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ORIGINAL ARTICLE
Volatile compounds associated to the loss of astringencyin persimmon fruit revealed by untargeted GC–MS analysis
Cristina Besada • Gerardo Sanchez •
Alejandra Salvador • Antonio Granell
Received: 13 March 2012 / Accepted: 18 May 2012
� Springer Science+Business Media, LLC 2012
Abstract High resolution volatile profiling (67 com-
pounds identified) of fruits from 12 persimmon cultivars
was established and used to characterize the different
astringency types of persimmon fruit before and after
deastringency treatment. Analysis of the volatile profile of
fruit enables us to differentiate between cultivars that at the
moment of harvest produced non-astringent fruit (Pollina-
tion Constant Non Astringent—PCNA-type) from astrin-
gent ones (non-PCNA-type). Fruit failing to accumulate
astringent compounds naturally (PCNA fruit) showed high
levels of 3(2H)-benzofuranone, while this compound was
not detected in any astringent type fruit (non-PCNA). In
addition to this, PCNA cultivars also showed at harvest
higher accumulation of benzeneacetaldehyde and lipid-
derived aldehydes (hexanal, heptanal, octanal and decanal)
than non-PCNA fruit. The application of postharvest
deastringency treatment to all non-PCNA cultivars resulted
on an important insolubilization of tannins. In general
the CO2-treatment enhanced the levels of acetaldehyde,
however those cultivars showing high levels of dihydro-
benzofuran at harvest did not present an increment of
acetaldehyde. In contrast, all non-PCNA cultivars exhib-
ited an important accumulation of lipid-derived aldehydes
due to CO2-treatment. Therefore, we propose that lipid-
derived aldehydes (mainly decanal, octanal and heptanal)
may be playing a role in the astringency loss. Our results
suggest that 3(2H)-benzofuranone, benzeneacetaldehyde
and lipid-derived aldehydes could be used as markers for
both natural and artificial loss of astringency.
Keywords Astringency � 3(2H)-Benzofuranone �Benzeneacetaldehyde � Decanal � Dihydrobenzofuran �Octanal � Tannins
1 Introduction
Fruit astringency is an important trait of persimmon fruit.
Astringency is due to a high concentration of soluble tan-
nins in the flesh that results in an unpalatable sensation of
dryness and is probably part of the evolutionary mecha-
nisms to deter frugivores from eating unripe fruit until
seeds are ready for dispersal. Persimmon varieties differ in
this astringency trait and are usually classified into four
groups (Bellini 1982; Sugiura 1983) according to the
astringency at harvest. Non-astringent cultivars when seeds
are present (Pollination Variant Non Astringent—PVNA-
type); non-astringent cultivars regardless of the presence of
seeds (Pollination Constant Non Astringent—PCNA-type);
astringent cultivars when fruit are seedless and mostly
astringent when seeds are present (Pollination Variant
Astringent—PVA type); astringent cultivars regardless of
the presence of seeds (Pollination Constant Astringent—
PCA-type).
It is known that fruit of all persimmon-types are very
astringent at the early and mid development. In the case of
PVA and PVNA cultivars, loss of astringency is also
associated to the presence of seeds, and this appears to be
Electronic supplementary material The online version of thisarticle (doi:10.1007/s11306-012-0436-2) contains supplementarymaterial, which is available to authorized users.
C. Besada � G. Sanchez � A. Granell
Instituto de Biologıa Molecular y Celular de Plantas (IBMCP),
Ingeniero Fausto Elio, s/n, 46022 Valencia, Spain
e-mail: besada_cri@gva.es
A. Salvador (&)
Instituto Valenciano de Investigaciones Agrarias (IVIA),
Carretera Moncada-Naquera, km 4,5, 46113 Valencia, Spain
e-mail: salvador_ale@gva.es
123
Metabolomics
DOI 10.1007/s11306-012-0436-2
due to the seed ability to produce acetaldehyde during fruit
development; however the production of this volatile
compound by seeds is almost null in PCA-type fruit, thus
the fruit are strongly astringent at harvest maturity (Sugiura
et al. 1979; Sugiura and Tomana 1983). The mechanism by
which the fruit of PCNA cultivars lose their astringency
during development while on the tree requires additional
considerations since first the production of acetaldehyde by
the seeds is null in most of PCNA fruit, and second the loss
of astringency takes place irrespective of seeds in parthe-
nocarpic fruits. Although early cessation of tannin cell
development is considered to be the main cause of natural
astringency loss in PCNA fruit this resulting in the dilution
of tannins concentration in the flesh as fruit grows
(Yonemori and Matsushima 1985, 1987), the mechanism
for losing astringency and associated soluble tannins on the
tree in PCNA cultivars has not been clarified completely.
To be ready for consumption fruit in the non-PCNA
groups (PVNA, PVA and PCA) are commercialized after
natural softening/ripening or after removal of astringency
in the packing house. An established common practice to
remove astringency from astringent fruit, is to expose the
fruit under anaerobic conditions at 95–98 % CO2 for
24–36 h. This treatment allows commercialization of non-
astringent fruit with a crispy texture.
It is believed that high CO2 results in the polymerization
of the soluble tannins to a non-astringent form and this is
mediated by acetaldehyde (Matsuo et al. 1991). Several
studies have shown that the rate of deastringency can be
positively correlated to the endogenous level of acetalde-
hyde accumulating in the fruit under anaerobic conditions
(Matsuo and Itoo 1977; Pesis and Ben-Arie 1984; Pesis
et al. 1987; Taira et al. 1989; Besada et al. 2010). Acet-
aldehyde would react with C-8 or C-6 of the proanthocy-
anidin A residues and would therefore by connecting two
proanthocyanidin residues result in tannin insolubilization
and a decrease in astringency. The covalent bonding of
acetaldehyde in insolubilized proanthocyanidins had been
supported by thiol degradation experiments, which nor-
mally degrade proanthocyanidins residues into their com-
ponent flavan-3-ol units. Thus, while thiol degradation of
the extract of the astringent persimmon fruits produced the
thioethers of flavan-3-ols, a direct treatment of the insol-
uble fraction of CO2 treated fruit released the bisthioethers
of the flavan-3-ol acetaldehyde adducts in addition to the
usual thioethers (Tanaka et al. 1994).
In addition to astringency, flavor and aroma are impor-
tant quality traits in persimmon. Flavor results mainly from
the combination of sweetness and sourness from sugars and
organic acids and aroma volatiles. The contribution of
aroma to the flavor quality of fresh produce has gained
increasing attention and breeders need more information
and analytical tools in order to select for flavor quality
(Baldwin 2004). Currently, characterization of persimmon
cultivars has been mainly focused on physico-chemical
fruit properties such as firmness, colour, size, tannin con-
centration (Romaguera et al. 2009) organic acids, sugars
content (Del Bubba et al. 2009), polyphenolics (Suzuki
et al. 2005) or carotenoids (Veberic et al. 2010). Very little
information is available about aroma volatile compounds
of persimmon as only two studies can be found in the
literature in which a low number of volatile compounds are
reported, 15 and 23 respectively (Horvat et al. 1991; Taira
et al. 1996). It is expected that as reported for many other
fruits the volatile complement of persimmon is richer than
this and therefore further work is needed to describe its
aroma profile in more detail.
It is known that deastringency treatments change the
volatile composition of persimmon fruit but most studies
have focused on the changes in acetaldehyde and ethanol
levels (Besada et al. 2010; Taira et al. 1992; Salvador et al.
2008). Only Taira et al. (1996) have provided a partial
description of the effect of deastringency treatment on the
volatile composition of persimmon.
To the best of our knowledge no information exists
about the influence of persimmon astringency type (PCNA,
PCA, PVA and PVNA) neither on volatile profile nor on
the changes in the volatile composition associated to
deastringency treatment. On the other hand, the possible
participation of volatile compounds in the natural loss of
astringency of PCNA-type persimmons has not been
studied. Rather it is assumed that this is regulated at the
levels of accumulation of phenolic compounds by the
downregulation of expression of structural genes of the
shikimate and flavonoid pathways and those of related
transferases (Akagi et al. 2009).
The objective of this study was to describe the volatile
complement of persimmon using state of the art analytical
techniques coupled to chemometrics analysis (Tikunov
et al. 2005; Zanor et al. 2009) and use this information to
evaluate the volatile profile of 12 cultivars of persimmon
covering [PCNA], [PCA], [PVA] and [PVNA] astringency
types. The influence of persimmon type on the volatile
profile, and the association of specific volatiles with the
desastringency mechanisms operating in PCNA and non-
PCNA fruits as well as the effect of desastringency treat-
ments applied to PCA, PVA and PVNA cultivars on vol-
atile composition is also described.
2 Materials and methods
2.1 Fruit material and postharvest treatment
Persimmon fruit (Diospyros kaki Thumb) from three cul-
tivars of each type PCA, PCNA, PVA and PVNA-type
C. Besada et al.
123
were evaluated and are listed in Table 1. In all cases
seedless (naturally parthenocarpic) fruits were collected at
maturity stage when showing crisp texture and commercial
colour from trees grown in the same orchard (39�34058.2200N,
0�23042.4900W; sandy-loam soil) under standard cultural
practices following Bellini (2002). All the cultivars are
part of the persimmon germplasm collection hosted by the
Instituto Valenciano de Investigaciones Agrarias (IVIA,
Valencia, Spain).
For PCNA-type cultivars, 15 fruit were collected and
evaluated at harvest. For non-PCNA-type cultivars, 30 fruit
were harvested and divided into two homogenous lots of 15
fruit; one lot was evaluated and analyzed at harvest and the
other one was treated with CO2 within the first 6 h in order
to remove astringency before evaluation/analysis.
The deastringency treatment consisted in exposing the
fruit to an open flow of 95 % CO2 at 20 �C and 90 % R.H.
for 24 h. After treatment fruit were stored at 15 �C (R.H.
90 %) for an additional 48 h period before analysis.
Volatile profile and soluble tannin content were ana-
lyzed in fruit just after harvest (for PCA, PCNA, PVA and
PVNA cultivars) and also in fruit after deastringency
treatment followed by 2 days at 15 �C (for PCA, PVA and
PVNA cultivars). Astringency level of the fruit was also
sensory evaluated.
2.2 Tannins determinations and astringency evaluation
Each lot of 15 fruit was divided into three samples (five
fruit per sample) and fruit were cut into four longitudinal
sections. Two of the opposite sections were sliced and
frozen at -20 �C to determine soluble tannins. Another set
of opposite sections from four of the five fruit per sample
were used for sensory evaluation. Opposite sections of the
remaining fruit were immediately frozen in liquid N2,
ground to powder in a precooled household coffee grinder,
and stored at -80 �C for volatile compounds analyses.
Volatiles were analyzed from three biological replicates.
Soluble tannins were evaluated using the Folin-Denis
method (Taira 1995), as described by Arnal and del Rıo
(2004), and results were expressed as % of fresh weight
(FW).
Sensory evaluation of astringency was performed using
composite samples of four fruit (peeled and sliced) per
replicate. A panel of 8–10 people who were trained for
persimmon fruit evaluation were asked to assess astrin-
gency in a 4-point scale, where 1 = very high astringent
fruit and 4 = no astringent. Samples were presented to
members of the panel in trays labeled with random 3-digit
codes and served at room temperature (25 ± 1 �C). The
members of the panel tasted 3 segments of each sample in
order to account for the biological variability of the
material. Milk was provided for palate-rinsing between
samples.
2.3 Volatile compounds analysis
2.3.1 Head space-solid phase microextraction (HS-SPME)
capture conditions
Volatile analysis was performed essentially as described by
Zanor et al. (2009), with minor modifications: 500 mg of
the frozen tissue powder was weighed in a 10 ml crimp cap
vial, capped and incubated at 30 �C for 10 min. Immedi-
ately after incubation 500 ll of 100 mM EDTA-NaOH (pH
7.5) solution and 1.1 g of CaCl2�2H2O were added to ter-
minate endogenous enzyme activity. Vials were closed,
mixed by vortex- agitation for 1 min and transferred to GC
autosampler for analysis. A 50/30 lm DVB/CAR/PDMS
(Supelco, Bellefonte, PA, USA) fiber was used for all the
Table 1 List of persimmon
cultivars analyzed in this study
PCA pollination constant
astringent, PVA pollination
variant astringent, PVNApollination variant non-
astringent, PCNA pollination
constant non-astringent.
Astringency at harvest:
A astringent, NA non-astringent
Cultivar Type Astringency
at harvest
Origin Nomenclature CV
Tomatero PCA A Spain CV1
Reus-6 PCA A Spain CV2
Rojo Brillante PCA A Spain CV3
Aizumishirazu-B PVA A Japan CV4
Reus-15 PVA A Spain CV5
Fuji PVA A Japan CV6
Constantı PVNA A Spain CV7
Kaki Tipo PVNA A Italy CV8
La Selva-14 PVNA A Spain CV9
Jiro (C24276) PCNA NA Japan CV10
O0Gosho PCNA NA Japan CV11
Hana-Fuyu PCNA NA Japan CV12
Volatile compounds of persimmon fruit
123
analysis. Preincubation and extraction time were 10 and
40 min, respectively at a temperature of 80 �C. Desorption
was performed at 250 �C for 1 min in splitless mode.
2.3.2 Gas chromatography–mass spectrometry (GC–MS)
conditions
Volatile organic compounds trapped on the fiber were
analysed by GC–MS using an autosampler COMBI PAL
CTC Analytics (Zwingen, Switzerland), a 6890 N GC
Agilent Technologies (Santa Clara, CA, USA) and a 5975B
Inert XL Mass Selective Detector Agilent, equipped with
an Agilent J&W Scientific DB-5 ms fused silica capillary
column (5 %-phenyl-95 %-dimethylpolysiloxane as sta-
tionary phase, 60 m length, 0.25 mm i.d., and 1 lm
thickness film). Oven temperature conditions were 40 �C
for 2 min, 5 �C/min ramp until 250 �C and then held iso-
thermally at 250 �C for 5 min. Helium was used as carrier
gas at 1.2 mL/min constant flow. Mass/z detection was
obtained by an Agilent mass spectrometer operating in the
EI mode (ionization energy, 70 eV; source temperature
230 �C). Data acquisition was performed in scanning mode
(mass range m/z 35–220; seven scans per second). Chro-
matograms and spectra were recorded and processed using
the Enhanced ChemStation software for GC–MS (Agilent).
2.3.3 Identification and relative quantification
of compounds
Compound identification was based on first the comparison
between the MS for each putative compound with those of
the NIST 2005 Mass Spectral library and in a selected
number of them by the consistency of GC retention time
and Mass Spectra information with that of a customized
library which have been generated using analytical grade
commercial compounds (Sigma-Aldrich Quımica, Spain).
A mixture of extracts representing the twelve varieties
analysed was injected at regular intervals interspersed
throughout the the injection series (every 6 samples) and
was used as a reference for correction of instrument vari-
ability and fiber aging.
A specific ion (m/z) was selected for relative quantifi-
cation of each compound (all this information can be found
in supplementary Table S1). The abundance signal of the
selected ion was related to that of the daily reference
(sample signal/reference signal).
2.4 Statistical analysis
For ANOVA analyses of variance, a multiple comparison
between means was done using the LSD test (P B 0.05)
using the Statgraphics Plus 5.1 software application
(Manugistics, Inc., Rockville, MD, USA).
When Principal Component, Hierarchical Cluster and
Pearson Correlation Analysis were used, data was first log 2
transformed (log 2 (sample signal/reference signal). For
these analyses, data were centred and the program Acuity 4.0
(Axon Instruments) was used. Pearson correlation coeffi-
cient was used as similarity metric and complete linkage as
linkage method for Hierarchical Cluster Analysis.
3 Results
3.1 Soluble tannins content and sensory evaluation
of astringency
A general assessment of the different genotypes in each
type of fruits was made. The content of soluble tannins of
fruit of the different cultivars was determined at harvest
and after postharvest deastringency treatment. This analy-
sis was combined with the sensory evaluation of astrin-
gency made by an expert panel as provided a global view
of the phenotypic variability under study.
To avoid variability due to the effect of seeds in non-
PCNA fruit all the collected samples were carefully
selected to be free of seeds. All persimmon cultivars of the
PCA and PVA-types showed similar levels of soluble
tannins (eliminated the effect of seeds all behave as
expected for astringent fruits), with concentrations close to
0.8 % FW while PVNA-type cultivars exhibited lower
soluble tannins with values around 0.5 % FW. As it was
expected, the concentration of soluble tannins of PCNA
cultivars at harvest was very low: 0.05–0.08 % (Fig. 1a).
Concentrations of soluble tannins of 0.5–0.8 % FW in
fruit from all the non-PCNA cultivars, are indicative of a
high level of astringency, which was confirmed by panel-
ists who evaluated these fruit as strongly astringent (sen-
sory value of 1). In contrast, fruits from PCNA cultivars
were qualified by the sensory panel as non-astringent
(sensory value of 4) consistent with their low content of
soluble tannins already observed at harvest.
Fruit from non-PCNA cultivars (PCA, PVA and PVNA-
type) showed a drastic decrease in the content of soluble
tannins with respect to those at harvest when submitted to
deastringency treatment with CO2 (Fig. 1a). Fruits from
‘Rojo Brillante’, ‘Constantı’, ‘Kaki Tipo’ and ‘La Selva-
14’ (CV3, CV7, CV8 and CV9) showed values lower than
0.04 %, ‘Aizumishirazu-B’ and ‘Fuji’ (CV4, CV6) values
of 0.05 %, ‘Tomatero’ and ‘Reus-6’ (CV1, CV2) presented
a content which was slightly over 0.06 % while the highest
content of soluble tannins after treatment was observed in
‘Reus-15’ (CV5) (0.08 % FW) (Fig. 1b). It is also inter-
esting to note the large standard deviation of the data from
‘Reus-15’ as compared to all the other cultivars, that may
suggest that the deastringency treatment in this cultivar is
C. Besada et al.
123
not as reliable as in the others. Consistent with that, sensory
evaluation of fruit treated with CO2 reported absence of
astringency (sensory value of 4) in all the cultivars except
on ‘Reus-15’, in which residual or medium astringency was
detected in the evaluated samples (sensory value of
2.1 ± 0.65).
3.2 Volatile compounds of persimmon: principal
components and hierarchical cluster analysis
The use of HS-SPME coupled with GC–MS for the anal-
ysis of persimmon fruits allowed the identification of 67
volatile compounds: 10 aldehydes, 5 phenylaldehydes, 7
ketones, 8 phenylketones, 10 esters, 7 carboxylic acids, 13
alcohols, 4 phenylalcohols, 2 phenylfuranes and carbon
dioxide. Compound identification was carried out by using
analytical grade commercial compounds when available
(unequivocally identification) or by matching their mass
spectra to the NIST library (putative identification) (Sup-
plementary Table S1). Among the total number of com-
pounds detected (74), seven of them remained not
identified.
Principal components analyses of the total volatile
dataset indicated that the first two principal components
explain 67 % of the total variance (Fig. 2a; Supplementary
Table S2). Clearly, the first two components separate per-
simmon samples into three groups: a first group of fruit
non-astringent naturally, that is PCNA-type cultivars at
harvest (CV10, CV11, CV12), a second group of fruit
astringent at harvest that includes PCA (CV1, CV2, CV3),
PVA (CV4, CV5, CV6), PVNA (CV7, CV8, CV9) type
cultivars and a third group including the fruit from astrin-
gent cultivars at harvest that lost the astringency after
subjected to deastringency treatment with CO2 (PCA: CV1,
Fig. 1 Soluble tannins content (% fresh weight) of the twelve
studied persimmon cultivars (abbreviations according to Table 1),
just after harvest (harvest) and after deastringency treatment (after
CO2) (a). Amplified figure of soluble tannin content after deastrin-
gency treatment (b). Vertical bars represent Standard Deviation
Fig. 2 Principal component analysis of volatile compounds of
persimmon fruit from the total data set. Score and loading plots for
the first, second and third principal components are shown. Three
individual fruit were analyzed for each cultivar at harvest and after
deastringency treatment. The code of colours is according to Fig. 3
(Color figure online)
Volatile compounds of persimmon fruit
123
CV2, CV3; PVA: CV4, CV5, CV6; PVNA-CV7, CV8,
CV9). It is interesting that according to the first component
(which explains 46 % of the variance) astringent fruits are
located in the middle and the two non-astringent groups are
in opposite directions from the astringent one, this indi-
cating that the natural and CO2 induced non-astringent fruit
differ significantly in their volatile profile. The first com-
ponent also separates the treated samples (to the left) from
untreated to the right.
The second component explained 21 % of the variance
and mainly separates fruit according to astringency, that is
non-astringent fruit (PCNA-type at harvest and non-
PCNA-type cultivars after deastringency treatment) from
astringent fruit (non-PCNA-type at harvest). The separa-
tion between PCNA at harvest and non-PCNA after CO2
also suggest that developmental/natural and postharvest-
induced deastringency may share some characteristics,
however they must have their own specific volatiles asso-
ciated to it, with more dramatic effects on PCNA fruits at
harvest.
The loadings plot reveals the main compounds respon-
sible of the separation among groups of samples (Fig. 2b;
Supplementary Table S2). The most relevant compounds
for the first component were ethanol, o-hydroybiphenil,
acetaldehyde and also benzeneacetaldehyde, 3(2H)-ben-
zofuranone and dihydrobenzofuran. The second component
was mainly defined by 3(2H)-benzofuranone, b-damasce-
none, a group of aldehydes (acetaldehyde, benzeneacetal-
dehyde, decanal, octanal, heptanal and hexenal) and the
following compounds dihydrobenzofuran, o-hydroxybi-
phenyl and ethanol. The loadings plot also indicates that
broadly speaking accumulation of 3(2H)-benzofuranone is
characteristic of PCNA fruit at harvest, and ethanol of fruit
treated with CO2.
Application of the Hierarchical Cluster Analysis to the
total data set showed the samples organized in two main
clusters, which have been named as cluster H and cluster D
(Fig. 3). Cluster H included samples for the 12 studied
cultivars at the moment of harvesting, while cluster D
included those samples submitted to deastringency treat-
ment with high concentrations of CO2. Subcluster H1
grouped those samples from cultivars non-astringent at
harvest (subcluster H1a: PCNA-type cultivars) and a group
of three astringent cultivars (subcluster H1b: CV1, CV2
and CV5) while subcluster H2 grouped samples from
PVNA-type cultivars and a group of three cultivars also
astringent at harvest (subcluster H2b: CV3, CV4 and CV6).
The cluster D, including all the fruit treated for deastrin-
gency, is divided in subcluster D1 which grouped cultivars
CV1, CV2 and CV5 and subcluster D2 where were inclu-
ded the rest of the treated samples.
3.3 Differences in the volatile profile among groups
of samples
In order to further explore the differences on individual
volatile compounds between the different groups of sam-
ples, Principal Component and ANOVA analyses were
applied to the following data sets: (1) PCNA cultivars and
non-PCNA cultivars at harvest, in order to reveal the dif-
ferences in volatile compounds associated to astringency/
non astringency at harvest (2) non-PCNA cultivars (PCA,
PVA, PVNA- cultivars) at harvest, to reveal the differences
in volatile profiles among the different astringent cultivars
(3) non-PCNA cultivars at harvest and non-PCNA cultivars
after deastringency treatment, to reveal the effect of the
deastringency treatment on the volatile profile (4) non-
PCNA cultivars after deastringency treatment application
and PCNA cultivars at harvest, to reveal the differences in
volatile profile between developmental deastrigency and
that induced by CO2 (Fig. 4; Tables 2, 3). Moreover, those
compounds revealed as especially relevant for separation
among the mentioned groups of samples, were submitted to
ANOVA analyses considering the total dataset (Fig. 5).
Fig. 3 Hierarchical cluster analysis of persimmon samples accord-
ing to their volatile profiles. Cultivar name nomenclature is according
to Table 1. Three biological replicate fruits (f1, f2 and f3) were
analyzed for each cultivar and stage including harvest (h) and after
deastringency treatment (d) (Color figure online)
C. Besada et al.
123
Fig. 4 Principal component analysis of volatile compounds of persim-
mon fruit. Score and loading plots for the first, second and third principal
components are shown. a, b Data set at harvest (PCNA and non-PCNA
cultivars). c, d Astringent cultivars (non-PCNA) at harvest. e, f non-
PCNA cultivars after submitted to deastringency treatment. Cultivar
nomenclatures are according to Table 1. Three individual fruit were
analyzed for each cultivar at harvest and after deastringency treatment.
The code of colours is according to Fig. 3 (Color figure online)
Volatile compounds of persimmon fruit
123
Table 2 ANOVA analysis
comparing the level of volatile
compounds among groups of
persimmon samples at harvest
Comparison of the relative
abundance (sample signal/
reference signal) among PCNA
and non-PCNA cultivars is
shown in the columns on the leftand comparison among
astringent cultivars (CV7, CV8,
CV9/CV3, CV4, CV5/CV1,
CV2,CV5) in the columns onthe right. Different letters in the
same row implies significant
differences (P value \ 0.05 %).
Only those compounds that
showed significant differences
are presented (– no significant
differences among the group of
samples). Bold values were used
in those compounds changing
two-fold or more among groups
nd non detected
At harvest Non-PCNA at harvest
PCNA Non-PCNA cvs. 7, 8, 9 cvs. 3, 4, 6 cvs. 1, 2, 5
Aldehydes
Hexanal 2.08b 0.82a 0.89b 0.93b 0.63a
Heptanal 1.69b 0.78a 0.73a 0.97b 0.66a
Octanal 2.37b 0.69a – – –
Decanal 2.10b 0.56a – – –
(E,E)-2,4-heptadienal – – 1.50b 1.77b 0.99a
(E)-2-octenal – – 1.02a 1.54b 0.95a
(Z)-2-nonenal – – 1.01a 1.24b 0.94a
Phenylaldehydes
Benzeneacetaldehyde 13.03b 2.52a 4.20b 1.03a 2.32a
Hexylcinnamaldehyde – – 0.85a 1.03ab 1.35b
Ketones
b-Damascenone 4.40b 1.43a 2.11c 0.76a 1.41b
6-Methyl-5-hepten-2-one 2.26b 1.09a 0.97a 1.11ab 1.20b
(-)-Bornan-2-one 1.41b 1.07a
2-Dodecanone – – 0.88a 1.08ab 1.21b
2-Undecanone – – 0.88a 1.06ab 1.14b
Geranyl acetone 1.12b 0.79a 0.82ab 0.68a 0.87b
Phenylketones
Hydroxycyclohexyl phenyl ketone 1.51b 1.08a – – –
Acetophenone – – 1.25b 1.08ab 1.04a
3-Ethylacetophenone 1.32b 0.98a – – –
2,4-Dimethylacetophenone 1.23b 1.00a 1.04b 0.91a 1.06b
3-Methylacetophenone 1.35b 1.13a – – –
Esters
Diisobutyl succinate 0.53a 0.81b – – –
Isopropyl decanoate 1.33b 0.75a – – –
Hexyl salicylate – – 0.92a 1.12ab 1.37b
Acids
Acetic acid 0.99b 0.63a 0.63a
2,2-Dimethylpropanoic acid 0.67a 1.06b 0.84a 1.26b 0.87a
Heptanoic acid 0.90a 1.08b 1.03a 1.20b 1.01a
Nonanoic acid 0.62a 0.82b – – –
Dodecanoic acid 0.61a 0.80b – – –
Alcohols
Ethanol 0.03a 0.07b 0.07ab 0.09b 0.04a
1-Hexanol 0.97a 1.14b 1.27b 1.13ab 1.00a
1-Dodecanol – – 0.83a 1.06ab 1.27b
Longiborneol – – 0.91a 1.07ab 1.20b
Phenylalcohols
Phenol – – 2.39b 0.88a 0.74a
o-Hydroxybiphenyl – – 0.22ab 0.19a 0.27b
5-Indanol – – 1.08a 1.07a 1.64b
Benzyl alcohol 1.21b 0.98a – – –
Phenylfuranes
Dihydrobenzofuran 2.37b 1.13a 0.36a 0.22a 2.81b
3(2H)-benzofuranone 138.8 nd – – –
C. Besada et al.
123
Table 3 ANOVA analysis comparing the level of volatile compounds among groups of persimmon samples at harvest and after CO2 treatment
Non-PCNA Non-astringent
Harvest After CO2 PCNA harvest Non-PCNA after CO2
Aldehydes
Acetaldehyde 0.51a 2.23b 0.68a 2.23b
(E)-(3,3-dimethylcyclohexylidene) acetaldehyde – – 1.32b 1.03a
Hexanal – – 2.08b 0.96a
Heptanal 0.78a 1.04b 1.69b 1.04a
Octanal 0.69a 1.16b 2.37b 1.16a
Decanal 0.56a 1.56b 2.10b 1.56a
(Z)-2-nonenal – – 1.40b 1.09a
(E)-2-decenal 0.88a 0.99b 1.32b 0.99a
Phenylaldehydes
3,4-Dimethyl-benzaldehyde 0.99b 0.77a – –
Benzeneacetaldehyde – – 13.03b 1.98a
Hexylcinnamaldehyde 1.07b 0.84a – –
Ketones
b-Damascenone 1.43a 6.40b – –
6-Methyl-5-hepten-2-one – – 2.26b 1.00a
(-)-Bornan-2-one – – 1.41b 1.02a
2-Dodecanone 1.06b 0.78a 0.99b 0.78a
2-Undecanone 1.03b 0.78a 1.02b 0.78a
Geranyl acetone 1.12b 0.82a
8,8,9-Trimethyl-deca-3,5-diene-2,7-dione 1.03b 0.86a 1.12b 0.86a
Phenylketones
Hydroxycyclohexyl phenyl ketone – – 1.51b 1.01a
4-Methylbenzophenone 1.04b 0.88a 1.20b 0.88a
3-Ethylacetophenone – – 1.32b 1.01a
2,4-Dimethylacetophenone 1.00b 0.92a 1.23b 0.92a
3-Methylacetophenone 1.13b 0.99a 1.36b 0.99a
Benzophenone 1.07b 0.95a 1.30b 0.95a
2,6-Di-tert-butylbenzoquinone 0.97b 0.67a 1.05b 0.67a
Esters
2-Ethylhexyl acetate 0.99b 0.84a 1.01b 0.84a
Ethyl methanoate 1.06a 1.36b 1.01a 1.36b
Octyl acrylate 1.00b 0.75a 0.99b 0.75a
Octyl propanoate 0.95b 0.73a 0.99b 0.73a
Diisobutyl succinate 0.81a 1.03b 0.53a 1.03b
Isopropyl decanoate 0.75a 1.09b – –
1-Methoxy-2-propanol acetate 1.20b 1.05a 1.23b 1.05a
Hexyl salicylate 1.14b 0.76a 0.89b 0.76a
Acids
Acetic acid 0.75a 1.33b – –
2,2-Dimethylpropanoic acid – – 0.68a 1.02b
Octanoic acid 0.92a 0.98b – –
Nonanoic acid 0.82b 0.67a – –
Dodecanoic acid 0.80a 1.07b 0.61a 1.07b
Alcohols
Ethanol 0.07a 3.34b 0.03a 3.34b
Volatile compounds of persimmon fruit
123
3.3.1 Analysis at harvest of volatile profile of fruits
from naturally non-astringent and astringent
cultivars
Principal component analyses of the dataset corresponding
to all fruits at harvest clearly separated non-astringent from
astringent fruit (Fig. 4a; Supplementary Table S3) and
revealed that being astringent or not at harvest was the
major source of variability (54 %).
Loadings plot (Fig. 4b; Supplementary Table S3) and
the comparison of PCNA with non-PCNA (PCA, PVA,
PVNA) cultivars at harvest by means of ANOVA analysis
(Table 2) revealed an important group of aldehydes,
ketones, phenylketones and phenylfuranes that accumulate
to higher levels in PCNA cultivars than in non-PCNA
cultivars. Among these compounds hexanal, heptanal,
octanal, decanal, benzeneacetaldehyde, b-damascenone,
6-methyl-5-hepten-2-one, dihydrobenzofuran and 3(2H)-
benzofuranone were those showing major differences
between PCNA and non-PCNA cultivars. The levels of
several organic acids, including heptanoic, nonanoic, do-
decanoic and 2,2-dimethylpropanoicacid were lower in
PCNA cultivars when compared to those in non-PCNA-
type fruit. No clear tendency was observed regarding esters
and alcohols, since levels of some of these compounds
(benzyl alcohol and isopropyl decanoate) were higher in
non-astringent fruit (PCNA-type) while other ones (etha-
nol, 1-hexanol and diisobutyl succinate) were higher in
astringent fruit (non-PCNA-type). The volatile compounds
showing the largest differences in accumulation between
PCNA and non-PCNA cultivars at harvest were benzene-
acetaldehyde and 3(2H)-benzofuranone. Both compounds
were present at higher levels in PCNA cultivars (Table 2;
Fig. 5a, b) and while the rest of volatile compounds are
present in all varieties and contribute to separation between
groups due to differential accumulation levels, 3(2H)-
benzofuranone is the only compound that was specifically
detected in a group of samples (Fig. 5a; Supplementary
Fig. S1). The identification of both compounds 3(2H)-
benzofuranone (Supplementary Fig. S2) and benzenacet-
aldehyde was confirmed by comparison of its retention
time and mass spectrum to the analytical grade commercial
compound.
As some volatiles such as acetaldehyde seem to partic-
ipate in the loss of astringency in a direct manner, corre-
lations among soluble tannins content and volatile
compounds levels at harvest were investigated. Results
indicated that strong inverse correlations (r \ -0.8) were
obtained for a number of compounds that included octanal,
benzeneacetaldehyde, decanal, 3(2H)-benzofuranone,
3-ethylacetophenone and hexanal (Supplementary Table
S4). These compounds showed accumulation levels much
higher at harvest in non-astringent fruits (low levels of
soluble tannins) than in astringent fruit, suggesting that
they could be used as markers for deastringency and at the
same time some of them could participate in the actual
developmental deastringency/tannin insolubilization in
PCNA fruits.
Table 3 continued
Non-PCNA Non-astringent
Harvest After CO2 PCNA harvest Non-PCNA after CO2
1-Hexanol – – 0.97a 1.15b
Neoisomenthol – – 1.11b 0.97a
3-Methyl-1-heptanol – – 1.17b 1.05a
1-Undecanol 1.00b 0.84a – –
Longiborneol 1.06b 0.78a – –
Phenylalcohols
Phenol – – 2.09b 0.91a
o-Hydroxybiphenyl 0.22a 1.55b 0.18a 1.55b
Benzyl alcohol 0.98b 0.76a 1.21b 0.76a
Phenylfuranes
Dihydrobenzofuran – – 2.37b 1.26a
3(2H)-benzofuranone – – 138.8 nd
Comparison of the relative abundance (sample signal/reference signal) among non-PCNA cutlivars at harvest and after submitted to deastrin-
gency CO2-treatment is shown in the columns on the left and comparison among PCNA cultivars at harvest and non-PCNA cultivars after
submitted to deastringency CO2-treatment in the columns on the right. Different letters in the same row implies significant differences
(P value \ 0.05 %). Only those compounds that showed significant differences are presented (– no significant differences among the group of
samples). Bold values were used in those compounds changing two-fold or more among groups
nd non detected
C. Besada et al.
123
Fig. 5 ANOVA analysis comparing the level of volatile compounds
among all the samples of persimmon fruit studied. The relative
abundance (sample signal/reference signal) of each compound was
compared among samples. Cultivar nomenclature, 1 to 12 in x axis, is
according to Table 1. It must be note that in this figure vertical barsrepresent the lower standard deviation, LSD, (P value \ 0.05 %)
when ANOVA was performed taking in account all the studied types
of samples
Volatile compounds of persimmon fruit
123
3.3.2 Intervarietal discrimination among astringent
cultivars at harvest
When principal component analyses was applied to the set
of data corresponding to astringent fruit at harvest (Fig. 4c;
Supplementary Table S5) it produced three separated
groups of samples: PVNA-type cultivars (CV7, CV8 and
CV9), a second group including CV3, CV4 and CV6 and a
third group showing together CV1, CV2 and CV5; this
separation is in accordance with the organization revealed
by the hierarchical cluster analysis from the total data set
(subcluster H2a, H2b and H1b respectively) (Fig. 3).
Loadings plot (Fig. 4d; Supplementary Table S5) and
ANOVA analysis (Table 2) revealed that PVNA-group
(CV7, CV8 and CV9) was characterized by high levels of
benzeneacetaldehyde, b-damascenone and phenol while
CV1, CV2 and CV5 shown increased levels of dihydro-
benzofuran respect on the other astringent fruit. In general
CV3, CV4 and CV6 showed the lowest levels of the
mentioned compounds as well as slightly higher level of
ethanol, 2,2-dimethylpropanoic acid and unsaturated alde-
hydes (E)-2-octenal and (Z)-2-nonenal (Table 2).
It is important to note that the levels of dihydrobenzo-
furan detected in CV1, CV2 and CV5 were similar to those
of non-astringent cultivars (PCNA) (Fig. 5c). Observation
of principal component analysis applied to the total set of
data at harvest (Fig. 4a) revealed that PCNA cultivars and
CV1, CV2 and CV5 may be grouped together and sepa-
rated from rest of cultivars according to the third compo-
nent (explaining 7.2 % of variance). Loadings plot showed
dihydrobenzofuran as the main compound closing PCNA
cultivars and CV1, CV2 and CV5 (Fig. 4b). According
with this, both groups of samples are clustered together
(Cluster H1) in the hierarchical cluster analysis (Fig. 3).
3.3.3 Effect of deastringency treatment
The effect of deastringency treatment on acetaldehyde and
ethanol production has been widely studied, since acetal-
dehyde produced under anaerobic conditions is directly
implied in the removing of astringency with high CO2
concentration (Pesis and Ben-Arie 1984; Taira et al. 1989;
Besada et al. 2010). An ANOVA analysis applied to the
subdataset corresponding to fruits of astringent cultivars at
harvest and after astringency removal by high CO2 con-
centration treatment, revealed the changes in volatile pro-
file associated to deastringency treatment application
(Table 3). The deastringency treatment resulted in the most
of the studied cultivars in dramatic increases in acetalde-
hyde and ethanol (Fig. 5d, e). Deastringency treatments
have additional effects and all treated non-PCNA fruit
showed higher concentrations of lipid-derived aldehydes,
mainly decanal but also octanal and heptanal than fruit at
harvest (Table 3; Fig. 5f–h); in contrast, phenylaldehydes
such as 3,4-dimethyl-benzaldehyde and hexylcinnamalde-
hyde levels were depressed after treatment (Table 3). Also
an important number of ketones and phenylketones showed
reduced levels after application of high concentration of
CO2, however level of the carotenoid-derived b-damasce-
none increased dramatically. Alcohols showed no clear
trend since some of them were reduced after treatment (1-
undecanol, longiborneol and benzyl alcohol) while others
were increased (ethanol and o-hydroxybiphenyl).
Principal component analysis on the subset of data from
fruit submitted to CO2 treatment (Fig. 4e; Supplementary
Table S6) separated the cultivars in two groups, the first
including CV1, CV2 and CV5 and the second group
including CV3, CV4, CV6, CV7, CV8 and CV9, which
correspond respectively with subclusters D1 and D2 gen-
erated by the hierarchical cluster of the total data set
(Fig. 3). Observation of the loadings plot (Fig. 4f; Sup-
plementary Table S6) reveals acetaldehyde, ethanol and
dihydrobenzofuran as the compounds mainly responsible
for separation among the both groups of cultivars (Fig. 4f).
Cultivars CV1, CV2 and CV5 (‘Tomatero’, ‘Reus-6’ and
‘Reus-15’ respectively) showed after deastringency treat-
ment higher concentration of dihydrobenzofuran and lower
of ethanol than the rest of treated cultivars while no
increment of acetaldehyde was observed, showing these
three compounds similar levels to those of PCNA cultivars
(Fig. 5c–e). It would be expected that those cultivars in
which acetaldehyde was not enhanced by CO2-treatment
would not show a decrease of soluble tannins. However,
despite the remained level of soluble tannins after treat-
ment was higher in CV1, CV2 and CV5 cultivars than in
the rest of the treated samples, these three cultivars showed
an important decrease in the levels of soluble tannins after
CO2-treatment when compared on content at harvest
(Fig. 1). Therefore, tannins insolubilization seems to be
non-acetaldehyde dependent in CV1, CV2 and CV5. This
suggests that these three cultivars and PCNA may share
some aspects of the volatiles associated to deastringency
process.
Correlation analysis of the concentrations of soluble
tannins and volatiles showed that the decrease in soluble
tannins following deastringency treatment was inversely
correlated to acetaldehyde (r = -0.84) and positively
correlated to dihydrobenzofuran levels (r = 0.86), this
indicating that high levels of acetaldehyde are associated
with low residual concentration of soluble tannins after
treatment, and that high levels of dihydrobenzofuran are
linked to high content of residual soluble tannins after
treatment application. Correlation analysis among volatile
compounds showed that acetaldehyde concentration after
deastringency process was positively correlated to ethanol
(r = 0.69) and as expected it was negatively correlated to
C. Besada et al.
123
dihydrobenzofuran (r = -0.78). This is in accordance with
the fact that CV1, CV2 and CV5 with the highest levels of
dihydrobenzofuran, not only after treatment but also at
harvest (Fig. 5c), were the only cultivars that did not
exhibited an increase in the concentration of acetaldehyde
after the fruit had been submitted to CO2-treatment
(Fig. 5d).
3.3.4 Differences between natural non-astringent fruit
and non-astringent fruit as result of deastringency
treatment
As mentioned above, principal components analysis of the
total data set (Fig. 2a) revealed that naturally-non-astrin-
gent fruit at harvest and fruit treated for deastringency are
in opposite directions from the astringent fruit at harvest,
this indicating important differences in the volatile com-
position of both groups of samples.
The largest differences between these two groups of non-
astringent fruit were observed in the levels of the following
volatiles: acetaldehyde, hexanal, octanal, benzeneacetalde-
hyde, 6-methyl-5-hepten-2-one, ethanol, phenol, o-hydrox-
ybiphenyl and 3-2H-benzofuranone (Table 3). Exposure to
CO2 resulted on high levels of acetaldehyde, ethanol and
o-hydroxybiphenyl in the most of the treated cultivars,
however fruit naturally-non-astringent showed low con-
centration of these volatiles (Fig. 5d, e; Table 3).
As mentioned, PCNA fruit exhibited high levels of
3(2H)-benzofuranone and benzeneacetaldehyde (Fig. 5a,
b; Table 3).
Lipid-derived aldehydes hexanal, heptanal, octanal and
decanal were increased after CO2-treatment however con-
centrations of these volatile compounds were even higher
in PCNA fruit (Fig. 5f–h; Table 3). An important number
of phenylketones and ketones, mainly 6-methyl-5-hepten-
2-one, were present at lower levels in CO2-treated fruit
respect to natural non-astringent fruit (Table 3).
4 Discussion
The analytical technique used in this work, HS-SPME
coupled with GC–MS, allowed the identification of 67
volatile compounds on persimmon samples. It is important
to note that the previous studies on persimmon volatile
profile reported only 15 (Horvat et al. 1991) and 23 com-
pounds (Taira et al. 1996), therefore HS–SPME–GC–MS is
confirmed here to be a powerful technique for the study of
volatiles in fruit and particularly in persimmon. Regarding
the previously described persimmon volatiles, Horvat et al.
(1991) reported bornyl acetate and (E)-2-hexenal as the
major volatile compounds identified from persimmon flesh,
while (E)-2-hexenol, benzeneacetaldehyde, phenylethyl
acetate, borneol, benzothiazole, neryl acetate, palmitic acid
and two saturated normal hydrocarbons (C23H48 and
C25H52) were identified as minor constituents. On the
other hand, Taira et al. (1996) found that n-butanol,
n-hexanol, (Z)-3-hexen-1-ol, 2-methyl hexanol, acetoin,
and acetic acid were compounds common to the three
varieties under their study (‘Hiratanenashi’, ‘Yokono’ and
‘Atago’). Among these compounds, in the present study
benzeneacetaldehyde, 1-hexanol and acetic acid were
identified in the cultivars of persimmon analyzed and we
also detected some related compounds such as (-)-bornan-
2-one as well as a high number of aliphatic aldehydes, both
saturated (hexanal, heptanal, octanal, decanal) and unsat-
urated ((E)-2-octenal, (Z)-2-nonenal, (E)-2-decenal, (E,E)-
2,4-heptadienal) and alcohols such as 2-ethyl-1-butanol,
2-ethyl-1-hexanol or 3-methyl-1-butanol. The fact that only
few of the volatile compounds previously reported to be
majority in persimmon flesh were not detected in our
samples is difficult to explain but it may be due to cultivar
specificity, extraction and analytical methods and even to
environmentally induced variability.
This is the first time that the volatile profile of fruit
covering the four types of persimmon cultivars according
to the astringency is studied. Our results show clearly a
different volatile profile at harvest of PCNA cultivars with
respect to non-PCNA cultivars (PCA, PVA, PVNA). Dif-
ferences in the volatile content of fruits that are non-
astringent naturally could give clues about the mechanism
responsible for PCNA fruit losing their astringency on tree.
This trait has become one of the most important breeding
objectives for persimmon in the last years. In this direction,
our results indicates that in contrast to fruits treated with
CO2 for deastringency, fruits of ‘Jiro (C24276)’, ‘O0Gosho’
and ‘Hana-Fuyu’ (CV10, CV11 and CV12 respectively)
that have lost astringency on the tree naturally do not
accumulate acetaldehyde or ethanol. These fruits however
accumulate high levels of benzenealdehyde and lipid-
derived aldehydes such as hexanal, heptanal, octanal and
decanal. The possibility exists for these compounds are
playing a role in natural deastringency of PCNA fruits in
the tree similar to that of the acetaldehyde in the loss of
astringency during postharvest deastringency treatment in
non-PCNA.
Another compound 3(2H)-benzofuranone accumulates
in PCNA fruit and it is not detected in any non-PCNA
cultivar (PCA, PVA and PVNA-type). Little information
exists regarding presence of 3(2H)-benzofuranone as nat-
ural product. It has been detected tentatively on faham
leaves (Cheong Sing and Smadja 1992) but it has never
been reported in fruit. On the other hand, the formation
of substituted 3(2H)-benzofuranone has been linked to
flavonoid oxidation process. In this way, 2-(3,4-di-
hydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone
Volatile compounds of persimmon fruit
123
and 2-(4-hydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzof-
uranone have been identified as oxidations products for
quercetin and kaempferol, respectively (Jorgensen et al.
1998). Similarly, Jungbluth et al. (2000) identified 2-(hy-
droxybenzoyl)-2-hydroxybenzofuran-3(2H)-one as product
of flavanol oxidation. It is know that the soluble tannins
responsible for astringency of non-PCNA-type cultivars are
basically polymers formed by flavan-3-ol subunits (Tanaka
et al. 1994; Matsuo and Itoo 1978). We are currently car-
rying out experiments in order to elucidate the mechanism
by which the accumulation of 3(2H)-benzofuranone in
PCNA cultivars may be linked to naturally astringency loss
in PCNA fruit.
Among the astringent cultivars at harvest, the volatile
profiles of PVNA fruit, ‘Constantı’, ‘Kaki Tipo’ and ‘La
Selva-14’ (CV7, CV8 and CV9 respectively), which showed
the lowest content of soluble tannins at harvest, were char-
acterized by high levels of benzeneacetaldehyde, again
indicating that this compound may be implied in natural loss
of astringency. The rest of the astringent cultivars are sepa-
rated according to their volatile profile into two groups,
‘Tomatero’, ‘Reus-6’ and ‘Reus-15’ (CV1, CV2 and CV5)
and ‘Rojo Brillante’, ‘Aizumishirazu-B’ and ‘Fuji’ (CV3,
CV4 andCV6). Cultivars CV1, CV2, CV5 were character-
ized by levels of dihydrobenzofuran higher than those of the
rest of astringent cultivar and similar to those of non-
astringent fruit. Recently, Naval et al. (2010) generated a
neighbor joining cladogram based on genetic markers for 71
cultivars of persimmon. Except for the cultivar CV3, paral-
lelism can be observed among the genetic tree and the one for
astringent cultivars at harvest based on to their volatile
profile. Naval et al. (2010) showed CV1, CV2 and CV5
closely related and different from the rest; similar groupings
were observed for CV7, CV8 and CV9 and for the cultivars
CV4 and CV6. It seems therefore that the volatile profile of
astringent fruit reflects the genetic divergence between cul-
tivars and therefore some of the volatiles could be used as
useful phenotypic markers.
We have also described for first time the changes in
volatile compounds associated to application of deastrin-
gency treatment to different type (PCA, PVA, PVNA) of
persimmon cultivars. When fruit from astringent cultivars
were submitted to CO2 treatment most of the cultivars
increased the levels of ethanol and acetaldehyde, however
cultivars, CV1, CV2 and CV5 (‘Tomatero’, ‘Reus-6’ and
‘Reus-15’) shown a different response, with very low
acetaldehyde and ethanol levels. Although sensory astrin-
gency was only detected in ‘Reus-15’ fruit, the residual
soluble tannins after deastringency process of these three
cultivars were slightly higher than in the rest of the vari-
eties. In the persimmon cultivar ‘Bull Heart’, it has been
also reported that the astringency removal under anaerobic
conditions was not linked to and increment of acetaldehyde
(Tsou and Lin 2009). On the other hand, our result shows
that the production of acetaldehyde after treatment is
inversely correlated to the level of dihydrobenzofuran at
harvest. In a recent study we have observed that when fruit
naturally-non-astringent (PCNA group), which also show
high levels of dihydrobenzofuran at harvest, were submit-
ted to the treatment with high concentrations of CO2 in the
same conditions described in this work, the level of acet-
aldehyde was not increased (data not published). It seems
therefore, that a link exists among high levels of dihydro-
benzofuran at harvest and the lack of accumulation of
acetaldehyde when the fruit is submitted to high concen-
tration of CO2 and that dihydrobenzofuran levels could be
used to predict the cultivar response to CO2-deastringency
treatment.
CO2-treatment resulted consistently in enhanced the
levels of lipid-derived aldehydes, mainly decanal but also
octanal and heptanal. Such increment of lipid-derived
volatiles is probably associated to the degradation of
membranes as consequence of CO2 application, as micro-
structural studies suggest (Salvador et al. 2007). An
increase of lipid-derived aldehydes as consequence of high
CO2 exposure has also been reported in ‘Fuji’ apples,
which showed higher concentrations of octanal, nonanal
and decanal after been exposed to high CO2 atmosphere,
besides C3-C6 alcohols that were inhibited by hypoxia
conditions (Argenta et al. 2004).
It have been reported the reaction of tannins from wine,
wood or tea with different class of aldehydes, such as fur-
furaldehyde (Nonier et al. 2006), ethyl-5-furfuraldehyde,
hydroxymethyl-furfuraldehyde, vanillin, and syringalde-
hyde (Vivas et al. 2008), cinnamaldehyde, formaldehyde,
trans-2-hexenal and citral, citronellal, coniferyl aldehyde or
sinapyl aldehyde (Tanaka et al. 2010). We suggest that lipid-
derived aldehydes (decanal, octanal and heptanal) increased
by deastringency treatment, may be playing a role similar to
that of acetaldehyde in the insolubilization of tannins of the
cultivars ‘Tomatero’, ‘Reus-6’ and ‘Reus-15’ (CV1, CV2
and CV5), in which accumulation of acetaldehyde was not
observed. We also propose that in PCNA fruits aldehydes
other than acetaldehyde may work for tannin insolubilization
and corresponding natural astringency loss.
5 Concluding remarks
In summary, among the 67 volatile compounds detected in
persimmon, in addition to acetaldehyde which is known to
be implicated in the artificial removal of astringency,
benzeneacetaldehyde, lipid-derived aldehydes (mainly
decanal, octanal and heptanal), and the two phenylfura-
nones (dihydrobenzofuran and 3(2H)-benzofuranone) have
been identified as candidates to play a role in the process of
C. Besada et al.
123
loss of astringency in persimmon. In addition to formulate
the hypothesis that other aldehydes appear to be involved
in astringency loss in persimmon, the identified volatile
organic compounds may be used as metabolite markers for
naturally and/or artificially astringency loss.
Acknowledgments This study has been supported by ‘Conselleria
d’Agricultura of Valencian Community’ and ‘Guarantee of Origin
Kaki Ribera del Xuquer’ (Valencia, Spain), the Spanish ‘Ministerio
de Educacion y Ciencia’ (Project INIA-RTA 2010-00080-00-00) and
Feder program from the EU. Cristina Besada is contracted by the
‘Conselleria d’Educacio of Valencian Community’. Gerardo Sanchez
holds a fellowship from INTA (Instituto Nacional de Tecnologıa
Agropecuaria, Argentine). The authors thank Ph.D. Marisa Badenes
for the supply of persimmon fruits. Metabolite profiling was con-
ducted at the Metabolomics Lab of IBMCP.
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