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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