3. Results
3.1. Volatile compounds
More than 70 compounds were identified in the headspace of
the two genotypes. The most abundant compounds are listed in Table
1. These included 31 esters (acetates and non-acetate esters), 8
sulphur-containing compounds, 10 alcohols, 8 aldehydes, 2 terpene
derivatives and 2 other compounds. Quantitative differences
were observed between the two maturity stages (immature (i) and
mature (m) fruit) and the two genotypes (medium shelf-life (MSL)
and long shelf-life (LSL)). Esters (acetates and non-acetate esters)
comprised more than 87% of the total volatiles collected from the
iMSL fruit, a percentage which increased to more than 93% in the
mMSL fruit. Similarly, the percentage of esters increased from
69% in the iLSL fruit to more than 77% in the mature fruit of the
same genotype. The most abundant esters identified were ethyl
acetate, 2-methylpropyl acetate, butyl acetate, 2-methylbutyl acetate
and ethyl butanoate. Wyllie et al. (1996) and Bauchot et al.
(2000) reported that these compounds were predominant in Makdimon
(C. melo var. reticulatus) and V้drantais (C. melo var. cantalupensis)
cultivars respectively. These compounds were also the
most abundant in a number of Charentais cantaloupe cultivars
(Aubert & Bourger, 2004) and in Jiashi muskmelon (var. reticulatus,
Hami melon) (Pang, Guo, Qin, Yao, Hu, & Wu, 2012).
Both immature fruits contained very few esters compared to
their respective mature fruit. Ten out of 13 acetates and 12 out
of 18 non-acetate esters were found significantly higher in the
mMSL fruit compared to the iMSL fruit. The same trend was observed
for the LSL fruits, but the levels were much lower and the
differences were not significant. However, the levels of ethyl esters
and particularly ethyl acetate, ethyl propanoate, ethyl 2-methylpropanoate,
ethyl butanoate and ethyl 2-methylbutanoate increased
4-fold for LSL and 26-fold for MSL with increasing
maturity.
Generally, the levels of esters were remarkably lower in the LSL
genotype, even in mLSL. Similar results were reported by
Lamikanra et al. (2003), where hybrids with long shelf-life and hybrids
with extended shelf-life presented significantly lower contents
of total volatile aromas than traditional shelf-life C. melo
var. reticulatus cv. Mission melons. Aubert and Bourger (2004),
who studied the volatile compounds of 15 Charentais melon cultivars,
reported the same trends: a reduction in a range of 43–77% of
total esters in LSL melons compared to MSL or wild melons. They
reported that these differences were more obvious for compounds
with low odour threshold values, such as ethyl 2-methylbutanoate
(0.006 lg/kg), ethyl butanoate (1 lg/kg), ethyl hexanoate (1 lg/
kg), butyl acetate (2 lg/kg) and hexyl acetate (2 lg/kg). Bauchot
et al. (1998) also noted that in transformed Charentais melons with
an ACC oxidase antisense gene, the total volatiles were 60–85%
lower than that of the nontransformed hybrids. They observed that
the reduction in volatiles in these melons was greater for ethyl esters
than for acetates, and since ethyl esters have lower odour
threshold values than acetates, the reduction of ethylene production
in these melons, had the greatest effect on the most potent
odorants (Bauchot et al., 2000).
Eight sulphur-containing compounds were identified in the
headspace of the samples including six thioether esters. Wyllie
and Leach (1992) reported that 2-(methylthio)ethyl acetate and
3-(methylthio)propyl acetate were the dominant sulphur
compounds in all melon cultivars studied, as was the case in the
Charentais melon under study, but only in mMSL fruit. Ethyl 2-
(methylthio)acetate was another important compound and again
present only in mMSL fruit. Generally, the sulphur-containing
esters were not detected in the LSL fruit and only two were detected
in the iMSL fruit. These compounds are very important in
the overall aroma profile of melons, because many are potent odorants
with low odour thresholds. A few authors have reported that
trace amounts of these compounds have a major impact on the
musky note of some melon aromas (Wyllie & Leach, 1992; Hayata,
Sakamoto, Kozuka, Sakamoto, & Osajima, 2002; Jordan, Shaw, &
Goodner, 2001; Wyllie & Leach, 1990; Wyllie, Leach, Wang, &
Shewfelt, 1994; Hayata, Sakamoto, Maneerat, Li, Kozuka, & Sakamoto,
2003). Aubert and Bourger (2004) also reported a considerable
reduction in the levels of these compounds in LSL cultivars,
whereas the total levels of them in wild or MSL cultivars were up
to 17 times higher than in LSL cultivars.
Besides esters and sulphur-containing compounds, some alcohols
and aldehydes were identified in the samples. The levels of
most alcohols increased with increasing maturity for both genotypes,
and this increase was significantly higher, particularly for
mMSL fruit. Regarding the aldehydes found, no significantly
changes were observed between the different samples except for
2-methyl-2-butenal and (Z)-6-nonenal. 2-Methyl-2-butenal was
significantly higher in mMSL fruit and (Z)-6-nonenal was significantly
higher in iMSL fruit. Terpenes like limonene, eucalyptol
and geranylacetone were also found, however, only eucalyptol
was found significantly higher in mMSL fruit. Finally, 2-methylbutanenitrile
and 3-methylbutanentrile were reported for the first
time in melons. These compounds were found to be significantly
higher in mMSL fruit.
To sum up, among all the volatiles identified, 30 compounds
were significantly affected by the maturity and 34 by the genotype,
supporting the hypothesis that both factors were very important.
The two-way ANOVA showed a clear trend, with many of the compounds
(mainly esters, sulphur-containing compounds and several
alcohols) showing a significant interaction between the two variables.
The combination of an MSL variety, and a fruit harvested
at maturity, produced a far greater increase in these compounds
than would have been predicted from a simple additive model.
This synergy is reflected in the GC–O data.
GC–olfactometry analysis of the samples yielded a total of 18
odorants in the chromatogram, which are presented in Table 2.
All but one of these compounds were identified in the GC–MS analysis,
the exception being (Z)-4-heptenal which was recognised by
its characteristic aroma and confirmed by comparison of its LRI
with that of the authentic sample. Quantitative differences were
observed between the two maturity stages and the two genotypes.
It is clearly illustrated in Table 2 that esters were the most important
contributors to the desirable sweet and fruity aroma of the
fruit. In particular, seven esters, including ethyl propanoate, propyl
acetate, ethyl 2-methylpropanoate, methyl 2-methylbutanoate,
ethyl butanoate, ethyl 2-methylbutanoate and butyl propanoate,
contributed to the fruity, pineapple-like and sweet aroma, particularly
of mMSL. Four of these esters were only detected in mMSL, and
the other three branched esters were also detected in the less mature
and the LSL fruits, but tended to have higher scores for mMSL.
Schieberle, Ofner, and Grosch (1990) studied the potent odorants
in muskmelons by aroma extraction dilution analysis (AEDA),
and they reported that indeed the volatile esters were responsible
for the fruity notes in the aroma of muskmelon and that methyl 2-
methylbutanoate and ethyl 2-methylbutanoate were the most intense
odorants in the ester fraction. Jordan et al. (2001) also found
that these two esters contributed to a fruity, sweet and cantaloupelike
aroma. Pang et al. (2012) studied the odour-active compounds
of Jiashi muskmelon using both detection frequency analysis (DFA)
and odour activity values (OAV). They reported that ethyl 2-methylpropanoate,
ethyl butanoate and ethyl 2-methylbutanoate were
the esters with the greatest relative importance and were characterised
as having fruity, sweet and cantaloupe-like odours. Hexanal,
which imparts a fresh green note (Schieberle et al., 1990),
and (Z)-3-hexen-1-ol, which imparts a herbal green note (Jordan
et al., 2001), were detected in these samples and described as having
green and grass notes, respectively. Eucalyptol, reported by
Schieberle et al. (1990), was another important odorant detected
only in mMSL samples. Kemp, Knavel, and Stoltz (1972), and Kemp,
Knavel, Stoltz, and Lundin (1974) concluded that (Z)-6-nonenal
and 3,6-nonadien-1-ol were two potent odorants contributing to
muskmelon flavour. These two compounds were also identified
in these samples, having a cucumber and green note, respectively.
(Z)-6-Nonenal was scored consistently higher in the immature
fruits, consistent with the greener notes of under-ripe fruit. These
compounds were also reported by Pang et al. (2012) in Jiashi muskmelons
and, along with 2,6-nonadienal and 2-nonenal, were the
important contributors for green and cucumber-like aromas. Pang
et al. (2012) also stated that although esters were superior in concentration
(86%), their contribution rate (OAV percentages) to the
aroma profile of Jiashi muskmelons was only 10%, whereas alcohols
and aldehydes were just the opposite. The contents of aldehydes
and alcohols were only 11 and 4% that of esters,
respectively, but their contribution rates were 56% and 34%
respectively.
Finally, of the eight sulphur compounds which were identified
in the headspace of the melons, four were detected by the assessors.
S-Methyl 2-methylbutanethioate had a sulphury odour,
whereas dimethyl trisulfide imparted a pickled onions and cabbage
odour. Ethyl 2-(methylthio)acetate and ethyl 3-(methylthio)propanoate
were only identified in mMSL and had an earthy but
slightly cucumber note and a cardboard but slightly green odour,
respectively. Overall, comparing the odours between the two
maturity stages and the two genotypes, it can be observed that
mMSL fruit presented the highest intensities, which resulted in a
more aromatic fruit compared to the others.
3. Results3.1. Volatile compoundsMore than 70 compounds were identified in the headspace ofthe two genotypes. The most abundant compounds are listed in Table1. These included 31 esters (acetates and non-acetate esters), 8sulphur-containing compounds, 10 alcohols, 8 aldehydes, 2 terpenederivatives and 2 other compounds. Quantitative differenceswere observed between the two maturity stages (immature (i) andmature (m) fruit) and the two genotypes (medium shelf-life (MSL)and long shelf-life (LSL)). Esters (acetates and non-acetate esters)comprised more than 87% of the total volatiles collected from theiMSL fruit, a percentage which increased to more than 93% in themMSL fruit. Similarly, the percentage of esters increased from69% in the iLSL fruit to more than 77% in the mature fruit of thesame genotype. The most abundant esters identified were ethylacetate, 2-methylpropyl acetate, butyl acetate, 2-methylbutyl acetateand ethyl butanoate. Wyllie et al. (1996) and Bauchot et al.(2000) reported that these compounds were predominant in Makdimon(C. melo var. reticulatus) and V้drantais (C. melo var. cantalupensis)cultivars respectively. These compounds were also themost abundant in a number of Charentais cantaloupe cultivars(Aubert & Bourger, 2004) and in Jiashi muskmelon (var. reticulatus,Hami melon) (Pang, Guo, Qin, Yao, Hu, & Wu, 2012).Both immature fruits contained very few esters compared totheir respective mature fruit. Ten out of 13 acetates and 12 outof 18 non-acetate esters were found significantly higher in themMSL fruit compared to the iMSL fruit. The same trend was observedfor the LSL fruits, but the levels were much lower and thedifferences were not significant. However, the levels of ethyl estersand particularly ethyl acetate, ethyl propanoate, ethyl 2-methylpropanoate,ethyl butanoate and ethyl 2-methylbutanoate increased4-fold for LSL and 26-fold for MSL with increasingmaturity.Generally, the levels of esters were remarkably lower in the LSLgenotype, even in mLSL. Similar results were reported byLamikanra et al. (2003), where hybrids with long shelf-life and hybridswith extended shelf-life presented significantly lower contentsof total volatile aromas than traditional shelf-life C. melovar. reticulatus cv. Mission melons. Aubert and Bourger (2004),who studied the volatile compounds of 15 Charentais melon cultivars,reported the same trends: a reduction in a range of 43–77% oftotal esters in LSL melons compared to MSL or wild melons. Theyreported that these differences were more obvious for compoundswith low odour threshold values, such as ethyl 2-methylbutanoate(0.006 lg/kg), ethyl butanoate (1 lg/kg), ethyl hexanoate (1 lg/kg), butyl acetate (2 lg/kg) and hexyl acetate (2 lg/kg). Bauchotet al. (1998) also noted that in transformed Charentais melons withan ACC oxidase antisense gene, the total volatiles were 60–85%lower than that of the nontransformed hybrids. They observed thatthe reduction in volatiles in these melons was greater for ethyl estersthan for acetates, and since ethyl esters have lower odourthreshold values than acetates, the reduction of ethylene productionin these melons, had the greatest effect on the most potentodorants (Bauchot et al., 2000).Eight sulphur-containing compounds were identified in theheadspace of the samples including six thioether esters. Wyllieand Leach (1992) reported that 2-(methylthio)ethyl acetate and3-(methylthio)propyl acetate were the dominant sulphurcompounds in all melon cultivars studied, as was the case in theCharentais melon under study, but only in mMSL fruit. Ethyl 2-(methylthio)acetate was another important compound and againpresent only in mMSL fruit. Generally, the sulphur-containingesters were not detected in the LSL fruit and only two were detectedin the iMSL fruit. These compounds are very important inthe overall aroma profile of melons, because many are potent odorantswith low odour thresholds. A few authors have reported thattrace amounts of these compounds have a major impact on themusky note of some melon aromas (Wyllie & Leach, 1992; Hayata,Sakamoto, Kozuka, Sakamoto, & Osajima, 2002; Jordan, Shaw, &Goodner, 2001; Wyllie & Leach, 1990; Wyllie, Leach, Wang, &Shewfelt, 1994; Hayata, Sakamoto, Maneerat, Li, Kozuka, & Sakamoto,2003). Aubert and Bourger (2004) also reported a considerablereduction in the levels of these compounds in LSL cultivars,whereas the total levels of them in wild or MSL cultivars were upto 17 times higher than in LSL cultivars.Besides esters and sulphur-containing compounds, some alcoholsand aldehydes were identified in the samples. The levels ofmost alcohols increased with increasing maturity for both genotypes,and this increase was significantly higher, particularly formMSL fruit. Regarding the aldehydes found, no significantlychanges were observed between the different samples except for2-methyl-2-butenal and (Z)-6-nonenal. 2-Methyl-2-butenal wassignificantly higher in mMSL fruit and (Z)-6-nonenal was significantlyhigher in iMSL fruit. Terpenes like limonene, eucalyptoland geranylacetone were also found, however, only eucalyptolwas found significantly higher in mMSL fruit. Finally, 2-methylbutanenitrileand 3-methylbutanentrile were reported for the firsttime in melons. These compounds were found to be significantlyhigher in mMSL fruit.To sum up, among all the volatiles identified, 30 compoundswere significantly affected by the maturity and 34 by the genotype,supporting the hypothesis that both factors were very important.The two-way ANOVA showed a clear trend, with many of the compounds(mainly esters, sulphur-containing compounds and severalalcohols) showing a significant interaction between the two variables.The combination of an MSL variety, and a fruit harvestedat maturity, produced a far greater increase in these compoundsthan would have been predicted from a simple additive model.This synergy is reflected in the GC–O data.GC–olfactometry analysis of the samples yielded a total of 18odorants in the chromatogram, which are presented in Table 2.All but one of these compounds were identified in the GC–MS analysis,the exception being (Z)-4-heptenal which was recognised byits characteristic aroma and confirmed by comparison of its LRIwith that of the authentic sample. Quantitative differences wereobserved between the two maturity stages and the two genotypes.It is clearly illustrated in Table 2 that esters were the most importantcontributors to the desirable sweet and fruity aroma of thefruit. In particular, seven esters, including ethyl propanoate, propylacetate, ethyl 2-methylpropanoate, methyl 2-methylbutanoate,ethyl butanoate, ethyl 2-methylbutanoate and butyl propanoate,contributed to the fruity, pineapple-like and sweet aroma, particularlyof mMSL. Four of these esters were only detected in mMSL, andthe other three branched esters were also detected in the less matureand the LSL fruits, but tended to have higher scores for mMSL.Schieberle, Ofner, and Grosch (1990) studied the potent odorantsin muskmelons by aroma extraction dilution analysis (AEDA),and they reported that indeed the volatile esters were responsible
for the fruity notes in the aroma of muskmelon and that methyl 2-
methylbutanoate and ethyl 2-methylbutanoate were the most intense
odorants in the ester fraction. Jordan et al. (2001) also found
that these two esters contributed to a fruity, sweet and cantaloupelike
aroma. Pang et al. (2012) studied the odour-active compounds
of Jiashi muskmelon using both detection frequency analysis (DFA)
and odour activity values (OAV). They reported that ethyl 2-methylpropanoate,
ethyl butanoate and ethyl 2-methylbutanoate were
the esters with the greatest relative importance and were characterised
as having fruity, sweet and cantaloupe-like odours. Hexanal,
which imparts a fresh green note (Schieberle et al., 1990),
and (Z)-3-hexen-1-ol, which imparts a herbal green note (Jordan
et al., 2001), were detected in these samples and described as having
green and grass notes, respectively. Eucalyptol, reported by
Schieberle et al. (1990), was another important odorant detected
only in mMSL samples. Kemp, Knavel, and Stoltz (1972), and Kemp,
Knavel, Stoltz, and Lundin (1974) concluded that (Z)-6-nonenal
and 3,6-nonadien-1-ol were two potent odorants contributing to
muskmelon flavour. These two compounds were also identified
in these samples, having a cucumber and green note, respectively.
(Z)-6-Nonenal was scored consistently higher in the immature
fruits, consistent with the greener notes of under-ripe fruit. These
compounds were also reported by Pang et al. (2012) in Jiashi muskmelons
and, along with 2,6-nonadienal and 2-nonenal, were the
important contributors for green and cucumber-like aromas. Pang
et al. (2012) also stated that although esters were superior in concentration
(86%), their contribution rate (OAV percentages) to the
aroma profile of Jiashi muskmelons was only 10%, whereas alcohols
and aldehydes were just the opposite. The contents of aldehydes
and alcohols were only 11 and 4% that of esters,
respectively, but their contribution rates were 56% and 34%
respectively.
Finally, of the eight sulphur compounds which were identified
in the headspace of the melons, four were detected by the assessors.
S-Methyl 2-methylbutanethioate had a sulphury odour,
whereas dimethyl trisulfide imparted a pickled onions and cabbage
odour. Ethyl 2-(methylthio)acetate and ethyl 3-(methylthio)propanoate
were only identified in mMSL and had an earthy but
slightly cucumber note and a cardboard but slightly green odour,
respectively. Overall, comparing the odours between the two
maturity stages and the two genotypes, it can be observed that
mMSL fruit presented the highest intensities, which resulted in a
more aromatic fruit compared to the others.
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