV
odor quality
group I
butyl acetate
genotype
TVsa
TOCVsa
TOCVs/TVs
fruity, pear/banana-like
pentyl acetate
hexyl acetate
3-methylbutyl acetate
3-methylbut-2-en-1-yl acetate
3-methylbut-3-en-1-yl acetate
3-methylbut-2-en-1-ol
group II
hexanal
(E)-hex-2-enal
(E)-non-2-enal
(Z)-non-6-enal
(E,Z)-nona-2,6-dienal
fruity, pineapple, banana oil-like
fruity, sweet, berry/pear-like
fruity, fresh, pear/banana-like
fruity, green apple, banana
fruity, banana
parents
Sm-4
Sm-23
Sm-26
Sm-29
47339 bcb
15730 a
58612 c
34218 b
11119 a
46498 c
31679 b
72
71
79
63
50413 bc
gassy fruity/green, lavender, fresh
hybrids
Sm-4 × Sm-23 (c4)
Sm-4 × Sm-23 (c5)
Sm-4 × Sm-23 (c16)
Sm-4 × Sm-29 (c30)
Sm-23 × Sm-26 (c25)
Sm-29 × Sm-26 (c27)
15749 a
32356 ab
20088 a
30238 ab
35028 ab
58155 c
10678 a
18849 a
10615 a
23059 ab
29649 b
44484 c
68
58
53
76
85
77
green
green, vegetable-like
penetrating, green, vegetable-like
green, melon/cucumber-like
powerful, green, vegetable-like
group III
â-damascenone
mesifurane
MP−H correlationc
0.789 NS
0.905 *
boiled apple/peach-like
caramel-like, burnt
a Average of samples of each clone. b Different letters indicate significant
differences between clones for the Student−Newman−Keuls test at P < 0.05.
c Correlation coefficient between hybrids and the respective midparent values, with
NS and * indicating nonsignificant and significant at P < 0.05, respectively.
γ-nonalactone
γ-decalactone
massoia lactone
creamy, coconut-like
creamy, peach-like
creamy, fatty, sweet, coconut-like
of which 3-methylbut-2-en-1-ol and 3-methylbut-3-en-1-ol have
been reported, with their respective acetates, as main constituents
of the volatile fraction of pepino (5, 6). Additionally, 4-nonan-
olide (γ-nonalactone), 4-decanolide (γ-decalactone), 5-dec-2-
enolide (massoia lactone), â-damascenone, nerolidol, linalool,
and 2,5-dimethyl-4-methoxy-3(2H)-furanone (mesifuran) (Table
2) have not been reported in pepino previously. All of them
were detected and identified by GC-MS. â-Damascenone could
be identified only in one sample by GC-MS. In the other samples
investigated by GC-olfactometry-MS â-damascenone was
identified tentatively by relating its characteristic odor (boiled
apple/peach) at the sniffing port and the corresponding retention
time with data of a reference sample. With its extremely low
odor threshold (16) â-damascenone is often recognized sen-
sorially far below its analytical detection limit during sniffing
analyses.
Volatile Compounds Contributing Significantly to Pepino
Fruit Aroma (OCVs). The sniffing qualitative analyses of the
four parents and the hybrid Sm-29 × Sm-26 (c27) revealed that
17 volatiles, of which 5 had not been reported previously in
pepino, are present in pepino fruits at levels high enough to be
detected by the human nose and, therefore, contribute signifi-
cantly to pepino aroma (OCVs).
Additionally, there is also some evidence that in some of the
samples 4-vinylphenol and 4-vinylguaiacol may also contribute
to the flavor. However, these phenols have to be regarded as
artifacts of distillation (17) and are therefore not further
considered. No reliable odor impressions were recorded for (Z)-
hex-3-enal and linalool, which were masked by coeluting
odoriferous compounds [hexanal and (Z)-non-6-enal, respec-
tively].
OCVs were grouped according to their odor quality (Table
3). Group I was related to a fruity and/or fresh odor and included
six acetates and 3-methylbut-2-en-1-ol. The volatiles of this
group are usually related to pear/banana-like flavors and are
naturally present in these two fruits (3). In this sense, 3-meth-
ylbutyl acetate is considered to be a key component of the
banana flavor (18).
tested, with the exception of (E)-hex-2-enal due to its lower
concentration. (E)-Non-2-enal, (Z)-non-6-enal, and (E,Z)-nona-
2,6-dienal showed strong and penetrating odors, which were
immediately related to cucumber and/or melon at the sniffing
port. In fact, they are considered as key components for the
flavor of these vegetables (3). Hexanal contributes with a grassy
note to the flavor of pepino fruits. These aldehydes are very
common volatiles in higher plants having a high lipoxygenase
activity and can be found in a wide range of fruits (3).
Group III is a miscellany of several volatiles that contribute
to pepino flavor with exotic and distinctive notes (Table 3).
The three lactones (γ-nonalactone, γ-decalactone, and massoia
lactone) were described at the sniffing port as having a creamy
odor resembling coconut oil/peach-like notes in agreement with
their descriptions in the literature (3). In fact, γ-nonalactone is
utilized in the formulation of synthetic coconut flavorings, and
γ-decalactone is an important component for the flavor of peach
and other Prunus fruits (19-21). Group III also includes two
volatiles that contribute with delicate and exotic notes to the
aroma of pepino: â-damascenone and mesifurane. The first one,
which was detected by the panelists in the five clones tested
(Table 2), is mainly responsible for the flavor of the essential
oil of rose (22) and has also been reported as an important flavor
compound in carotene-rich fruits such as tomato (23, 24). The
second is a relevant compound in the aroma of cultivated
Fragaria (25, 26) and pineapple (27).
The two most common names by which S. muricatum is
known in English are pepino (Spanish word for cucumber) and
melon-pear (28). Probably, the fact that the flavor of pepino
fruit is mainly the result of volatiles reported to be relevant to
the flavor of cucumber, melon, and pear, was the main reason
for giving such names to this crop.
Quantitative and Qualitative Variation of Volatiles be-
tween Clones. There were important differences between clones
for total concentration of volatiles and total concentration of
OCVs (Table 4). The highest levels of total volatiles were found
in parents Sm-26 (close to 60000 µg kg-1) and Sm-29 (50000
µg kg-1) and their hybrid Sm-26 × Sm-29 (c27). The lowest
levels were found in parent Sm-23 and hybrid Sm-4 × Sm-23
(c4) (Table 4).
Group II is constituted by five C6 and C9 linear aldehydes,
which contribute to pepino flavor with herbaceous/green notes
(Table 3). These volatiles have been previously reported in
pepino (5) and were detected by the panelists in the five clones
OCVs represent >50% of total volatiles, ranging from 53%
in Sm-4 × Sm-23 (c16) to 85% in Sm-23 × Sm-26 (c25) (Table

Volatiles of Pepino Clones
J. Agric. Food Chem., Vol. 52, No. 18, 2004 5667
Table 5. Intervals of Concentration (Micrograms per Kilogram) of Odor-Contributing Volatiles (OCVs) in the Volatile Fraction of Parental and Hybrid
Clonesa
hybrids
parents
Sm-26
Sm-4 ×
Sm-4 ×
Sm-4S ×
Sm-4 ×
Sm-23 ×
Sm-29 ×
OCV
Sm-4
500
Sm-23
Sm-29
Sm-23 (c4) Sm-23 (c5) Sm-23 (c16) Sm-29 (c30) Sm-26 (c25) Sm-26 (c27)
group I
butyl acetate
pentyl acetate
284−350
NQ
1908−1961
NQ
4658−5581
23−50
785−2462 2900
NQ
660−1000
NQ
2351
NQ
1091−1135 2384−3176
NQ
a
NQ
NQ
85−95
hexyl acetate
3-methylbutyl acetate
130
188
217−413
123−238
210−301
537−798
7680−8653
613−680
1010−1038
8403−9722
136−318
79−370
836−3473 300−327
115−126
100−145
93−94
53
137−394
186
222
5499
7390
1302
233−268
247−272
9639
5213−9853 15102−17370
524−849 2118−2823
293−350
966−990
12577−12738
3-methylbut-2-en-1-yl acetate 9415 503−565
3-methylbut-3-en-1-yl acetate 17267 3804−4692 18500−21900 11500−12300 1851−4377 2182−4104 5009−6287
3-methylbut-2-en-1-ol
group II
1062 333−493
1442−2075
2698−3158
161−1210 722−996
323−394
hexanal
241
156
475
50
2070
4085
1513
1643
168−225
0−42
760−1506 441−1153 832−1115
696
1003−4703 650−677
2700−9386 3272−3856
1915−3268 1647−3308
2181−2782 976−1794
(E)-hex-2-enal
(E,Z)-nona-2,6-dienal
(E)-non-2-enal
(Z)-non-6-enal
569−1586
2616−9506
1086−4835
2447−4014
105−406
50−419
0−272
50−60
1469
569
445
498
3501−4965 1271−2413
1723−4518 278
1012−1846 NQ
2322
1306
676
4734 122−361
2182−4211 378−498
1059
372−777
525−926
group III
â-damascenone
mesifurane
γ- nonalactone
γ-decalactone
massoia lactone
b
c
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
10
NQ
NQ
NQ
NQ
NQ
NQ
58−80
NQ
20
121−134
−
−
−
−
−
NQ
NQ
NQ
NQ
50
NQ
NQ
NQ
30
NQ
NQ
NQ
NQ
NQ
NQ
NQ
50
NQ
NQ
NQ
20
40−50
NQ
20
100
a Two samples per clone were analyzed except for Sm-4 and Sm-4 × Sm-29 (c30) in which only one sample was utilized. Each sample was prepared from three fruits,
each from a different plant. b NQ ) not quantified because concentration was too low during GC analysis. c Not investigated by GC−olfactometry−MS.
4). Similar to the total volatile fraction, significant differences
were found between clones for the total concentration of OCVs.
Parent Sm-26 and hybrid Sm-29 × Sm-26 (c27) showed the
highest values (∼45000 µg kg-1), followed by Sm-4, whereas
the lowest values were found in Sm-23 and the three hybrids
between this clone and Sm-4 (<20000 µg kg-1) (Table 4).
Several facts suggest that the differences among clones for
both total volatiles and total OCVs might have an important
genetic component. Hybrids [with the exception of Sm-4 × Sm-
29 (c30)] showed concentrations intermediate or not significantly
different from that of one or both parents. In addition, a high
positive correlation between hybrids and mid-parent value was
found for odor-contributing compounds (Table 4), indicating
that the greater the average of a couple of parents, the higher
the value of the respective hybrid.
Differences in the flavor of the parental clones can be
explained by quantitative and qualitative differences in indi-
vidual OCVs. Sm-29, with a fruity and exotic flavor, showed
higher concentrations of compounds of group I than Sm-23,
characterized by a green/vegetable-like flavor (Table 5). In fact,
compounds of group I represented the most important fraction
of the total concentration of volatiles (55%) and total concentra-
tion of OCVs (90%) in Sm-29 (Tables 4 and 5), and levels of
3-methylbut-2-en-1-yl acetate were 15 times higher in Sm-29
than in Sm-23. Moreover, all of the compounds of group III
detected here were present in Sm-29, and this contributed to
the final aroma of its fruits with exotic notes of coconut and
peach. On the contrary, only â-damascenone was found to
contribute significantly to the aroma of Sm-23 fruits (Table
5). Differences in the levels of linear aldehydes contribute further
to explain differences between both clones, as Sm-23 showed
higher levels than Sm-29 for the five aldehydes. In this way,
hexanal levels in Sm-23 were 50 times higher than that in Sm-
29 (Table 5).
the concentrations of volatiles from other groups. The range of
compounds of group III in Sm-26 is not as wide as in Sm-29
and, furthermore, Sm-26 has a greater concentration of alde-
hydes than Sm-29 and even higher than Sm-23 in the case of
(E,Z)-nona-2,6-dienal and (Z)-non-6-enal (Table 5). This greater
concentration of group II compounds may mask the flavor of
volatiles of group I.
In the case of Sm-4, only high values of 3-methylbut-2-en-
1-yl acetate and 3-methylbut-3-en-1-yl acetate (similar to Sm-
26 and Sm-29) and (Z)-non-6-enal were found (Table 5). In
the case of the other OCVs, low levels were found in this clone
(many of them among the lowest of the four parents) (Table
5). Thus, despite ranking third for the total OCVs, with values
even higher than those of Sm-29 (Table 4), the flavor of this
clone is the least intense of the four parents. This fact suggests
that the presence of certain volatiles at adequate concentrations
may be more important than the value of total volatiles (or even
total OCVs) for the intensity of flavor in pepino fruit.
Hybrids, as parents, showed quantitative differences for
OCVs. In addition, a strong relationship between parents and
hybrids for the concentration of individual volatiles was found
(Table 5). This suggests that the flavor has a complex genetic
control, as it is inherited as a combination of different traits
rather than as a single trait. Thus, hybrids involving parents
Sm-26 and Sm-29 showed, in general, higher levels of group I
volatiles than the three hybrids from crossing Sm-4 × Sm-23
(Table 5). In the case of aldehydes we found that in almost all
cases the hybrid with the highest value of a particular aldehyde
is always derived from the parent with the highest value (Table
5). Furthermore, individual compounds of group III were found
only in hybrids derived from parents in which such compounds
had also been identified (Table 5).
The hypothesis of a parental effect in the constitution of the
flavor in the hybrids is also supported by the range of volatiles
found in Sm-29 × Sm-26 (c27). This hybrid, selected from the
crossing between parents with the highest concentrations of
compounds of groups I and III, also showed the highest values
of these compounds among the hybrids (Table 5).
Although Sm-26 showed higher values of total OCVs than
Sm-29, and similar levels of compounds of group I (higher in
3-methylbut-3-en-1-yl acetate) (Table 5), its flavor is not as
fruity and attractive as that of Sm-29. This may be related to

5668 J. Agric. Food Chem., Vol. 52, No. 18, 2004
Rodr´ıguez-Burruezo et al.
Table 6. Correlation Coefficients between Odor-Contributing Volatiles (OCVs) of Groups I and IIa
compoundb
BA
PA
HA
PA
HA
3-MBA 3-M-2-BA 3-M-3-BA 3-M-2-Bol hexanal (E)-2-hexenal (E)-2-nonenal (Z)-6-nonenal (E,Z)-2,6-nonadienal
0.483NS 0.743*** 0.763*** 0.489NS
0.485NS 0.842*** 0.814**
0.768*** 0.475NS
0.287NS
0.856*** −0.326NS
−0.365NS
−0.193NS
−0.339NS
−0.317NS
−0.476NS
−0.339NS
−0.379NS
0.868***
−0.113NS
0.026NS
0.065NS
0.187NS
0.127NS
0.338NS
0.094NS
0.265NS
0.387NS
−0.316NS
−0.385NS
−0.548*
−0.172NS
−0.085NS
−0.187NS
0.128NS
−0.014NS
0.360NS
0.035NS
0.067NS
0.285NS
0.909***
0.547*
0.888*** 0.777**
−0.225NS
0.298NS
0.689**
0.753*** −0.035NS
0.950 *** −0.269NS
3-MBA
0.787***
−0.246NS
−0.182NS
0.244NS
3-M-2-BA
3-M-3-BA
3-M-2-Bol
hexanal
(E)-hex-2-enal
(E)-non-2-enal
(Z)-non-6-enal
0.791*** 0.769*** −0.440NS
0.680**
−0.263NS
−0.314NS
−0.192NS
−0.250NS
−0.003NS
0.221NS
a NS, *, **, and *** indicate nonsignificant and significant at P < 0.05, 0.01, and 0.001, respectively. b BA, butyl acetate; PA, pentyl acetate; HA, hexyl acetate; 3-MBA,
3-methylbutyl acetate; 3-M-2-BA, 3-methylbut-2-en-1-yl acetate; 3-M-3-BA, 3-methylbut-3-en-1-yl acetate; 3-M-2-Bol, 3-methylbut-2-en-1-ol.
Examples of the typical segregation of the hybrid offsprings
of pepino (10) were also found. As clones of this species are
usually highly heterozygous, the hybrid population derived from
a crossing between two different clones includes genetically
different individuals (29). As a consequence, a high degree of
variation (segregation) can be found among hybrids of the same
parents and, even, some individuals may show higher levels
than any parent (transgressive) for many traits (10, 30). Thus,
differences among the three hybrids derived from Sm-4 and
Sm-23 for total volatiles and total OCVs (Table 4) and
individual volatiles (Table 5) may be explained by the het-
erozygous nature of parents. On the other hand, hybrid Sm-29
× Sm-26 (c27) was transgressive to their respective parents for
3-methylbut-2-en-1-yl acetate, hexanal, and (E)-hex-2-enal,
whereas Sm-4 × Sm-23 hybrids were transgressive for butyl
acetate (Table 5). Obtention of transgressive individuals for
flavor compounds is a very important point for the genetic
improvement of the flavor, because it indicates that selection
of individuals with improved flavor resulting from specific
combinations of genes from parental clones can be obtained.
Correlations between Odor-Contributing Volatiles. Only
OCVs of groups I and II were considered in the estimate of
phenotypic correlations. Compounds of group III were not
included because their concentrations were not quantified or
were very low. Nineteen significant correlations between
compounds of groups I and II were found. All significant
correlations but one were positive (within a range of 0.547-
0.950) and were detected between compounds from the same
group (most of them between compounds of group I) (Table
6), whereas the only negative correlation was found between
hexyl acetate (group I) and (Z)-non-6-enal (group II). This
suggests that development of clones for either dessert or salad
use will be facilitated because simultaneous selection for high
concentration of fruity volatiles and low concentration of
vegetable-like flavors is possible in the first case, whereas the
contrary is possible in the second case.
a highly divergent metabolic pathway, called the phytooxylipin
pathway. In this pathway, lipoxygenase produces 9- and 13-
hydroperoxides of both linoleic and linolenic acids. Then,
hydroperoxide lyase releases C6 and C9 linear aldehydes from
13-hydroperoxides and 9-hydroperoxydes, respectively. In the
case of C6 aldehydes, hexanal (from linoleic acid) and (Z)-hex-
3-enal (linolenic acid) are found, and the latter is isomerized to
(E)-hex-2-enal, which has been reported in strawberry and
tomato (34-36) and may account for the positive correlation
found between hexanal and (E)-hex-2-enal.
The study of a clonal collection representative of different
flavors in S. muricatum has allowed the identification of a wide
spectrum of volatile constituents in this species. These volatiles
can be assigned to three groups according to their odor quality:
fruity/fresh (acetates and prenol), green (C6 and C9 aldehydes)
and exotic (lactones, mesifuran, and â-damascenone). Quantita-
tive and qualitative differences between clones for these
compounds are clearly related to differences in their final flavor.
In this way, fruits with higher levels of acetates, prenol, and
constituents with exotic notes and low levels of aldehydes are
more suited for use as a dessert fruit, whereas the contrary is
true for fruits destined to be used in salads. The levels of
volatiles of hybrids are greatly influenced by the parents, and
this offers opportunities for selection and development of clones
with improved flavor, as it is possible to select clones with more
intense fruity (or green) flavor from planned crossings.
ACKNOWLEDGMENT
We thank B. Zimmermann and E. Schu¨tz for skilful assistance
in the experimental work.
LITERATURE CITED
(1) Heiser, C. B. Origin and variability of the pepino (Solanum
muricatum): A preliminary report. Baileya 1964, 12, 151-158.
(2) Nuez, F.; Ruiz, J. J. El Pepino Dulce y su CultiVo; FAO: Rome,
Italy, 1996; pp 18-20.
(3) Morton, I. D.; MacLeod, A. J. Food FlaVors, Part C. The FlaVor
of Fruits; Elsevier: New York, 1990.
(4) Cunningham, D. G.; Acree, T. E.; Barnard, J.; Butts, R. M.;
Braell, P. A. Charm analysis of apple volatiles. Food Chem.
1985, 19, 137-147.
(5) Shiota, H.; Young, H.; Paterson, V. J.; Irie, M. Volatile aroma
constituents of pepino fruit. J. Sci. Food Agric. 1988, 43, 343-
354.
Many correlations can be explained because most of these
compounds are metabolites of related pathways, so that they
have precursors in common. Thus, it was found that concentra-
tions of 3-methylbutyl acetate, 3-methylbut-2-en-1-yl acetate,
3-methylbut-3-en-1-yl acetate, and 3-methylbut-2-en-1-ol (pre-
nol) were positively correlated (Table 6). The conversion of
hexanoate (precursor of hexyl acetate) into butyrate (precursor
of butyl acetate) by â-oxidation (31) may explain the positive
correlation between butyl acetate and hexyl acetate.
Higher plants (e.g., cucumber, melon, pear) can form C6 and
C9 aldehydes from unsaturated C18 fatty acids such as linoleic
or linolenic acid through sequential reactions by lipoxygenase
and fatty acid hydroperoxide lyase (32, 33). This is a branch of
(6) Ruiz-Bevia´, F.; Font, A.; Garc´ıa, A. N.; Blasco, P.; Ruiz, J. J.
Quantitative analysis of the volatile aroma components of pepino
fruit by purge-and-trap and gas chromatography. J. Sci. Food
Agric. 2002, 82, 1182-1188.

Volatiles of Pepino Clones
J. Agric. Food Chem., Vol. 52, No. 18, 2004 5669
(7) Buttery, R. G. Quantitative and sensory aspects of flavor of
tomato and other vegetable and fruits. In FlaVor Science:
Sensible Principles and Techniques; Acree, T. E., Teranishi, R.,
Eds.; American Chemical Society: Washington, DC, 1993; pp
259-286.
(8) Saliba-Colombani, V.; Causse, M.; Langlois, D.; Philouze, J.;
Buret, M. Genetic analysis of organoleptic quality in fresh market
tomato. 1. Mapping QTLs for physical and chemical traits. Theor.
Appl. Genet. 2001, 102, 259-272.
(9) Spooner, D. M.; Anderson, G. J.; Jansen, R. K. Chloroplast DNA
evidence for the interrelationships of tomatoes, potatoes, and
pepinos (Solanaceae). Am. J. Bot. 1993, 80, 676-688.
(10) Rodr´ıguez-Burruezo, A.; Prohens, J.; Nuez, F. Performance of
hybrid segregating populations of pepino (Solanum muricatum)
and its relation to genetic distance among parents. J. Hortic. Sci.
Biotechnol. 2003, 78, 911-918.
(11) Prohens, J.; Ruiz, J. J.; Nuez, F. Growing cycles for a new crop,
the pepino, in the Spanish Mediterranean. Acta Hortic. 2000,
523, 53-60.
(23) Buttery, R. G.; Teranishi, R.; Ling, L. C.; Turnbaugh, J. G.
Quantitative and sensory studies on tomato paste volatiles. J.
Agric. Food Chem. 1990, 38, 336-340.
(24) Buttery, R. G.; Takeoka, G. R.; Ling, L. C. Furaneol: odor
threshold and importance to tomato aroma. J. Agric. Food Chem.
1995, 43, 1638-1640.
(25) Schieberle, P.; Hofmann, T. Evaluation of the character impact
odorants in fresh strawberry juice by quantitative measurements
and sensory studies on model mixtures. J. Agric. Food Chem.
1997, 45, 227-232.
(26) Wein, M.; Lewinsohn, E.; Schwab, W. Metabolic fate of isotopes
during the biological transformation of carbohydrates to 2,5-
dimethyl-4-hydroxy-3(2H)-furanone in strawberry fruits. J. Agric.
Food Chem. 2001, 49, 2427-2432.
(27) Berger, R. G.; Drawert, F.; Kollmannsberger, H.; Nitz, S.;
Schraufstetter, B. Novel volatile in pineapple fruit and their
sensory properties. J. Agric. Food Chem. 1985, 33, 232-235.
(28) Prohens, J.; Ruiz J. J.; Nuez, F. The pepino (Solanum muricatum,
Solanaceae): a “new” crop with a history. Econ. Bot. 1996, 50,
355-368.
(29) Wricke, G.; Weber, E. QuantitatiVe Genetics and Selection in
Plant Breeding; de Gruyter: Berlin, Germany, 1986; pp 1-2.
(30) Prohens, J.; Nuez, F. Strategies for breeding a new greenhouse
crop, the pepino (Solanum muricatum Aiton). Can. J. Plant Sci.
1999, 79, 269-275.
(12) Rodr´ıguez-Burruezo, A.; Prohens, J.; Nuez, F. Genetic analysis
of quantitative traits in pepino (Solanum muricatum) in two
growing seasons. J. Am. Soc. Hortic. Sci. 2002, 127, 271-278.
(13) Van der Dool, H.; Kratz, P. D. A generalization of the retention
index system including linear temperature programmed gas-
liquid partition chromatography. J. Chromatogr. 1963, 11, 463-
471.
(31) Flores, F.; El Yahyaoui, F.; de Billerbeck, G.; Romojaro, F.;
Latche´, A.; Bouzayen, M.; Pech J. C.; Ambid, C. Role of
ethylene in the biosynthetic pathway of aliphatic ester aroma
volatiles in charentais cantaloupe melons. J. Exp. Bot. 2002, 53,
201-206.
(14) Kollmannsberger, H.; Lorenz, M.; Weinreich, B.; Nitz, S. The
flavour composition of “Manzanilla de la Montan˜a” (Helenium
aromaticum) from Chile. AdV. Food Sci. 1998, 20, 122-131.
(15) Wheeler, J. W.; Shamin, M. T.; Brown, P.; Duffield, R. M.
Sesquiterpenoid esters from the venom of the European hornet
(Vespa crabro). Tetrahedron Lett. 1983, 24 (52), 5811-5814.
(16) Grosch, W. Evaluation of the key odorants of foods by dilution
experiments, aroma models and omission. Chem. Senses 2001,
26, 533-545.
(17) Tressl, R.; Kossa, T.; Renner, R.; Ko¨ppler, H. Gaschromatog-
raphisch-massenspektrometrische untersuchungen u¨ber die bil-
dung von phenolen und aromatischen kohlenwasserstoffen in
lebensmitteln. Z. Lebensm. Unters. Forsch. 1976, 162, 123-
130.
(32) Ble´e, E. Phytooxylipins and plant defense reactions. Prog. Lipid
Res. 1998, 37, 33-72.
(33) Noordermeer, M. A.; Veldink, G. A.; Vliegenthart, J. G. F. Fatty
acid hydroperoxide lyase: a plant cytochrom P450 enzyme
involved in wound healing and pest resistance. ChemBioChem
2001, 2, 494-504.
(34) Riley, J. C. M.; Willemot, C.; Thompson, J. E. Lipoxygenase
and hydroperoxide lyase activities in ripening tomato fruit.
PostharVest Biol. Technol. 1996, 7, 97-107.
(18) Berger, R. G.; Drawert, F.; Kollmannsberger, H. Biotechnological
production of flavour compounds II. PA-storage for the com-
pensation of flavour losses in freeze-dried banana slices. Z.
Lebensm. Unters. Forsch. 1986, 183, 169-171.
(19) Takeoka, G. R.; Flath, R. A.; Mon, T. R.; Teranishi, R.; Guentert,
M. Volatile constituents of apricots (Prunus armeniaca). J. Agric.
Food Chem. 1990, 38, 471-477.
(35) Pe´rez, A. G.; Sanz, C.; Olias, R.; Olais, J. M. Lipoxygenase and
hydroperoxide lyase activities in ripening strawberry fruits. J.
Agric. Food Chem. 1999, 47, 249-253.
(36) Alexander, L.; Grierson, D. Ethylene biosynthesis and action in
tomato: a model for climacteric fruit ripening. J. Exp. Bot. 2002,
53, 2039-2055.
(20) Chapman, G. W., Jr.; Horvat, R. J.; Forbus, W. R., Jr. Physical
and chemical changes during the maturation of peaches (cv.
Majestic). J. Agric. Food Chem. 1991, 39, 867-870.
(21) Horvat, R. J.; Chapman, G. W., Jr.; Senter, S. D.; Robertson, J.
A.; Okie, W. R.; Norton, J. D. Comparison of the volatile
compounds from several commercial plum cultivars. J. Sci. Food
Agric. 1992, 60, 21-23.
Received for review March 3, 2004. Revised manuscript received June
1, 2004. Accepted June 6, 2004. A.R.-B. thanks MECD (Spain) for a
predoctoral grant and economic support for his stay at the Technische
Universita1t Mu1nchen. This work has been partially financed by the
Generalitat Valenciana (GV01-92).
(22) Ohloff, G. Scent and Fragances, the Fascination of Odors and
Their PerspectiVes; Springer-Verlag: Berlin, Germany, 1994;
pp 154-158.
JF040107W