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Modificaciones en factores relacionados con el
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J. Agric. Food Chem. 2011, 59, 335–341 335
DOI:10.1021/jf1035959
Preharvest Calcium Sprays Improve Volatile Emission at

Commercial Harvest of ‘Fuji Kiku-8’ Apples
ABEL ORTIZ,† JORDI GRAELL,‡ AND ISABEL LARA*,†
†
Departament de Quı́mica, Unitat de Postcollita-XaRTA, Universitat de Lleida, Alcalde Rovira Roure
191, 25198 Lleida, Spain, and ‡Departament de Tecnologı́a d’Aliments, Unitat de Postcollita-XaRTA,
Universitat de Lleida, Alcalde Rovira Roure 191, 25198 Lleida, Spain
Apple (Malus domestica Borkh.) fruit intended for long-term storage are frequently harvested com-
mercially before becoming fully ripe, often resulting in poor aroma development. Since postharvest
calcium dips have proved effective for the enhancement of flavor-related volatile esters after cold
storage of apples, this study was undertaken in order to assess whether preharvest calcium sprays
(7 weekly applications at 1.6%, w/v, 81-123 days after full bloom) could also aid in improving this
important attribute at harvest. This procedure significantly increased calcium content in treated fruit.
The emission of aroma-related volatile esters by untreated and calcium-treated ’Fuji’ apples was then
monitored during maturation and ripening over two months prior to commercial harvest. Results
indicate that most of the compounds contributing to overall flavor in ripe fruit were enhanced in
response to preharvest calcium applications, suggesting that this procedure may be suitable for the
improvement of fruit aroma at harvest. The emission of acetate esters was particularly favored,
consistent with higher acetaldehyde contents in treated fruit. These effects arose apparently from
increased pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) activities, possibly leading
to a better supply of alcohols and acyl CoAs for ester biosynthesis.
KEYWORDS: Alcohol o-acyltransferase; alcohol dehydrogenase; apple; aroma; preharvest calcium

sprays; pyruvate decarboxylase; volatile esters
INTRODUCTION applications in ‘Fuji’ (10) and ‘Golden Reinders’ (11) apple fruit
Because most apple (Malus domestica Borkh.) production is improve the emission of aroma volatile compounds after mid-
aimed for mid- or long-term storage, it is a common practice to term cold storage under either air or controlled atmosphere,
harvest fruit before full ripeness with the purpose of improving particularly of those compounds having the most impact on
storage potential and resistance to postharvest handling proce- overall flavor. Therefore, the question arises whether preharvest
dures. Yet, this practice is not free from drawbacks, as the volatile calcium treatments might be a feasible procedure to avoid
profile emitted by apple fruit changes continuously throughout undesirable effects on aroma quality when apple fruit is harvested
maturation and ripening (1, 2), and the emission of flavor- before full ripeness, at a maturity stage suitable for long-term
contributing volatile compounds during the postharvest period storage. Here we report the production of aroma volatile com-
is dependent upon the developmental stage at harvest. Conse- pounds by untreated and calcium-treated ’Fuji’ apples during
quently, fruit often fails to develop full flavor after harvest if it maturation and ripening over two months prior to commercial
is picked before optimal maturity (3-5). Since flavor is a key harvest.
attribute for sensory quality and consumer acceptance of apple
fruit (6), disregard of these aspects often causes unsatisfactory MATERIALS AND METHODS
eating quality in spite of benefits in terms of firmness and external Plant Material, Calcium Treatment, and Standard Quality Anal-
appearance. Therefore, the improvement of aroma-related vola- ysis. ‘Fuji’ apple (Malus domestica Borkh.) fruit, growing on 7-year-old
tile production has become an important challenge for the fruit trees grafted on M-9 EMLA rootstocks at an experimental orchard in
industry. Mollerussa (NE Spain), were sprayed weekly with CaCl2 (1.6%, w/v).
Treatment period was 23 June to 4 August 2008, corresponding to 81 and
Calcium treatment of apples is a widely used practice, which
123 days after full bloom (dafb), respectively. Uniform and defect-free
has been demonstrated to be useful for delaying or reducing fruit samples from treated and untreated trees were then picked weekly
softening rates (7), physiological disorders (8), and fungi-caused over two months (11 August to 22 October), covering a dafb range of
decay (9). In contrast, little information has been reported to date 130-202. Samples were coded H1-H10, corresponding to successive
on the effects of calcium treatments on fruit flavor. Interestingly, picking dates. Commercial harvest at the producing area took place at
recent work has shown that postharvest calcium chloride (CaCl2) 195 dafb (H9 stage). At each sampling date, 15 apples per treatment were
assessed individually for standard quality parameters. Firmness (N ) was
*Corresponding author. E-mail: lara@quimica.udl.cat. Tel: þ34 measured on two opposite sides of each fruit, using an Effegi penetrometer
973 702526. Fax: þ34 973 238264. equipped with an 11-mm diameter convex tip. Soluble solids content (SSC)
© 2010 American Chemical Society Published on Web 12/02/2010 pubs.acs.org/JAFC

336 J. Agric. Food Chem., Vol. 59, No. 1, 2011 Ortiz et al.
and titratable acidity (TA) were measured in juice pressed from the whole Table 1. Standard Quality Parameters and Calcium Content of ‘Fuji Kiku-8’
fruit. SSC was determined with a hand-held refractometer (Atago, Tokyo, Apples around the Commercial Harvesta
Japan), and results were expressed as % sucrose in an equivalent solution. parameter H8 H9 H10
TA was determined by titrating 10 mL of juice with 0.1 N NaOH to pH 8.1
using 1% (v/v) phenolphthalein; results were given as g of malic acid L-1. firmness (N) control 68.93 a 63.96 b 62.29 b
Starch hydrolysis (SI) was rated visually using a 1-10 EUROFRU (LSD = 4.70) calcium 73.44 a 69.95 a 68.77 a
(CTIFL, France) scale (1, full starch; 10, no starch), after dipping cross- SSC (%) control 14.72 a 15.21 a 15.47 a
sectional fruit halves in 0.6% (w/v) I2-1.5% (w/v) KI solution for 30 s. (LSD = 1.93) calcium 14.25 a 15.07 a 15.29 a
Chemicals. The chemicals used were of the highest quality available, TA (g L-1) control 2.94 b 2.85 b 2.61 b
supplied by Sigma-Aldrich (Steinheim, Germany) unless otherwise indi- (LSD = 0.52) calcium 3.51 a 3.56 a 3.43 a
cated. Ethyl acetate, tert-butyl propanoate, propyl acetate, 1-propanol, SI (1-10)b control 6.7 a 8.1 a 9.0 a
ethyl butanoate, ethyl 2-methylbutanoate, butyl acetate, 1-butanol, pentyl (LSD = 0.91) calcium 6.5 a 7.1 b 7.8 b
acetate, 2-methyl-1-butanol, hexyl acetate, 1-hexanol, and hexyl 2-methyl- calcium content (mg 100 g-1) control 3.27 b 3.10 b 2.92 b
butanoate were obtained from Fluka (Buchs, Switzerland). Ethanol was (LSD = 0.42) calcium 4.16 a 3.92 a 3.89 a
purchased from Panreac Quı́mica, S.A. (Castellar del Valles, Spain). a
2-Methylpropyl acetate was obtained from Avocado Research Chemicals Values represent means of 15 (standard quality) or three (calcium content)
Ltd. (Madrid, Spain). replicates. Means followed by different letters within the same column for a given
parameter are significantly different at P e 0.05 (LSD test). b EUROFRU 1-10
Determination of Calcium Content. Lyophilized tissue (1 g) was
scale (1, full starch; 10, no starch).
ashed in a muffle furnace at 500 C for 2 h. Ashes were digested thereafter
with 4 mL of HCl/distilled water (1:1, v/v) and heated at 70 C until 180 C, detector temperature 220 C. Acetaldehyde was identified and
complete sample dehydration as described in a previous work (10). Dried quantified by comparison with external standards (Merck, Darmstadt,
material was then resuspended in 2 mL of HCl/distilled water (1:1, v/v) for Germany), and the results were expressed as μL L-1.
15 min, filtered through ‘Whatman 40 Ashless’, and the filtrate was diluted Extraction and Assay of Aroma Volatile-Related Enzyme Activ-
to 50 mL in distilled water. Samples were then analyzed by inductively ities. Samples of skin and flesh tissues were taken separately at each
coupled plasma emission spectroscopy (ICP-OES) in a ‘Horiba Jobin Yvon picking date (2 apples/replicate 3 replicates), frozen in liquid nitrogen,
ACTIVA’ spectrometer, and results were expressed as mg 100 g FW-1. freeze-dried, powdered, and kept at -80 C until processing. One hundred
Analysis of Volatile Compounds. Eight kilograms of intact apples milligrams of lyophilized powdered tissue was used for each determina-
(2 kg/replicate 4 replicates) were taken for the extraction of volatile tion. Extraction and assay of lipoxygenase (LOX; EC 1.13.11.12), pyru-
compounds according to the method of dynamic headspace as described vate decarboxylase (PDC; EC 4.1.1.1), alcohol dehydrogenase (ADH;
previously (10). Briefly, intact fruit from each treatment were placed EC 1.1.1.1), and alcohol o-acyltransferase (AAT; EC 2.3.1.84) activities on
into an 8-L Pyrex container, and an air-stream (900 mL min-1) was passed crude enzyme extracts were performed as described elsewhere (13). Hydro-
through for 4 h. The effluent was then recovered in an adsorption tube peroxyde lyase (HPL; EC 4.1.2.-) activity was extracted and assayed
(ORBO-32; SUPELCO, Bellefonte, PA, USA) filled with 100 mg of according to ref (14). Total protein content in the enzyme extract was
activated charcoal (20/40 mesh), from which volatile compounds were determined with the Bradford method (15), using BSA as a standard. In all
desorbed by agitation for 40 min with 0.5 mL of diethyl ether. Identifica- cases, one activity unit (U) was defined as the variation in one unit of
tion and quantification of volatile compounds were achieved on a Hewlett- absorbance per minute. Each determination was done in triplicate, and
Packard 5890 series II gas chromatograph equipped with a flame ioniza- results were expressed as specific activity (U mg protein-1).
tion detector and a cross-linked free fatty acid phase (FFAP; 50 m Statistical Analysis. A multifactorial design with calcium treatment
0.2 mm i.d. 0.33 μm) capillary column. The injection volume was 1 μL and sampling time as factors was used to statistically analyze the results.
from each extract in all the analyses. The oven program was set at 70 C All data were tested by analysis of variance (GLM-ANOVA procedure)
(1 min) and the temperature was initially raised by 3 C min-1 to 142 C with the SAS System 9.0 program package (SAS Institute, Cary, NC,
and then by 5 C min-1 to 225 C. It was then kept constant for 10 min at 2002). Means were separated by the Fisher’s LSD test at P e 0.05.
this final temperature. Helium was used as the carrier gas at a flow rate of Partial least-squares regression (PLSR) was used additionally as a
0.8 mL min-1 (42 cm s-1), with a split ratio of 40:1, in the presence of air predictive method to relate a matrix of dependent variables (Y ) to a set of
(400 mL min-1) and H2 (32 mL min-1). The injector and detector were explanatory variables (X ) in a single estimation procedure, with full cross-
held at 220 and 240 C, respectively. A second capillary column (SGE, validation as a validation procedure. The Unscrambler 6.11a software
Milton Keynes, UK) with 5% phenyl polysilphenylene-siloxane as the package (CAMO ASA, 1997) was used for developing these models.
stationary phase (BPX5, 30 m 0.25 mm i.d. 0.25 μm) was also used for
compound identification under the same operating conditions as described RESULTS AND DISCUSSION
above. Volatile compounds were identified by comparing retention indexes Significant increases in calcium content were found in the flesh
with those of standards and by enriching apple extract with authentic of treated fruit around the commercial harvest date (Table 1),
samples. The quantification was made using butylbenzene (assay>99.5%, showing that exogenous calcium was actually incorporated and
Fluka) as the internal standard, run with each added standard aside from
that it penetrated into the inner tissues. SSC and TA levels of H8,
the matrix to develop standard curves for each volatile analyzed. A GC-MS
system (Agilent Technologies 6890N - 5973N) was used for compound H9, and H10 fruit (Table 1) were suitable for storage according
confirmation, in which the same capillary column was used as in the GC to local recommendations (TA<4 g L-1, SSC>13%), but only
analyses. Mass spectra were obtained by electron impact ionization at H9 samples showed SI values within the optimal range (7-8),
70 eV. Helium was used as the carrier gas (42 cm s-1), according to the and untreated H9 and H10 fruit were slightly less firm than
same temperature gradient program as described above. Spectrometric recommended (68.5-78.5 N). SSC was apparently unaffected by
data were recorded (MSD Chemstation D.03.00.611) and compared with calcium applications. Contrarily, treated fruit showed lower
those from the NIST NBS75A original library mass spectra. The concen- starch index at harvest, together with higher firmness and acidity
tration of each volatile compound was expressed as μg per kg of fruit. levels, indicative of delayed ripening (Table 1). This delay in the
Analysis of Acetaldehyde Concentration. Juice samples (5 mL) ripening process may be favorable for storage of produce;
obtained individually from 15 fruit were introduced in 10-mL test tubes however, it may also have exacerbated the lack of aroma devel-
and incubated 1 h at 65 C for the analysis of acetaldehyde content as
opment often encountered when apple fruit are picked before
described elsewhere (12). A 1-mL headspace gas sample was taken and
injected into a Hewlett-Packard 5890 Series II gas chromatograph, being fully ripe. Therefore, we focused on this important attribute
equipped with a column containing Carbowax (5%) on Carbopack for the eating quality of fruit.
(60/80, 2 m 2 mm i.d.) as the stationary phase, and a flame ionization Preharvest Calcium Sprays Enhanced the Emission of Key Vola-
detector. Nitrogen was used as the carrier gas (24 cm s-1), and operating tile Esters by Ripe Fruit. Volatile esters are reportedly the most
conditions were as follows: oven temperature 80 C, injector temperature important contributors to apple aroma (reviewed in ref 16),
Article J. Agric. Food Chem., Vol. 59, No. 1, 2011 337
a
Table 2. Emission ( μg kg ) of Straight- (A) and Branched-Chain (B) Esters by ‘Fuji Kiku-8’ Apples during On-Tree Maturation
-1
compound RIb RIc OTHd H1 H2 H3 H4 H5 H6 H7 H8 H9 H10
methyl acetate 854 - 8300 control 19.0 a 19.4 a 31.7 a 25.1 a 26.8 a 20.7 a 21.6 a 27.6 a 26.5 a 25.4 a
(LSD = 3.7) calcium 16.7 a 21.9 a 30.2 a 30.2 a 24.8 a 19.7 a 23.4 a 27.7 a 25.7 a 25.7 a
ethyl acetate 882 609 5000 control 28.1 a 43.2 b 84.8 b 82.6 b 87.1 b 79.4 b 113.7 b 117.7 b 73.4 a 83.3 b
propyl acetate 945 649 2000 control - - - - - 1.0 1.2 a 1.5 a 4.2 a 17.4 b
(LSD = 3.3) calcium - - - - - - 2.1 a 2.6 a 6.8 a 25.2 a
methyl butanoate 955 656 5 control - - - - - 2.1 a 2.6 a 2.1 a 4.7 a 6.8 a
(LSD = 1.5) calcium - - - - - 2.0 a 2.1 a 2.4 a 4.5 a 7.1 a
ethyl butanoate 1002 803 1 control 1.3 a 1.7 a 1.6 a 0.6 a 2.5 a 1.2 a 5.1 a 5.0 a 4.5 a 4.7 a
propyl propanoate 1008 809 57 control - - - - - - - - 2.2 a 12.0 a
(LSD = 1.9) calcium - - - - - - - - 2.7 a 9.8 a
butyl acetate 1040 813 10 control 1.5 2.2 3.8 1.6 4.2 a 5.6 a 7.5 a 14.8 b 67.8 b 110.7 b
(LSD = 12.3) calcium - - - - 0.5 a 4.7 a 7.1 a 27.8 a 91.6 a 133.1 a
butyl propanoate 1123 910 25 control - - - - - - - 3.6 a 19.8 a 53.0 a
(LSD = 3.6) calcium - - - - - - - 4.7 a 16.8 a 47.8 b
pentyl acetate 1161 914 5 control 18.1 a 14.7 a 14.2 a 9.0 a 7.0 a 5.5 a 6.6 a 8.2 a 14.2 a 17.8 a
(LSD = 4.0) calcium 10.2 b 7.6 b 6.6 b 6.8 a 6.4 a 6.2 a 5.5 a 9.4 a 13.2 a 18.9 a
butyl butanoate 1218 1000 100 control - - - - - - - 3.1 a 17.6 a 26.7 a
(LSD = 3.1) calcium - - - - - - - 3.9 a 16.2 a 25.3 a
hexyl acetate 1292 1015 2 control 15.8 a 14.2 a 15.5 a 10.0 a 9.3 a 5.2 a 6.3 a 10.6 b 35.6 b 60.4 b
(LSD = 3.2) calcium 6.5 b 6.9 b 6.1 b 5.5 b 5.1 b 3.4 a 5.2 a 14.5 a 45.9 a 81.8 a
propyl hexanoate 1360 1099 - - - - - - - - - - 3.3 a 13.6 b
(LSD = 1.7) calcium - - - - - - - - 2.0 a 15.5 a
hexyl propanoate 1379 1109 8 control 6.5 a 6.6 a 9.4 a 10.2 a 11.7 a 12.9 a 12.6 a 15.8 b 20.1 b 31.2 b
butyl hexanoate 1473 1196 250 control 11.4 b 12.2 b 12.4 b 13.6 b 17.8 b 20.4 b 20.2 b 21.9 b 41.9 b 54.4 b
hexyl butanoate 1477 1197 250 control 21.3 b 20.4 b 21.9 b 17.9 a 16.2 a 7.8 a 6.0 a 8.5 b 20.4 b 29.2 b
ethyl octanoate 1502 1201 92 control 8.5 b 4.8 b 4.2 b 3.8 b 1.9 b 1.5 b 0.7 a - - -
(LSD = 3.1) calcium 17.3 a 16.6 a 14.0 a 12.5 a 11.4 a 5.7 a 3.4 a - - -
pentyl hexanoate 1590 1293 - control - - - - - - - - 6.6 a 7.6 a
(LSD = 1.3) calcium - - - - - - - - 6.7 a 7.9 a
hexyl hexanoate 1687 1392 6400 control 23.5 a 18.7 b 14.4 b 9.2 b 11.5 b 9.3 b 8.4 b 11.5 b 31.2 a 31.4 a
(LSD = 4.1) calcium 25.9 a 26.2 a 25.3 a 27.3 a 30.2 a 26.4 a 22.7 a 22.8 a 26.0 b 27.2 b
butyl octanoate 1690 1394 - control - - - - - - - 0.8 a 5.3 a 4.4 a
(LSD = 2.0) calcium - - - - - - - 2.7 a 5.6 a 4.7 a
B
b c d
compound RI RI OTH H1 H2 H3 H4 H5 H6 H7 H8 H9 H10
tert-butyl propanoate 928 717 19 control 15.4 a 14.3 b 16.2 a 16.3 a 19.7 a 15.7 a 11.6 a 11.4 a 11.3 a 9.3 a
2-methylpropyl acetate 976 691 5 control 1.0 4.5 8.4 8.7 10.4 4.5 5.7 a 5.5 a 6.2 b 8.7 b
(LSD = 2.2) calcium - - - - - - - 3.6 a 9.5 a 13.0 a
ethyl 2-methylbutanoate 1015 845 0.006 control 8.1 3.2 4.5 3.3 1.2 <0.5 - - - -
(LSD = 2.1) calcium - - - - - - - - - -
2-methylpropyl propanoate 1046 865 - control - - - - - - - - 3.2 a 4.1 a
(LSD = 1.4) calcium - - - - - - - - 2.9 a 4.2 a
2-methylbutyl acetate 1096 876 5 control 2.2 a 4.8 a 14.1 a 17.2 a 45.7 a 44.0 a 92.8 a 165.7 b 287.8 b 401.6 b
butyl 2-methylpropanoate 1129 1009 80 control - - - - - - - - - 0.9 a
(LSD = 0.2) calcium - - - - - - - - - 0.7 a
2-methylpropyl butanoate 1138 954 - control 4.4 a 4.3 a 5.3 a 4.2 a 4.0 a 0.9 a <0.5 - - -
(LSD = 1.5) calcium 5.4 a 4.2 a 5.9 a 3.1 a 3.5 a 1.0 a 0.6 - - -
2-methylbutyl propanoate 1180 950 19 control 4.5 a 4.7 a 4.6 a 4.4 a 4.2 b 4.0 b 4.9 b 6.3 b 9.1 b 19.9 b
2-methylbutyl 2-methylpropanoate 1190 1016 - control 17.0 a 12.9 a 5.8 a 4.5 a 1.9 a 2.5 a - - - -
(LSD = 2.1) calcium 17.8 a 12.6 a 6.2 a 4.6 a 2.1 a 1.6 a - - - -
butyl 2-methylbutanoate 1235 1042 17 control 4.1 a 6.4 a 11.0 a 10.1 a 10.4 a 11.3 a 10.9 a 12.2 a 24.7 a 43.9 a
(LSD = 4.9) calcium 0.8 a 1.0 b 0.8 b 1.9 b 1.6 b 3.7 b 3.2 b 7.8 a 16.7 b 34.8 b
2-methylbutyl 2-methylbutanoate 1324 1106 - control 36.3 a 37.7 a 35.2 a 29.4 a 11.1 a 4.9 a 2.2 a 2.5 a 3.1 a 12.1 a
Table 2. Continued
compound RIb RIc OTHd H1 H2 H3 H4 H5 H6 H7 H8 H9 H10
hexyl 2-methylbutanoate 1488 1239 6 control 22.1 b 18.4 b 20.5 b 20.3 b 27.3 b 26.9 b 30.2 b 32.8 b 37.2 b 61.0 b
a
Values are the means of four samples obtained each from 2 kg of apples after 4h of collection (-: non-detected). For a given ester, means within the same column followed by
different letters are significantly different at P e 0.05 (LSD test). b Kovats retention index in a cross-linked FFAP column. c Kovats retention index in a BPX5 column (-: eluted with
the solvent). d Odor thresholds (μg kg-1) in water as reviewed in ref 11 (-: not found).
both in quantitative and qualitative terms. Nineteen straight- Table 3. Log10 of Odor Unit Value (Concentration/Odor Threshold) of Volatile
chain and 12 branched-chain esters were identified in the volatile Esters Contributing to Overall Flavor of ‘Fuji Kiku-80 Apples around the
fraction emitted by fruit during the experimental time, although Commercial Harvesta
not all of them were detected at all sampling dates considered compound H8 H9 H10
(Table 2). Some of these volatile esters were apparently unaffected
methyl butanoate control <0 <0 0.14 a
by treatment, while significant differences between treated and
calcium <0 <0 0.15 a
untreated fruit were observed in other instances. In some cases,
2-methylpropyl acetate control 0.04 0.09 b 0.24 b
treatment effects were dependent upon the maturity stage of calcium <0 0.28 a 0.41 a
samples (Table 2). Since an objective of this work was to assess ethyl butanoate control 0.70 a 0.66 a 0.67 a
whether preharvest calcium sprays might be useful for the im- calcium 0.71 a 0.67 a 0.73 a
provement of aroma quality of fruit at harvest, particular attention butyl acetate control 0.17 b 0.83 b 1.04 b
was placed on the latter phases of fruit maturation. The emission calcium 0.44 a 0.96 a 1.12 a
of eight straight-chain esters (ethyl acetate, propyl acetate, butyl 2-methylbutyl acetate control 1.52 b 1.76 b 1.90 b
acetate, propyl hexanoate, hexyl acetate, hexyl propanoate, butyl calcium 1.63 a 1.90 a 2.03 a
hexanoate, and hexyl butanoate) and of four branched-chain pentyl acetate control 0.21 a 0.45 a 0.55 a
calcium 0.27 a 0.42 a 0.58 a
esters (2-methylpropyl acetate, 2-methylbutyl acetate, 2-methyl-
butyl propanoate control <0 <0 0.33 a
butyl propanoate, and hexyl 2-methylbutanoate) was increased
calcium <0 <0 0.28 b
significantly in treated fruit around the commercial harvest date 2-methylbutyl propanoate control <0 <0 0.02 b
(Table 2). In contrast, the production of butyl propanoate, hexyl calcium <0 <0 0.13 a
hexanoate, and butyl 2-methylbutanoate decreased in response to butyl 2-methylbutanoate control <0 0.16 0.41 a
treatment. calcium <0 <0 0.31 b
The question arose whether the alterations in ester production hexyl acetate control 0.72 b 1.25 b 1.48 b
observed in response to treatment were relevant for the aroma calcium 0.86 a 1.36 a 1.61 a
profile of fruit at harvest. Therefore, ester production must be hexyl propanoate control 0.29 b 0.42 b 0.59 b
considered not only in quantitative, but also in qualitative terms. calcium 0.41 a 0.56 a 0.77 a
hexyl 2-methylbutanoate control 0.74 b 0.79 b 1.01 b
Twelve out of the 31 volatile esters identified during the experi-
calcium 0.83 a 0.89 a 1.09 a
mental period were found to have log odor units (OU)>0 by the
a
time of commercial harvest (Table 3) and thus deemed as likely to Values are the means of four samples obtained each from 2 kg of apples after
have an impact on overall flavor (17). Most of these contributing 4 h of collection. For a given ester, means within the same column followed by
different letters are significantly different at P e 0.05 (LSD test).
compounds, with the exception of 2-methylpropyl and pentyl
acetates, have been shown to be also important for the aroma of
‘Fuji’ apples after cold storage under air or ULO conditions (10), not modified in response to treatment and illustrates the relevance
some of them (ethyl butanoate, 2-methylbutyl acetate, hexyl of alcohol supply for ester production. Accordingly, the emission
acetate) reportedly providing fruity odors to apple aroma (18). and thus the log OU value at harvest of hexyl and 2-methylbutyl
Interestingly, many of these compounds, particularly those show- propanoates, as well as of hexyl 2-methylbutanoate, were also
ing the highest log OU values and thus putatively having the most enhanced in response to treatment. However, results also show
impact on fruit aroma, were enhanced in treated samples, that additional factors may play an important role in ester
suggesting that preharvest calcium applications have a potential production: for instance, the log OU values for butyl propanoate
to improve this attribute at harvest. and butyl 2-methylbutanoate were lower in calcium-treated fruit
The impact of treatment was dependent upon the chemical in spite of the higher availability of 1-butanol (Table 4) and
nature of each ester. Butanoate esters were apparently unaffected, contrarily to the observations for butyl acetate (Table 3), which
while log OU values of acetate esters were higher in treated fruit, suggests that acetyl CoA was the preferred acyl CoA substrate for
with the exception of pentyl acetate (Table 3). This is consis- the AAT isoforms present in the tissues.
tent with the observation of increased acetaldehyde content in Preharvest Calcium Sprays Increased the Availability of Specific
calcium-treated samples (Figure 1A), as acetaldehyde can be used Precursors for Ester Biosynthesis. AAT activity is necessary for
by plant tissues as a precursor for the biosynthesis of acetyl ester production (20), and detectable levels were found through-
CoA (19), one of the substrates required for the biosynthesis of out the experimental time (Table 5). However, the emission of
acetate esters by AAT action, and indeed a good correlation was volatile esters (Table 2) did not appear to parallel AAT dynamics.
found between acetaldehyde content and the emission of acetate The highest AAT activity levels in the flesh were found at the H7
esters (Figure 1B). An alcohol moiety is the second substrate stage, the only sampling point for which significant differences
necessary for AAT-catalyzed ester production, and data show were observed between treated and untreated samples (Table 5).
that the emission of 1-butanol, 2-methyl-1-butanol, and 1-hex- Contrarily, AAT activity in the skin tissue was altered signifi-
anol was higher in treated fruit, while that of 1-pentanol was cantly in response to treatment throughout the experimental
unaffected (Table 4), which might explain why pentyl acetate was period. Untreated fruit displayed a maximum at the H8 stage,
one week before commercial harvest, which in treated samples alcohol precursors (X variables), this model explaining up to 80%
was more moderate and advanced by approximately one month. of total variability in ester emission during the whole two-month
These results agree with previous observations for ‘Fuji’ period considered (data not shown). If only advanced maturity
apples (10, 21) suggesting that, provided a minimum level of stages were considered in the model (H6-H10), 87% of vari-
AAT activity is present in the tissues, an adequate supply of ability could be accounted for, calcium-treated fruit displaying
precursors is the actual key factor accounting for ester biosyn- higher levels of important precursors such as 1-butanol, 2-methyl-
thesis. In agreement with those reports, a partial least-squares 1-butanol, 1-hexanol, and acetaldehyde.
regression (PLSR) model developed for flavor-contributing esters This observation highlights the relevance of upstream enzymes
(Y variables) revealed a strong relationship to acetaldehyde and providing these intermediates for ester biosynthesis, and therefore
an additional PLSR model was developed in order to have an
overview of the possible involvement of different volatile-related
enzyme activities in improved availability of these substrates in
mature (H6-H10) fruit. The loadings plot for this model
(Figure 2) showed that the production of most alcohols, with
the exception of ethanol, was related to PDC and ADH activities,
which suggests that these activities were relevant for the observed
increase in the emission of volatile esters. Indeed, higher PDC and
ADH activities were found for calcium-treated fruit during the
last stages of fruit maturation both in the skin and in the flesh
(Figure 3). In the case of PDC activity, higher activity levels in the
skin of treated samples were observed throughout the whole
experimental period. This is consistent with previous reports
for ‘Fuji’ fruit, indicating that postharvest CaCl2 treatments
enhanced the biosynthesis of some impact compounds after
mid-term storage through an increase in PDC and ADH activities
associated with better supply of acetaldehyde and alcohol pre-
cursors (10). The calcium-related increase in these enzyme activ-
ities has been attributed to increased O2 gradients across apple
tissues in response to the treatment, due to higher difficulty for O2
diffusion (22) and to augmented internal CO2 levels (23-25),
causing hypoxia-like induction of PDC and ADH.
PDC uses a 2-oxoacid to render CO2 and an aldehyde, which is
metabolized further to either the corresponding alcohol by ADH-
catalyzed reduction, or to an acyl-CoA by aldehyde dehydrogen-
ase (ALDH, EC 1.2.1.5) (26). Both alcohols and acyl-CoA
moieties are the required substrates for AAT-mediated ester
formation. The observation that ethanol was apparently unre-
lated to ADH activity suggests that acetaldehyde was being
diverted preferentially to the synthesis of acetyl-CoA, necessary
Figure 1. Acetaldehyde content (A) and correlation to the emission of for the production of acetate esters, and agrees with the general
acetate esters (B) by ‘Fuji Kiku-8’ apples during on-tree maturation. In increase in the emission of acetate esters by treated fruit (Tables 2
panel A, asterisks indicate significant differences between treated and and 3). Therefore, ADH may have used aldehydes other than
untreated fruit at P e 0.05 (LSD test). Vertical bar indicates LSD. Points acetaldehyde for obtaining the required alcohols. This is inter-
represent means of 15 replicates. esting in the light of results showing increased HPL activity in the
Table 4. Emission of Alcohols ( μg kg-1) by ‘Fuji Kiku-8’ Apples during On-Tree Maturationa
compound RIb RIc H1 H2 H3 H4 H5 H6 H7 H8 H9 H10
ethanol 912 - control 24.7 b 30.3 b 58.1 b 56.4 b 56.0 b 59.0 a 65.0 a 62.1 a 55.3 a 50.8 a
(LSD = 4.6) calcium 30.5 a 35.0 a 68.5 a 66.8 a 66.5 a 38.1 b 54.7 b 55.9 b 42.5 b 40.7 b
1-propanol 992 - control - - - - - - - - 3.3 a 11.3 b
(LSD = 3.5) calcium - - - - - - - - 5.1 a 15.0 a
1-butanol 1119 626 control - - <0.5 - 0.8 1.1 a 2.5 a 5.1 a 19.3 b 23.9 b
(LSD = 3.8) calcium - - 0.5 0.5 - 2.2 a 3.1 a 8.4 a 24.8 a 28.3 a
2-methyl-1-butanol 1199 667 control - - - - 4.6 a 3.9 a 8.7 a 14.5 b 32.2 b 45.7 b
(LSD = 5.2) calcium - - - - 5.4 a 4.7 a 12.2 a 20.6 a 46.5 a 59.5 a
1-pentanol 1262 688 control 1.6 a 1.8 - - - - - - - -
(LSD = 0.3) calcium 1.8 a - - - - - - - - -
1-hexanol 1392 869 control - - - - - - - 1.8 a 4.3 b 5.3 b
(LSD = 2.5) calcium - - - - - - - 3.9 a 11.1 a 14.8 a
2-ethyl-1-hexanol 1565 1031 control 46.1 b 35.4 b 21.1 b 21.5 b 16.8 b 11.8 b 7.8 b 10.0 a 6.8 a 3.7 a
a
Values are the means of four samples obtained each from 2 kg of apples after 4 h of collection (-: non-detected). For a given alcohol, means within the same column followed
by different letters are significantly different at P e 0.05 (LSD test). b Kovats retention index in a cross-linked FFAP column. c Kovats retention index in a BPX5 column (-: eluted
with the solvent).
a
Table 5. Flavor-Related Enzyme Activities (U mg protein ) in ‘Fuji Kiku-8’ Apples during On-Tree Maturation
-1
tissue activity H1 H2 H3 H4 H5 H6 H7 H8 H9 H10
skin LOX control 109.13 a 128.85 a 63.04 a 72.23 a 85.03 a 84.05 a 102.32 a 84.83 a 84.59 a 74.11 a
(LSD = 16.1) calcium 99.34 b 104.93 b 54.83 a 54.60 b 67.91 b 67.09 b 76.95 b 67.89 b 73.75 a 59.81 a
HPL control 61.05 a 65.41 b 43.78 b 39.70 b 57.25 b 57.55 b 54.77 a 49.55 a 52.12 a 62.50 a
(LSD = 7.5) calcium 65.95 a 101.92 a 59.67 a 57.43 a 76.00 a 73.01 a 51.52 a 41.75 b 42.57 b 52.94 b
AATb control 54.56 b 58.21 b 81.24 b 90.26 b 90.85 b 89.74 a 104.78 a 128.54 a 62.25 a 51.79 a
(LSD=14.2) calcium 92.22 a 90.78 a 107.05 a 106.98 a 107.80 a 75.34 b 81.61 b 71.02 b 41.98 b 41.19 a
flesh LOX control 9.59 a 6.44 a 4.85 a 4.77 a 12.25 a 11.65 a 46.05 a 34.25 a 31.21 a 35.67 a
(LSD = 5.5) calcium 7.22 a 11.30 a 4.21 a 6.14 a 11.18 a 10.80 a 36.17 b 30.09 a 28.39 a 28.50 b
HPL control 39.47 a 37.93 a 19.86 a 18.02 a 11.56 a 20.76 a 15.93 a 14.91 a 15.45 a 19.05 a
(LSD = 5.9) calcium 35.76 a 31.45 b 18.41 a 17.88 a 14.11 a 17.39 a 13.11 a 11.70 a 10.55 a 13.66 a
AATb control 16.72 a 17.82 a 15.25 a 12.69 a 17.35 a 22.50 a 26.56 a 21.02 a 18.48 a 17.07 a
(LSD = 3.7) calcium 14.67 a 17.68 a 14.51 a 12.14 a 14.96 a 21.37 a 21.81 b 20.18 a 18.10 a 14.04 a
a
Values are the means of three replicates. Different letters within the same column for a given enzyme activity indicate significant differences at P e 0.05 (LSD test). b AAT
activity data are given as mU mg protein-1.
Figure 2. Loadings plot of PC1 versus PC2 corresponding to a PLSR

model for emission of alcohols and acetaldehyde content (Y variables) vs
volatile-related enzyme activities (X variables) in mature (H6 to H10) ‘Fuji
Kiku-8’ apples (AA, acetaldehyde; etOH, ethanol; prOH, 1-propanol; bOH,
1-butanol; 2mbOH, 2-methyl-1-butanol; hOH, 1-hexanol; 2ehOH, 2-ethyl-
1-hexanol). For enzyme labels, the suffix ‘S’ or ‘F’ refers to the activity in the
skin or the flesh, respectively.
skin tissue of calcium-treated samples up to H6 maturity stage

(Table 5). HPLs catalyze the cleavage of fatty acid hydroper-
oxides generated by LOX action to aldehydes and oxoacids, the Figure 3. Pyruvate decarboxylase (A) and alcohol dehydrogenase (B) in
aliphatic aldehydes hexanal and 3-hexenal being major products the skin and flesh tissues of ‘Fuji Kiku-8’ apples during on-tree maturation.
of its action on 13-hydroperoxy linoleic or linolenic acids, res- Asterisks indicate significant differences between treated and untreated
pectively (27). In this work, 1-hexanol was the alcohol showing fruit at P e 0.05 (LSD test). Vertical bars indicate LSD. Points represent
the highest dependence on ADH activity (Figure 2), and it has means of three replicates.
been reported that hexanal and hexyl acetate are produced mainly
in the skin of apple fruit (28). LOX activity in the skin was on aroma development often encountered when fruit are picked
generally lower in treated than in untreated samples, consistent too early.
with the protective role exerted by calcium on structural integrity
of membranes (29). Although this may seem in contradiction ABBREVIATIONS USED
with enhanced production of straight-chain esters, generally AA, acetaldehyde; AAT, alcohol o-acyltransferase; ADH,
considered to arise from lipid metabolism through the LOX alcohol dehydrogenase; HPL, hydroperoxide lyase; ICP-OES,
pathway (21), it has been hypothesized that the protective inductively coupled plasma emission spectroscopy; LOX, lipoxy-
role of calcium on membranes might allow better regulation of genase; OTH, odor threshold; OU, odor unit; PDC, pyruvate
LOX activity and hence higher straight-chain ester emission in decarboxylase; PLSR, partial least-squares regression; SI, starch
spite of lower LOX activity levels (10). In contrast, no sig- index; SSC, soluble solids content; TA, titratable acidity.
nificant differences in LOX or HPL activities between treated
and untreated samples were observed in general for the flesh LITERATURE CITED
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recipient of an FPU grant from the Ministerio de Ciencia e
(16) Lara, I. Changes in flavour-related volatile production during post-
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76-84. AGL2006-00345/ALI project, financed by the Ministerio de Educación
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and other vegetables and fruits. In Flavor Science: Sensible Principles technical assistance.

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J. Agric. Food Chem. 2009, 57, 4931–4938 4931
DOI:10.1021/jf9003576
Calcium Dips Enhance Volatile Emission of Cold-Stored

‘Fuji Kiku-8’ Apples
ABEL ORTIZ, GEMMA ECHEVERRÍA, JORDI GRAELL, AND ISABEL LARA*
Area de Post-Collita, XaRTA, UdL-IRTA, Alcalde Rovira Roure 191, 25198 Lleida, Spain
Despite the relevance of volatile production for overall quality of apple (Malus domestica Borkh.)
fruit, only a few studies have focused on the effects of calcium treatments on this quality attribute.
In this work, ‘Fuji Kiku-8’ apples were harvested at commercial maturity, dipped in calcium chloride
(2%, w/v), stored at 1 C and 92% relative humidity for 4 or 7 months under either air or ultralow
oxygen (ULO; 1 kPa of O2/2 kPa of CO2), and placed subsequently at 20 C. Ethylene production,
standard quality parameters, emission of volatile compounds, and the activities of some related
enzymes were assessed 7 days thereafter. Calcium concentration was higher in CaCl2-treated than
in untreated fruit, suggesting that the treatment was effective in introducing calcium into the tissues.
Higher calcium contents were concomitant with higher flesh firmness and titratable acidity after
storage. Furthermore, calcium treatment led to increased production of volatiles in middle-term
stored apples, probably arising from enhanced supply of precursors for ester production as a
consequence of increased pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH)
activities. After long-term storage, higher volatile emission might have arisen also from the
enhancement of alcohol o-acyltransferase (AAT) activity, which was increased as a result of calcium
treatment. In addition to storage period, the effects of calcium treatment were also partially
dependent on storage atmosphere and more noticeable for fruit stored in air.
KEYWORDS: Alcohol o-acyltransferase; alcohol dehydrogenase; calcium applications; controlled

atmosphere; pyruvate decarboxylase; volatile compounds
INTRODUCTION Although fruit flavor depends upon taste and aroma, the latter
Cold storage of apple fruit, under either air or hypoxic condi- is considered to play a dominant role (7 ). The aroma profile of a
tions, is a widespread technology used to delay many ripening- fruit is complex and depends on the combination of all volatile
related modifications and, thus, to extend the commercial life of compounds emitted, in addition to the concentration and odor
produce. In addition, calcium chloride (CaCl2) has been widely threshold of each individual emitted compound. Volatile produc-
used as a preservative and firming agent in the fruit and vegetable tion in fruits is a process under tight control, involving enzymes as
industry for whole as well as for fresh-cut produce. Prestorage cal- well as substrates and energy supplied from many pathways.
cium treatment of apples has been shown to reduce the incidence of In particular, metabolism of fatty acids through both β-oxidation
physiological disorders (1 ), softening rates (2 ), and decay caused and the lipoxygenase (LOX; EC 1.13.11.12) pathway has been
by fungi (3 ). Moreover, calcium treatment of fruit may have ben- reported to be the principal source of precursors for the produc-
eficial side effects on the nutritional quality of produce, as a bulk of tion of those volatile compounds responsible for the aroma of
findings link dietary calcium deficiency to some chronic diseases, most fruits (8 ). β-Oxidation is generally considered to be the main
including osteoporosis, hypertension, and colon cancer (4 ). metabolic pathway producing primary aroma in fruits, whereas
The ultimate objective of calcium applications, as of any other the LOX system may account for the widest assortment of lipid-
postharvest treatment, is to enhance consumer acceptance of the derived precursors of aroma compounds in disrupted plant
commodity and/or to maintain it for as long as possible. It has tissues. Besides, cell walls and membranes become more perme-
been found that calcium infiltration of apples significantly able to different substrates in the course of ripening, and thus the
increased sensory hardness and overall acceptability scores (5 ). role of the LOX pathway in the biosynthesis of volatiles becomes
In addition to texture, also flavor is a key attribute determining more significant (8 ).
consumer acceptance of apples (6 ). Thus, to better understand Although the relationships between calcium treatments and
how consumer acceptance of fruit may be affected by calcium aroma volatile production are of interest, only a few works on this
applications, more information is needed on the alterations in subject have been published. Calcium has been shown to play
flavor induced by the treatments. an important role in maintaining structural integrity not only
of cell walls but also of cell membranes, thus delaying lipid
*Corresponding author (e-mail: lara@quimica.udl.cat; telephone: catabolism (9 ). Therefore, the production of lipid-derived pre-
+34 973 702526; fax: +34 973 238264). cursors of volatiles, and hence of aroma-related volatile
© 2009 American Chemical Society Published on Web 05/18/2009 pubs.acs.org/JAFC

compounds, might be modified in response to calcium treatments. samples were then analyzed by inductively coupled plasma emission
The purpose of this work was to investigate the suitability of spectroscopy (ICP-OES) in a Horiba Jobin Yvon ACTIVA spectrometer.
calcium applications for preservation of the overall quality of Each determination was done in triplicate, and results were expressed as
apple fruit during the poststorage period, with special focus on milligrams per 100 g of fresh weight (FW).
the biosynthesis of volatile compounds through the LOX path- Determination of Ethylene Production. Ethylene production was
way. To simulate the usual commercial procedures for apples measured at harvest date and 7 days after cold storage. Six apples per batch
(storage period atmosphere calcium treatment) were weighed, placed
before marketing and to assess possible interactions, CaCl2-
into 3 L respiration jars, and continuously aerated with humidified air at
treated and untreated samples were cold-stored under either air a rate of 5 L h-1. Gas samples of the effluent air from the respiration jars
or ultralow oxygen (ULO) conditions. were taken with a 1 mL syringe and injected into a gas chromatograph
(Agilent Technologies 6890N) equipped with a flame ionization detector
and an alumina column (1.5 m 3 mm). Gas analyses were conducted
MATERIALS AND METHODS
isothermically at 100 C. N2 carrier gas, air, and H2 flows were 45, 400, and
Plant Material, Calcium Treatment, and Storage Conditions. 45 mL min-1, respectively. The injector and detector were held at 120 and
Apple (Malus domestica Borkh. cv. Fuji Kiku-8) fruit were harvested in 180 C, respectively. Results were expressed as microliters of ethylene per
2006 at commercial maturity (187 days after full bloom), from 5-year-old kilogram and hour.
trees grafted on M-9 EMLA rootstocks at the IRTA-Experimental Station Analysis of Volatile Compounds. Eight kilograms of intact apples
in Mollerussa, in the area of Lleida (northeastern Spain). Immediately (2 kg/replicate 4 replicates) were taken for extraction and analysis
after harvest, fruits were randomly divided into six lots, three of which of volatile compounds. The extraction was performed according to the
were dipped in a 2% (w/v) CaCl2 solution at ambient temperature for method of dynamic headspace as described in ref (10), with some
5 min. After treatment, CaCl2-treated and untreated apples were stored at modifications. Briefly, intact fruits from each treatment were placed into
1 C and 92% relative humidity (RH) in cold rooms under either air or an 8 L Pyrex container, and an air stream (900 mL min-1) was passed
ULO conditions (1 kPa of O2/2 kPa of CO2). The experimental chambers through for 4 h. The effluent was then recovered in an adsorption tube
(20 m3) at the UdL-IRTA research center were used for storage of fruit. (ORBO-32; Supelco, Bellefonte, PA) filled with 100 mg of activated
The O2 and CO2 concentrations were monitored continuously and charcoal (20/40 mesh), from which volatile compounds were desorbed
corrected automatically using N2 from a tank and by scrubbing off excess by agitation for 40 min with 0.5 mL of diethyl ether. Identification and
CO2 with a charcoal system. A humidifier was used to maintain RH to quantification of volatile compounds were achieved on a Hewlett-Packard
constant levels. Fruit samples were taken from each storage atmosphere 5890 series II gas chromatograph equipped with a flame ionization
after 4 or 7 months of storage and placed at 20 C to simulate commercial
detector and a cross-linked free fatty acid phase (FFAP; 50 m 0.2 mm
shelf life and final quality of fruit that reach potential consumers. Unless
i.d. 0.33 μm) capillary column. The injection volume was 1 μL from each
stated otherwise, analyses were carried out after 7 days at 20 C.
extract in all of the analyses. The oven program was set at 70 C (1 min),
Chemicals. The chemicals obtained were of the highest quality and the temperature was initially raised by 3 C min-1 to 142 C and then
available and were supplied by Sigma-Aldrich (Steinheim, Germany)
by 5 C min-1 to 225 C. It was then kept constant for 10 min at this
unless otherwise indicated. Ethyl acetate, tert-butyl propanoate, propyl
final temperature. Helium was used as the carrier gas at a flow rate of
acetate, 1-propanol, ethyl butanoate, ethyl 2-methylbutanoate, butyl
0.8 mL min-1 (42 cm s-1), with a split ratio of 40:1, in the presence of air
acetate, 2-methyl-1-propanol, 1-butanol, pentyl acetate, 2-methyl-1-buta-
(400 mL min-1) and H2 (32 mL min-1). The injector and detector were
nol, hexyl acetate, 1-hexanol, and hexyl 2-methylbutanoate were obtained
held at 220 and 240 C, respectively. A second capillary column (SGE,
from Fluka (Buchs, Switzerland). Ethanol was purchased from Panreac
Milton Keynes, U.K.) with 95% dimethyl-5% diphenylpolysiloxane as the
Quı́mica, S.A. (Castellar del Valles, Spain). 2-Methylpropyl acetate was
obtained from Avocado Research Chemicals Ltd. (Madrid, Spain). stationary phase (BPX5, 30 m 0.25 mm i.d. 0.25 μm) was also used for
compound identification under the same operating conditions as described
Analysis of Standard Quality Parameters. Fifteen apples per batch
above. Volatile compounds were identified by comparing retention indices
(storage period atmosphere calcium treatment) were used individually
with those of standards and by enriching apple extract with authentic
for the analysis of flesh firmness, soluble solids content (SSC), titratable
acidity (TA), and skin color. Fruits were analyzed at harvest and 7 days samples. Quantification was made using butylbenzene (assay > 99.5%,
after removal from cold storage as described above. Flesh firmness was Fluka) as the internal standard, run with each added standard aside from
measured on opposite sides of each fruit with a penetrometer (Effegi, the matrix to develop standard curves for each volatile analyzed. A GC-
Milan, Italy) equipped with an 11-mm diameter plunger tip; results were MS system (Agilent Technologies 6890N-5973N) was used for compound
expressed in newtons. SSC and TA were measured in juice pressed from the confirmation, with the same capillary column as used in the GC analyses.
whole fruit. SSC was determined with a hand-held refractometer (Atago, Mass spectra were obtained by electron impact ionization at 70 eV. Helium
Tokyo, Japan), and results were expressed as percent of sucrose in an was used as the carrier gas (42 cm s-1), according to the same temperature
equivalent solution. TA was determined by titrating 10 mL of juice with gradient program as described above. Spectrometric data were recorded
0.1 N NaOH to pH 8.1 using 1% (v/v) phenolphthalein; results were given (MSD Chemstation D.03.00.611) and compared with those from the NIST
as grams of malic acid per liter. Fruit epidermis color was determined NBS75A original library mass spectra. The concentration of each volatile
with a portable tristimulus colorimeter (Chroma Meter CR-200, Minolta compound is expressed as micrograms per kilogram of fruit.
Corp., Osaka, Japan) using CIE illuminant D65 and with an 8 mm mea- Analysis of Acetaldehyde Concentration. A 5 mL sample of juice
suring aperture diameter. Skin color was measured at two points on the obtained individually from 15 fruits per batch (storage period atmo-
equator of each fruit that were 180 apart: one on the side exposed to sphere calcium treatment) was introduced in a 10 mL test tube and
sunlight (ES) and the other on the shaded side (SS). Hue angle was frozen at -20 C until analysis of acetaldehyde content as described
measured on both the side exposed to the sun and the shaded side, and the previously (11 ). Test tubes closed with a rubber cap and containing frozen
resulting values were respectively used as measurements of superficial and juice from each fruit were thawed and incubated at 65 C for 1 h.
background color. Thereafter, a 1 mL headspace gas sample was taken with a syringe
Determination of Calcium Content. To determine calcium content, and injected into a Hewlett-Packard 5890 series II gas chromatograph,
samples of pulp tissue were taken from five apples per batch, frozen in equipped with a column containing Carbowax (5%) on Carbopack
liquid nitrogen, freeze-dried, powdered, and kept at -80 C until proces- (60/80, 2 m 2 mm i.d.) as the stationary phase and a flame ionization
sing. Weight loss after lyophilization was consistently around 80%. One detector. Nitrogen was used as the carrier gas (24 cm s-1), and operating
gram of lyophilized powdered tissue was ashed in a muffle furnace at 500 conditions were as follows: oven temperature, 80 C; injector temperature,
C for 2 h. Ashes were digested thereafter with 4 mL of HCl/distilled water 180 C; detector temperature, 220 C. Acetaldehyde was identified and
(1:1, v/v) and heated at 70 C until complete dehydration of sample. quantified by comparison with external standards (Merck, Darmstadt,
Subsequently, dried material was resuspended in 2 mL of HCl/distilled Germany), and the results were expressed as microliters per liter.
water (1:1, v/v) for 15 min and passed through a Whatman 40 ashless filter. Extraction and Assay of Aroma Volatile-Related Enzyme Activ-
Finally, the filtrate was diluted to 50 mL in distilled water. Prepared ities. Samples of both peel and pulp tissue were taken separately from
Table 1. Meaning of X, Y, and Z Values for the Generic Sample Labels Did Calcium-Treated Fruit Actually Incorporate Calcium? To
1 2
check the actual incorporation of calcium into the tissues after
treatment and storage, flesh samples were analyzed by ICP-OES.
X a
4 7 Dipping of apples in 2% (w/v) CaCl2 resulted in significantly
Yb air 1:2 increased concentrations of calcium in the pulp of fruit (Table 3).
Zc 0 2 Range of increases observed was 16-39.6%, indicating that
a
Storage period at 1 C (months) + 7 days at 20 C. b Storage atmosphere CaCl2 treatment was efficient in incorporating calcium into fruit
conditions (O2/CO2). c Calcium treatment (% CaCl2, w/v). tissues. Calcium content was generally higher in fruit stored for
7 months than after 4 months, which is in agreement with earlier
five apples per batch (storage period atmosphere calcium treatment), reports on calcium penetration rates into the flesh of apples from
frozen in liquid nitrogen, lyophilized, powdered, and kept at -80 C until the epidermis. Calcium can penetrate the pulp through different
processing. One hundred milligrams of lyophilized powdered tissue feasible ways, including the lenticels as well as the microsco-
was used for each determination. Extraction and assay of LOX, pyru- pic fissures present in fruit cuticle and epidermis (22 ). Because
vate decarboxylase (PDC; EC 4.1.1.1), alcohol dehydrogenase (ADH;
fissures and other irregularities have been shown to become wider
EC 1.1.1.1), and alcohol o-acyltransferase (AAT; EC 2.3.1.84) activities
on crude enzyme extracts were performed as described elsewhere (12 ).
and deeper as storage duration increases (23 ), calcium ions could
Hydroperoxide lyase (HPL) activity was extracted and assayed accord- be taken up by fruit at higher rates. Thus, extending the storage
ing to the method in ref (13). Total protein content in the enzyme period may allow easier and faster calcium incorporation into
extract was determined with the Bradford method (14 ), using BSA as a fruit pulp from the epidermis.
standard. In all cases, one activity unit (U) was defined as the variation Standard Quality Parameters and Emission of Volatile Com-
in one unit of absorbance per minute. Each determination was done pounds after Cold Storage. Table 4 shows values for standard
in triplicate, and results were expressed as specific activity (U mg-1 of quality parameters in ‘Fuji Kiku-8’ apples after cold storage plus
protein). an additional period of 7 days at 20 C, which simulated
Statistical Analysis. A multifactorial design with storage period, commercial life and final quality of the fruits that reach potential
storage atmosphere, and calcium treatment as factors was used to stati- consumers. Similarly to previous reports (2 ), calcium treatment
stically analyze the results. All data were tested by analysis of variance
enhanced air-stored fruit quality by retarding flesh softening and
(GLM-ANOVA procedure) with the SAS program package (SAS In-
stitute, Cary, NC, 1988) (15 ). Means were separated by the Fisher’s LSD reducing the rate of titratable acid decline. Moreover, air-stored,
test at P e 0.05. Partial least-squares regression (PLSR) was used as a CaCl2-treated fruit also had higher superficial red color. Super-
predictive method to relate a matrix of a dependent variable (Y) to a set of ficial color is an important issue to take into account in the
explanatory variables (X) in a single estimation procedure. Sample names marketing of ‘Fuji’ apples, because insufficient red color devel-
were coded as XYZ, where X, Y, and Z refer to storage period, storage opment is generally associated with low visual consumer accept-
atmosphere, and calcium treatment, respectively, and take values of 1, 2, or ability and has been reported as the most important instrumental
3 as indicated in Table 1. Unscrambler version 6.11a software (CAMO quality parameter influencing apple purchasing patterns (24 ).
ASA, 1997) (16 ) was used for developing these models. As a pretreatment, Keeping fruit in ULO conditions also resulted in higher fruit firm-
data were centered and weighed by the inverse of the standard deviation of ness and TA content, regardless of calcium treatment. Further-
each variable to avoid dependence on measured units. Full cross-valida-
more, the combination of ULO storage and calcium treatment
tion was run as a validation procedure.
more effectively slowed the loss of fruit firmness and TA than
either alone, suggesting that ULO and calcium treatment had an
RESULTS AND DISCUSSION additive effect in slowing the loss of fruit firmness and TA.
Standard Quality and Emission of Volatile Compounds at Besides instrumental quality parameters, aroma is also a key
Harvest. Firmness of fruit at harvest averaged 71.2 N, the soluble attribute determining consumer acceptance of apples (6 ), and
solids content was 17.6%, and the titratable acidity was 3.5 g L-1, indeed the relevance of certain volatile compounds for consumer
thus indicating a suitable stage of maturity for long-term cold acceptability of ‘Fuji’ apples has been previously reported (19, 25).
storage, according to recommendations for this cultivar (17 ). Therefore, we were interested specifically in assessing the effects
Thirty-six compounds were identified and quantified in the of a calcium treatment, alone or in combination with storage in
volatile fraction emitted by ‘Fuji Kiku-8’ apples at harvest, ULO, on the emission of volatile compounds by ‘Fuji Kiku-8’
composed of 28 esters (9 acetates, 7 propanoates, 1 2-methylpro- fruit during shelf life at 20 C subsequent to cold storage. The
panoate, 4 butanoates, 3 2-methylbutanoates, and 4 hexanoates) emission of volatile compounds exhibiting positive log odor units
and 8 alcohols (Table 2). Esters were quantitatively prominent (OU) either at harvest or after storage in air, and thus considered
among the volatiles produced, accounting for 73 and 89% to have an impact on the overall flavor of fruit (18 ), is shown in
of total volatiles emitted at harvest and after 1 week at 20 C, Table 5. Butyl butanoate was also included due to its quantitative
respectively. Ethyl butanoate, ethyl 2-methylbutanoate, 2-meth- importance in the volatile fraction emitted by air-stored samples
ylbutyl acetate, butyl 2-methylbutanoate, pentyl propanoate, as well as ethyl acetate as an indicator of possible fermentative
hexyl acetate, hexyl propanoate, and hexyl 2-methylbutanoate processes in stored fruit.
were found to have log odor units (OU) > 1 at harvest. Therefore, Results showed that ULO-stored apples exhibited reduced
these molecules are suggested as having an impact on overall aroma volatile production, which is in accordance with previous
flavor (18 ). Ethyl butanoate, ethyl 2-methylbutanoate, 2-methyl- reports in ‘Fuji’ apples (19, 25). Although controlled atmosphere
butyl acetate, and hexyl acetate have been previously identified as (CA) storage has many beneficial effects on fruit quality, it has
compounds that contribute to ‘Fuji’ flavor at harvest (19, 20), been reported to cause a decrease in the production of volatiles
providing fresh-green and fruity odors (10, 21). The number of due partially to a lack of lipid-derived precursors for ester
putative flavor-contributing compounds increased in fruit kept at biosynthesis (20, 26). This reduction in the emission of vola-
20 C for 7 days after harvest, as the log OU of four more esters tile compounds by ULO-stored apples may decrease the value
(tert-butyl propanoate, butyl acetate, butyl propanoate, and of produce. However, results reported herein suggest that
2-methylbutyl propanoate) also became positive, suggesting an prestorage CaCl2 treatments caused enhanced emission of
enrichment of the aroma profile of apples in comparison to that some impact compounds (Table 5). After 4 months of cold
observed immediately after harvest. storage, the emissions of ethyl acetate, ethyl 2-methylbutanoate,
Table 2. Emission of Aroma Volatile Compounds ( μg kg ) by ‘Fuji Kiku-8’ Apples 0 (H) and 7 (H+7) Days after Harvest
-1
compounda RIb RIc OTHd He log OUf H+7e log OUf codeg
methyl acetate 854 - 8300 (a) 21.21 a -2.59 48.25 a -2.24

ethyl acetate 882 609 13500 (b) 23.38 a -2.76 32.93 a -2.61 ea
ethanol 912 - 100000 (c) 204.34 a -2.69 36.34 a -3.44 etOH
tert-butyl propanoate 928 717 19 (a) 16.02 a -0.07 35.46 a 0.27 tercbpr
propyl acetate 945 649 2000 (b) 13.88 b -2.16 27.70 a -1.86
methyl butanoate 955 656 5 (d) <0.5 0.57 mb
2-methylpropyl acetate 976 691 65 (e) 5.79 a -1.05 9.79 a -0.82
1-propanol 992 - 9000 (c) 9.06 b -3.00 22.66 a -2.60
ethyl butanoate 1002 803 1 (f) 4.46 b 0.65 11.16 a 1.05 eb
propyl propanoate 1008 809 57 (c) 12.39 b -0.66 28.25 a -0.30
ethyl 2-methylbutanoate 1015 845 0.006 (e) 24.00 a 3.60 41.31 a 3.84 e2mb
butyl acetate 1040 813 66 (b) 45.36 b -0.16 98.37 a 0.17 ba
2-methylpropyl propanoate 1046 865 - <0.5 1.88
2-methyl-1-propanol 1054 996 250 (g) 2.56 a -1.99 4.83 a -1.71
2-methylbutyl acetate 1096 876 11 (e) 360.33 a 1.52 653.09 a 1.77 2mba
1-butanol 1119 626 500 (c) 18.23 b -1.44 53.99 a -0.97 bOH
butyl propanoate 1123 910 25 (c) 16.83 b -0.17 42.70 a 0.23 bpr
butyl 2-methylpropanoate 1129 1009 80 (h) 12.90 b -0.79 38.81 a -0.31
pentyl acetate 1161 914 43 (b) 11.54 a -0.57 22.46 a -0.28
2-methylbutyl propanoate 1180 950 19 (a) 12.60 a -0.18 45.10 a 0.38 2mbpr
2-methyl-1-butanol 1199 667 250 (f) 48.40 b -0.71 135.81 a -0.26 2mbOH
butyl butanoate 1218 1000 100 (h) 10.26 b -0.99 32.47 a -0.49 bb
butyl 2-methylbutanoate 1235 1042 17 (h) 21.31 b 0.10 70.37 a 0.62 b2mb
pentyl propanoate 1247 969 1 (e) 1.89 b 0.28 5.25 a 0.72 ppr
1-pentanol 1262 688 4000 (g) 1.47 b -3.43 4.47 a -2.95 pOH
hexyl acetate 1292 1015 2 (g) 42.78 b 1.33 98.35 a 1.69 ha
hexyl propanoate 1379 1109 8 (i) 15.72 b 0.29 58.28 a 0.86 hpr
1-hexanol 1392 869 500 (g) 1.69 b -2.47 6.99 a -1.85 hOH
2-methylpropyl hexanoate 1399 1153 - 1.65 b 23.90 a
butyl hexanoate 1473 1196 700 (h) 23.62 b -1.47 94.90 a -0.87
hexyl butanoate 1477 1197 250 (e) 9.01 b -1.44 38.10 a -0.82
hexyl 2-methylbutanoate 1488 1239 6 (h) 58.67 b 0.99 169.53 a 1.45 h2mb
octyl acetate 1549 1215 12 (f) 1.20 a -1.00 6.51 a -0.27
2-ethylhexanol 1565 1031 - 1.48 a 3.73 a
pentyl hexanoate 1590 1293 - 3.39 a 7.98 a
hexyl hexanoate 1687 1392 6400 (j) 8.99 b -2.85 29.62 a -2.33
a b
Compounds identified on the basis of a comparison of mass spectrometric data and retention indices with authentic reference compounds. Kovats retention index in cross-
linked FFAP column. c Kovats retention index in BPX5 column; -, eluted with the solvent. d Odor threshold (μg kg-1) in water reported in ref (a) (34 ), (b) (35 ), (c) (36 ), (d) (37 ), (e)
(38 ), (f) (39 ), (g) (18 ), (h) (40 ), (i) (41 ), (j) (42 ); -: not found. e Values are the means of four samples obtained each from 2 kg of apples after 4 h of collection. Means within the
same row followed by different lower case letters are significantly different at P e 0.05 (LSD test). f Log10 of odor unit value = log10 (amount/OTH). g Codes used for multivariate
analysis of data.
Table 3. Calcium Content (mg 100 g-1 FW) in the Flesh of ‘Fuji Kiku-8’ Apples applications prior to cold storage of apples under hypoxic
after 7 Days at 20 C following Cold Storagea conditions might also be useful as a means for partial regenera-
storage period tion of aroma quality during the commercial life of fruit,
storage atmosphere treatment 4 months 7 months particularly after middle-term cold storage. These results are in
agreement with earlier findings (27 ), when it was observed that
air untreated 2.88 Bb 4.14 Ba calcium-treated ‘Golden Delicious’ apples produced the same or
CaCl2 3.89 Ab 4.83 Aa higher total flavor-associated volatile levels in comparison to
ULO untreated 3.15 Bb 5.08 Ba
untreated fruit when stored for at least 4 months.
CaCl2 4.40 Ab 5.89 Aa
After fruits were stored for 7 months, the effects of calcium
a
Data represent means of three replicates. Means within the same column for a treatment on the production of volatile compounds were different
given storage atmosphere followed by different capital letters are significantly for the two storage atmospheres considered. The emission of
different at P e 0.05 (LSD test). Means in the same row followed by different lower
case letters are significantly different at P e 0.05 (LSD test). some aroma volatile compounds, namely, ethyl acetate, tert-butyl
propanoate, 2-methylbutyl acetate, 2-methylbutyl propanoate,
2-methylbutyl acetate, butyl propanoate, butyl butanoate, and butyl butanoate, butyl 2-methylbutanoate, and hexyl 2-methyl-
pentyl propanoate were higher in CaCl2-treated than in untreated butanoate, was higher in CaCl2-treated fruit stored in air than in
apples, regardless of storage atmosphere, which suggests that untreated control fruit, whereas no such enhancement was found
calcium applications might help to improve aroma quality in this in apples stored under ULO (Table 5). Furthermore, decreased
cultivar after middle-term storage. Indeed, sensory analysis by productions of butyl acetate, 2-methylbutyl acetate, butyl
means of a consumer panel (data not shown) indicated higher 2-methylbutanoate, and hexyl 2-methylbutanoate were observed
acceptance scores for CaCl2-treated fruit. For ULO-stored sam- in fruit stored in ULO, which partially agrees with previous work
ples, calcium treatments enhanced the production of butyl on ‘Fuji’ apples (19 ), when significantly reduced total aroma
acetate, butyl 2-methylbutanoate, hexyl acetate, and hexyl volatile emission was found after storage for 7 months in ULO.
propanoate after 4 months of cold storage. Therefore, CaCl2 The reduction in total volatile emission after long-term storage of
a
Table 4. Maturity and Quality Parameters of ‘Fuji Kiku-8’ Apples at Harvest and after 7 Days at 20 C following Cold Storage
storage period
4 months 7 months
at harvest atmosphere untreated CaCl2 untreated CaCl2
ethylene production (μL kg-1 h-1) 0.1 air 14.6 Aa 16.6 Aa 12.9 Ba 21.7 Aa
ULO 0.5 Ab 0.5 Ab 1.3 Ab 1.4 Ab
acetaldehyde content (μL L-1) 0.3 air 0.9 Ba 1.4 Aa 1.0 Aa 0.7 Ba
ULO 0.7 Bb 1.0 Ab 0.7 Ab 0.4 Ba
firmness (N) 71.2 air 64.3 Bb 70.2 Ab 55.3 Bb 63.1 Ab
ULO 71.1 Ba 76.5 Aa 72.4 Aa 72.3 Aa
TA (g L-1) 3.5 air 1.5 Bb 2.1 Ab 0.9 Bb 1.2 Ab
ULO 2.7 Aa 2.5 Aa 7.7 Ba 2.0 Aa
SSC (%) 17.6 air 16.8 Aa 16.9 Aa 16.1 Ba 16.6 Aa
ULO 16.8 Aa 17.0 Aa 15.5 Bb 16.1 Ab
hue (SS) 101.9 air 89.1 Aa 91.3 Aa 90.4 Aa 75.1 Bb
ULO 80.1 Aa 78.6 Ab 85.4 Aa 87.7 Aa
hue (ES) 44.8 air 54.3 Aa 44.5 Ba 58.0 Aa 45.4 Ba
ULO 46.3 Ab 41.7 Aa 48.0 Ab 42.3 Aa
a
Data represent means of 3 (ethylene production) and of 15 (acetaldehyde content and standard quality parameters) replicates. Means in the same row for a given storage
period showing different capital letters are significantly different at P e 0.05 (LSD test). Means followed by different lower case letters within a column for a given parameter are
significantly different at P e 0.05 (LSD test).
Table 5. Emission ( μg kg-1) of Volatile Esters Contributing to Overall Flavor of ‘Fuji Kiku-8’ Apples after 7 Days at 20 C following Cold Storagea
storage period
4 months 7 months
atmosphere untreated CaCl2 untreated CaCl2
ethyl acetate air 34.1 (-2.6) Ba 53.8 (-2.4) Aa 10.1 (-3.1) Ba 20.0 (-2.8) Aa
ULO 16.4 (-2.9) Bb 35.5 (-2.5) Ab 13.9 (-2.9) Aa 16.4 (-2.9) Aa
tert-butyl propanoate air 31.6 (0.2) Aa 33.4 (0.2) Aa 6.0 (-0.5) Ba 13.1 (-0.1) Aa
ULO 5.9 (-0.5) Ab 9.4 (-0.3) Ab 4.0 (-0.7) Aa 4.5 (-0.6) Ab
methyl butanoate air 19.9 (0.6) Aa 19.5 (0.6) Aa 9.9 (0.3) Aa 12.7 (0.4) Aa
ULO 1.1 (-0.6) Bb 0.8 (-0.8) Bb 0.5 (-1.0) Ab 0.2 (-1.4) Ab
ethyl butanoate air 44.5 (1.6) Aa 41.1 (1.6) Aa 14.2 (1.2) Aa 17.5 (1.2) Aa
ULO 9.0 (1.0) Ab 14.7 (1.2) Ab 5.3 (0.7) Ab 1.0 (0.0) Ab
ethyl 2-methylbutanoate air 151.5 (4.4) Ba 179.8 (5.5) Aa 48.0 (3.9) Aa 52.3 (3.9) Aa
ULO 48.4 (3.9) Bb 82.7 (4.1) Ab 18.9 (3.5) Ab 11.6 (3.2) Ab
butyl acetate air 203.2 (0.5) Aa 232.2 (0.5) Aa 96.6 (0.2) Aa 98.7 (0.2) Aa
ULO 70.9 (0.0) Bb 142.8 (0.3) Ab 64.6 (0.0) Aa 18.7 (-0.5) Bb
2-methylbutyl acetate air 536.6 (1.7) Ba 708.7 (1.8) Aa 247.6 (1.4) Bb 334.3 (1.5) Aa
ULO 456.2 (1.6) Ba 706.8 (1.8) Aa 459.7 (1.6) Aa 167.9 (1.2) Bb
butyl propanoate air 62.2 (0.4) Ba 96.5 (0.6) Aa 46.8 (0.3) Aa 50.0 (0.3) Aa
ULO 22.1 (0.0) Bb 48.2 (0.3) Ab 21.4 (-0.1) Ab 9.8 (-0.4) Ab
2-methylbutyl propanoate air 29.0 (0.2) Aa 46.3 (0.4) Aa 12.8 (-0.2) Ba 22.0 (0.1) Aa
ULO 7.3 (-0.4) Ab 20.4 (0.0) Ab 12.7 (-0.2) Aa 6.9 (-0.4 Ab
butyl butanoate air 161.9 (0.2) Ba 194.0 (0.3) Aa 79.6 (-0.1) Ba 124.8 (0.1) Aa
ULO 35.1 (-0.5) Bb 64.4 (-0.2) Ab 30.8 (-0.5) Ab 17.9 (-0.7) Ab
butyl 2-methylbutanoate air 324.5 (1.3) Aa 369.4 (1.3) Aa 107.6 (0.8) Ba 167.5 (1.0) Aa
ULO 61.2 (0.6) Bb 134.0 (0.9) Ab 49.0 (0.5) Ab 15.9 (0.0) Bb
pentyl propanoate air 4.9 (0.7) Ba 6.9 (0.8) Aa 1.8 (0.3) Aa 2.3 (0.4) Aa
ULO 2.2 (0.3) Bb 4.6 (0.4) Ab 1.2 (0.1) Aa 0.8 (-0.1) Ab
hexyl acetate air 245.0 (2.1) Aa 258.0 (2.1) Aa 113.7 (1.8) Aa 142.0 (1.9) Aa
ULO 154.7 (1.9) Bb 204.5 (2.0) Ab 98.4 (1.7) Aa 49.6 (1.4) Ab
hexyl propanoate air 154.6 (1.3) Aa 148.0 (1.3) Aa 47.7 (0.8) Ba 70.6 (0.9) Aa
ULO 54.7 (0.8) Bb 87.9 (1.0) Ab 39.6 (0.7) Aa 25.7 (0.5) Ab
hexyl 2-methylbutanoate air 966.5 (2.2) Aa 687.8 (2.0) Ba 255.1 (1.6) Ba 363.8 (1.8) Aa
ULO 273.6 (1.6) Ab 359.2 (1.8) Ab 187.3 (1.5) Aa 85.0 (1.2) Bb
a
Data represent means of four replicates obtained each from 2 kg of apples after 4 h of collection. Numbers between brackets stand for log OU, where OU = concentration/ odor
threshold. Means in the same row for a given storage period showing different capital letters are significantly different at P e 0.05 (LSD test). Means followed by different small
letters within a column for a given compound are significantly different at P e 0.05 (LSD test).
apples in CA has been suggested to arise from partial inhibition of Volatile-Related Enzyme Activities after Cold Storage. A PLSR
some volatile-related enzyme activities (20, 26). Therefore, the model was developed for the emission of selected volatile esters,
effects of calcium treatment and of storage conditions considered their alcohol precursors, and acetaldehyde content (Y variables),
herein on several volatile-related enzyme activities were also with LOX, HPL, PDC, ADH, and AAT activities as the X
analyzed. variables. The model accounted for 84% of total variability
Table 6. Specific Activities (U mg protein) of Volatile-Related Enzymes in
-1
the Pulp of ‘Fuji Kiku-8’ Apples after 7 Days at 20 C following Cold Storagea
storage period
4 months 7 months
atmosphere untreated CaCl2 untreated CaCl2
LOX air 5.519 Aa 4.381 Ba 3.875 Aa 2.925 Bb

ULO 4.234 Aa 4.103 Aa 4.121 Aa 4.403 Aa
PDC air 50.946 Ba 85.420 Aa 41.085 Aa 40.079 Aa
ULO 29.304 Ba 55.962 Ab 25.079 Ab 24.815 Ab
ADH air 34.961 Ba 56.833 Aa 42.488 Aa 42.257 Aa
ULO 19.428 Bb 28.818 Ab 35.086 Ab 38.125 Aa
AAT air 0.009 Aa 0.011 Aa 0.030 Ba 0.036 Aa
ULO 0.011 Aa 0.013 Aa 0.022 Ab 0.023 Ab
a
Data represent means of three replicates obtained each from 2 kg of apples
after 4 h of collection. Means in the same row for a given storage period showing
different capital letters are significantly different at P e 0.05 (LSD test). Means
followed by different lower case letters within a column for a given enzyme activity
are significantly different at P e 0.05 (LSD test).
to produce acetaldehyde, which can be processed subsequently by

ADH or by aldehyde dehydrogenase (ALDH, EC 1.2.1.5) to
render ethanol or acetyl-CoA, respectively. In turn, acetyl-CoA
acts as the acylating agent for acetate ester formation (28 ).
Although all of these products can accumulate during fruit
ripening even under aerobic conditions, PDC and ADH are
generally associated with anaerobic metabolism. Consequently,
PDC and ADH activities can be induced by low O2 levels in fruit
tissues. Previous work (29 ) has shown that gradients in O2
Figure 1. Scores (A) and loadings (B) plots of PC1 versus PC2 corre-
concentration across pulp tissue of apples were increased by
sponding to a PLSR model for emission of volatile compounds (Y variables)
vacuum infiltration of CaCl2, due to decreased O2 diffusivity in
versus volatile-related enzyme activities (X variables) in ‘Fuji Kiku-8’ apple
pulp tissues and increased skin resistance to gas diffusion. In
fruit after cold storage under different conditions. Samples and volatile
addition, applications of calcium can also increase internal CO2
compounds are labeled as indicated in Tables 1 and 2, respectively
levels in apples (30, 31). Thus, dipping of fruit in a CaCl2 solution
(AA, acetaldehyde).
could have lessened O2 levels and caused CO2 accumulation
observed in the production of volatile compounds (Figure 1), inside the fruit, which would explain enhanced PDC and ADH
which suggests a positive link to the enzyme activities included in activities observed in CaCl2-treated apples stored for 4 months
the regression analysis. Samples distributed along the first princi- (Table 6), possibly leading to improved availability of substrates
pal component (PC1) of the corresponding scores plot according for AAT-catalyzed ester production during the shelf life period.
to storage period (Figure 1A). In turn, samples stored for Indeed, hypoxia-induced acetaldehyde and ethanol accumulation
4 months separated according to storage atmosphere (PC1) and has been shown to have the potential to increase the production of
according to calcium treatment for each storage atmosphere volatile esters in apple (32 ). Accordingly, the emission of alcohol
(PC2). With regard to fruit stored for 7 months, no clear precursors and acetaldehyde content in the samples were asso-
differentiation was observed between samples. The loadings plot ciated with the emission of volatile esters as shown by PLSR
(Figure 1B) showed that samples stored for 4 months were analysis (Figure 1B). Actually, an increase in the production of
characterized by higher emission of all volatile compounds some alcohol precursors (ethanol, 1-butanol, 2-methyl-1-butanol,
included in the model. Higher emissions were associated with and 1-hexanol) due to CaCl2 treatment was observed in apples
higher PDC, ADH, and, to a lesser extent, LOX activities in the stored for 4 months (Table 7). Acetaldehyde content in CaCl2-
pulp tissue, particularly for fruit treated with CaCl2 and stored in treated fruit was also higher than in untreated apples (Table 4).
air. The plot also indicated higher volatile production for CaCl2- This was possibly related to enhanced ADH and PDC activities,
treated fruit stored in ULO in comparison to untreated fruit, respectively, in the pulp of these apples (Table 6). In fact, good
linked likewise to higher levels of LOX, PDC, and ADH correlations were found between the emission of some specific
activities. The model reflected the diminished capacity for bio- ester families and their corresponding alcohol precursors. The
synthesis of volatile compounds after long-term storage of fruit plots of the selected ethyl and butyl esters emitted versus ethanol
(Table 5), possibly arising from a lessening in these enzyme and 1-butanol are given as examples (panels A and B, respectively,
activity levels. of Figure 2), showing R2 values of 0.74 and 0.94, correspondingly,
AAT activity is directly responsible for the production of suggesting the importance of substrate supply for AAT-catalyzed
volatile esters by linking an alcohol to an acyl-CoA (8 ). However, ester production.
the regression model showed no apparent relationship between LOX activity, which has been found to be essential for the
ester production and this enzyme activity (Figure 1B), suggesting recovery of the ability to synthesize volatile esters after cold
the relevance of an adequate supply of substrates for the storage of ‘Fuji’ apples (26 ), was inhibited by calcium treatment
AAT-catalyzed reaction by enzymes located upstream in the in air-stored fruit (Table 6). During fruit ripening, cell walls and
biosynthetic pathway. This would explain the strong association membranes become more permeable to different substrates,
observed between PDC and ADH activities in the pulp tissue and increasing the possibility of a LOX-mediated cleavage of
the emission of volatile esters after storage. PDC uses pyruvic acid fatty acids, but this chance of reaction might be delayed in
Table 7. Emission ( μg kg ) of Alcohol Precursors for Volatile Ester
-1
of most volatile esters selected than those stored for 4 months
Biosynthesis by ‘Fuji Kiku-8’ Apples after 7 Days at 20 C following Cold (Table 5). This may be related to lower levels of their alcohol
Storagea precursors. Anyhow, air-stored samples dipped in CaCl2 emitted
storage period higher levels of some volatile compounds than untreated fruit.
4 months 7 months After 7 months of cold storage, CaCl2-treated apples stored in air
were characterized by higher AAT activity in the pulp tissue
atmosphere untreated CaCl2 untreated CaCl2 (Table 6) and, indeed, showed enhanced production of some
ethanol air 24.2 Ba 41.3 Aa 11.7 Aa 13.9 Aa volatile esters (Table 5). These results indicate that, although not
ULO 15.1 Ba 33.9 Aa 9.4 Aa 10.3 Aa sufficient, AAT activity was necessary for the production of
1-butanol air 168.0 Ba 214.0 Aa 38.2 Ba 53.7 Aa volatile esters after cold storage and that not only substrate
ULO 37.4 Bb 69.7 Ab 23.7 Cb 3.2 Db supply but also ester-forming capacity itself may be compromised
2-methyl-1-butanol air 133.6 Ba 211.3 Aa 92.8 Aa 111.6 Aa after long-term storage of ‘Fuji Kiku-8’ apples. Calcium dips
ULO 68.3 Bb 124.5 Ab 109.6 Aa 59.7 Bb prior to cold storage in air were thus shown to potentially have
1-pentanol air 4.2 Aa 3.8 Aa 2.3 Aa 2.5 Aa beneficial effects on the aroma quality of ‘Fuji Kiku-8’ apples,
ULO 1.9 Ab 2.4 Ab 1.7 Aa 1.6 Aa even after a long period of cold storage. The observed increase in
1-hexanol air 5.8 Ba 8.5 Aa 2.6 Ba 6.0 Aa
AAT activity, which was concomitant with higher ethylene
ULO 4.8 Ba 8.1 Aa 4.7 Aa 1.8 Bb
production (Table 4) in CaCl2-treated fruit after long-term
a
Data represent means of four replicates obtained each from 2 kg of apples after storage, may be in agreement with previous work reporting
4 h of collection. Means in the same row for a given storage period showing different the activity of AAT to be ethylene-dependent (33 ). However,
capital letters are significantly different at P e 0.05 (LSD test). Means followed by
different lower case letters within a column for a given compound are significantly inhibition of AAT activity in ULO-stored samples (Table 6) was
different at P e 0.05 (LSD test). not reversed by calcium treatments, and indeed the beneficial
effects of calcium applications on the emission of volatile
esters after long-term storage were restricted to fruit stored in
air (Table 5).
In summary, calcium treatment generally increased production
of volatile esters after storage of ‘Fuji Kiku-8’ apples, probably as
a consequence of enhanced PDC and ADH activities with
concomitantly better supply of acetaldehyde and alcohol pre-
cursors in fruit stored for 4 months. Treatment also increased
AAT activity in samples stored in cold air for a longer period,
leading to higher emission of volatile esters. Inhibition of volatile
ester emission after long-term storage in ULO was apparently not
recoverable by calcium applications. Thus, postharvest calcium
treatments have the potential to improve aroma quality of cold-
stored ‘Fuji Kiku-8’ apples, with interesting implications from the
commercial point of view: these treatments have no damaging
effects on the environment, are considerably more economical
and simple than CA technology, and may have beneficial side
effects on the nutritional quality of produce.
ABBREVIATIONS USED
AA, acetaldehyde; AAT, alcohol o-acyltransferase; ADH,
alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; CA,
controlled atmosphere; ES, exposed side; HPL, hydroperoxide
lyase; LOX, lipoxygenase; OTH, odor threshold; OU, odor unit;
Figure 2. Correlation between the emissions of ethanol (A) and 1-butanol PC1, first principal component; PC2, second principal compo-
(B) and flavor-contributing ethyl and butyl esters, respectively, emitted nent; PDC, pyruvate decarboxylase; PLSR, partial least-squares
by cold-stored ‘Fuji Kiku-8’ apples. Points represent the means of four regression; SS, shaded side; SSC, soluble solids content; TA,
replicates. titratable acidity; ULO, ultralow oxygen atmosphere.
CaCl2-treated fruit as a consequence of the beneficial role of this ACKNOWLEDGMENT

mineral in maintaining the structural integrity of membranes (9 ). We are indebted to P. Sope~na and A. Latorre for technical
The observation that the production of several straight-chain assistance.
esters, generally considered to arise from lipid metabolism
through the LOX pathway (8 ), was enhanced in CaCl2-treated
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Postharvest Biology and Technology 57 (2010) 114–123
Contents lists available at ScienceDirect
Postharvest Biology and Technology

journal homepage: www.elsevier.com/locate/postharvbio
The emission of flavour-contributing volatile esters by ‘Golden Reinders’ apples is

improved after mid-term storage by postharvest calcium treatment
Abel Ortiz a , Gemma Echeverría c , Jordi Graell b , Isabel Lara a,∗
a
Departament de Química, Unitat de Postcollita-XaRTA, Universitat de Lleida, Rovira Roure 191, 25198 Lleida, Spain
b
Departament de Tecnologia d’Aliments, Unitat de Postcollita-XaRTA, Universitat de Lleida, Rovira Roure 191, 25198 Lleida, Spain
c
IRTA Lleida, Unitat de Postcollita-XaRTA, Rovira Roure 191, 25198 Lleida, Spain
a r t i c l e i n f o a b s t r a c t
Article history: Calcium treatment of apple (Malus × domestica Borkh.) fruit is a widely used practice aimed mainly at
Received 10 December 2009 avoiding the development of bitter pit. However, very few studies have reported the effects of such
Accepted 16 March 2010 treatments on the production of flavour-related volatile compounds, despite the relevance of aroma
and taste for overall quality. In this study, commercially mature ‘Golden Reinders’ apples were dipped
Keywords: in 2% (w/v) calcium chloride prior to storage at 1 ◦ C and 92% RH under either air, standard controlled
Alcohol dehydrogenase
atmosphere (SCA; 3 kPa O2 :2 kPa CO2 ) or ultra-low oxygen atmosphere (ULO; 1 kPa O2 :2 kPa CO2 ) for
Apple
19 or 31 weeks, and subsequent removal to 20 ◦ C for 7 d, after which the emission of aroma-related
Aroma
Calcium dips
volatile compounds and a number of maturity and standard quality parameters were assessed. Calcium
Controlled atmosphere treatment notably enhanced the production of aroma volatile compounds after mid-term storage under
Pyruvate dehydrogenase air and, to a lesser extent, under SCA. Retention of titratable acidity (TA) was also improved in air-
Volatile esters stored fruit in response to calcium treatment, while no effects on firmness were observed. Although
ULO-stored samples showed the highest firmness and TA values under storage, aroma volatile production
was severely depleted, and calcium treatment could not overcome this inhibition. It is therefore suggested
that calcium applications allow improving aroma quality while preserving adequate levels of key standard
quality parameters after mid-term storage of ‘Golden Reinders’ fruit, and are thus a simple and economical
alternative to CA storage of this apple cultivar.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction quality (Goldberg, 1984) and in preservation of firmness and cell

wall structure (Saftner et al., 1998; Abbott et al., 2000) has been
The apple cultivar ‘Golden Reinders’ originated from a mutation explored in previous work, little information has been reported to
of ‘Golden Delicious’. It exhibits an excellent aptitude for long-term date on the effects of calcium treatments on fruit flavour.
postharvest storage, particularly under controlled atmospheres Although perception of fruit flavour results from the combina-
(CA), and early harvest time improves the marketing possibilities tion of mouth feel, taste and aroma, the latter is often considered
in comparison to other cultivars. While these fruit show good resis- to play a dominant role (Kader, 2008). Apple aroma results from
tance to russeting and cracking, they are susceptible to bitter pit, a complex mixture of a large number of odour-active volatile
a corking disorder characterised by discrete sunken lesions that compounds including carboxylic esters as the predominant chem-
can develop just prior to harvest or during storage. As bitter pit ical species together with alcohols, aldehydes, ethers and ketones
is generally associated with low calcium content in the fruit flesh (Dimick and Hoskin, 1983; Goff and Klee, 2006), and depends
(Ferguson and Watkins, 1989), calcium dips have become a stan- upon the combination, concentration and odour threshold (OTH)
dard practice in the fruit industry. Yet besides appearance, other of volatiles emitted (Buttery, 1993; Dixon and Hewett, 2000). The
factors driving desirability and final consumer preference for apples impact of an individual compound on the volatile profile of apples
also include nutritional value, texture and flavour (Harker et al., can be measured using the odour activity value (OAV) (Rothe and
2008). Whereas the role of calcium in the promotion of nutritional Thomas, 1963; Guadagni et al., 1966). This value is defined as the
ratio of concentration of the volatile compound in the food to
its odour threshold, and it is generally accepted that compounds
displaying log OAV > 0 are likely to contribute actively to overall
∗ Corresponding author at: Departament de Química, Unitat de Postcollita-XaRTA
flavour (Plotto et al., 2000; Mehinagic et al., 2006; Ryan et al., 2008).
Universitat de Lleida, Rovira Roure 191, 25198 Lleida, Spain. Tel.: +34 973 702526;
fax: +34 973 238264. Fruit aroma is cultivar-specific (Pérez and Sanz, 2008) and,
E-mail address: lara@quimica.udl.cat (I. Lara). accordingly, differences in the contribution of each individual com-
0925-5214/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.postharvbio.2010.03.006
A. Ortiz et al. / Postharvest Biology and Technology 57 (2010) 114–123 115
pound to overall flavour and consumer acceptance have been 2.3. Analysis of standard quality parameters
observed among apple cultivars (Holland et al., 2005; Komthong et
al., 2006; Mehinagic et al., 2006; Echeverría et al., 2008; Villatoro Fifteen apples per batch (storage period × atmosphere ×
et al., 2009). Additionally, CA storage of apples, widely used for calcium treatment) were used individually for the analysis of
fruit quality preservation and shelf-life extension, has been shown flesh firmness, soluble solids content (SSC), titratable acidity (TA)
consistently to reduce the production of some volatiles, with and skin colour. Flesh firmness was measured on two opposite
concomitant detrimental effects on flavour (Streif and Bangerth, sides of each fruit with a hand-held penetrometer (Effegi, Milan,
1988; Dixon and Hewett, 2000; Mattheis et al., 2005). It would Italy) equipped with an 11-mm diameter plunger tip; results were
be therefore highly advisable to identify optimal postharvest han- expressed in N. SSC and TA were measured in juice pressed from
dling procedures for each apple cultivar, which may allow supply the whole fruit. SSC was determined with a hand-held refractome-
of better-tasting fruit, hence favouring enhanced consumption. ter (Atago, Tokyo, Japan), and results expressed as % sucrose in
Flavour is particularly important in this regard. It has been observed an equivalent solution. TA was determined by titrating 10 mL of
recently that calcium treatment led to increased production of juice with 0.1N NaOH to pH 8.1 using 1% (v/v) phenolphthalein;
volatiles after mid-term storage of ‘Fuji’ apples (Ortiz et al., 2009). results were given as g malic acid L−1 . Fruit epidermis colour was
However, these results may be not the same for ‘Golden Rein- determined with a portable tristimulus colorimeter (Chroma Meter
ders’ fruit, which differ notably in their postharvest behaviour, CR-200, Minolta Corp, Osaka, Japan) using CIE illuminant D65 and
including storage potential and textural properties. Therefore, the with an 8-mm measuring aperture diameter.
purpose of this work was to assess the responses of ‘Golden Rein-
ders’ apples to the calcium applications widely used for cultivars 2.4. Determination of ethylene production
in the ‘Golden’ group, with special focus on the biosynthesis of
flavour-contributing volatile compounds. To simulate commer- The rate of ethylene production was determined (3 repli-
cial procedures before marketing of produce, CaCl2 -treated and cates × 2 apples/replicate) by placing fruit in 3-L respiration flasks,
untreated fruit were stored under either air or CA. continuously aerated with humidified air at a flow rate of around
1.5 L h−1 . Samples of the effluent air were taken with a 1-mL
syringe, and injected into a gas chromatograph (Agilent Tech-
2. Materials and methods
nologies 6890N) equipped with a flame ionisation detector and
an alumina column (1.5 m × 3 mm). Gas analyses were conducted
2.1. Plant material, calcium treatment and storage conditions
isothermally at 100 ◦ C. N2 carrier gas, air and H2 flows were 45,
400 and 45 mL min−1 , respectively. The injector and detector were
Apple (Malus × domestica Borkh., cv. Golden Reinders) fruit were
held at 120 and 180 ◦ C, respectively. Results were expressed as mL
harvested in 2007 at commercial maturity (139 d after full bloom),
ethylene kg−1 h−1 .
from 7-year-old trees grafted on M-9 EMLA rootstocks at the IRTA-
Experimental Station in Mollerussa, in the area of Lleida (NE, Spain).
2.5. Analysis of acetaldehyde concentration
Ethylene production at harvest was 1.4 mL kg−1 h−1 , when firm-
ness averaged 72.3 N, starch index was 5.3 (1, full starch; 10, no
A 5-mL sample of juice obtained individually from 15 fruit
starch), soluble solids content 13.3% and titratable acidity 6 g L−1 .
per batch (storage period × atmosphere × calcium treatment) was
Immediately after harvest, fruit were randomly divided into six
placed in a 10-mL test tube closed with a rubber cap and incu-
lots, three of which were dipped in a CaCl2 solution (2%, w/v, in
bated at 65 ◦ C for 1 h (Ke et al., 1994). A 1-mL headspace gas sample
deionised water) at ambient temperature for 5 min. After treat-
was taken thereafter with a syringe and injected into a gas chro-
ment, CaCl2 -treated and untreated apples were stored at 1 ◦ C and
matograph (Agilent Technologies 6890N) equipped with a column
92% RH under either air, standard controlled atmosphere (SCA;
containing Carbowax (5%) on Carbopack (60/80, 2 m × 2 mm i.d.)
3 kPa O2 :2 kPa CO2 ) or ultra-low oxygen atmosphere (ULO; 1 kPa
as the stationary phase, and a flame ionisation detector. Nitrogen
O2 :2 kPa CO2 ). O2 and CO2 concentrations were monitored contin-
was used as the carrier gas (45 mL min−1 ), and operating conditions
uously, and corrected automatically using N2 from a tank and by
were as follows: oven temperature 80 ◦ C, injector temperature
scrubbing off excess CO2 with a charcoal system. A humidifier was
180 ◦ C, detector temperature 220 ◦ C. Acetaldehyde was identified
used to maintain RH to constant levels. Fruit samples were taken
and quantified by comparison with external standards, and results
after 19 or 31 weeks of storage, and placed at 20 ◦ C to simulate
were expressed as mL L−1 .
commercial shelf-life and final quality of fruit reaching potential
consumers. Unless stated otherwise, analyses were carried out 7 d
2.6. Analysis of volatile compounds
thereafter.
Eight kg of intact apples (2 kg/replicate × 4 replicates) were

2.2. Determination of calcium content taken for the extraction and analysis of volatile compounds. The
extraction was performed using the dynamic headspace method.
Samples of flesh tissue (outer cortex) were taken (3 repli- Briefly, intact fruit from each treatment were placed into an
cates × 2 apples/replicate), frozen in liquid nitrogen, freeze-dried, 8-L Pyrex container, and an air stream (900 mL min−1 ) was passed
powdered, and kept at −80 ◦ C until processing. One gram of through for 4 h. The effluent was then recovered in an adsorption
lyophilised powdered tissue was ashed in a muffle furnace at 500 ◦ C tube (ORBO-32TM ; SUPELCO, Bellefonte, PA, USA) filled with 100 mg
for 2 h. Ashes were digested thereafter with 4 mL HCl:distilled of activated charcoal (20/40 mesh), from which volatile compounds
water (1:1, v/v) and heated at 70 ◦ C until complete sample dehy- were desorbed by agitation for 40 min with 0.5 mL of diethyl
dration. Dried material was then resuspended in 2 mL HCl:distilled ether. Identification and quantification of volatile compounds were
water (1:1, v/v) for 15 min, filtered through ‘Whatman 40 Ashless’ achieved on a Hewlett Packard 5890 series II gas chromatograph
paper, and the filtrate diluted to 50 mL in distilled water. Sam- equipped with a flame ionisation detector and a cross-linked free
ples were then analysed by inductively coupled plasma emission fatty acid phase (FFAP; 50 m × 0.2 mm i.d. × 0.33 mm) capillary col-
spectroscopy (ICP-OES) in a ‘Horiba Jobin Yvon ACTIVA’ spectrom- umn. The injection volume was 1 mL from each extract in all the
eter. Each determination was done in triplicate, and results were analyses. The oven program was set at 70 ◦ C (1 min) and the temper-
expressed as mg 100 gFW−1 . ature was initially raised by 3 ◦ C min−1 to 142 ◦ C, then by 5 ◦ C min−1
116 A. Ortiz et al. / Postharvest Biology and Technology 57 (2010) 114–123
to 225 ◦ C, and then kept constant for 10 min at this final tem- Table 1
Calcium content (mg 100 gFW−1 ) in the flesh of ‘Golden Reinders’ apples after 7 d at
perature. Helium was used as the carrier gas at a flow rate of
20 ◦ C following cold storage.
0.8 mL min−1 , with a split ratio of 40:1, in the presence of air
(400 mL min−1 ) and H2 (32 mL min−1 ). The injector and detector Storage atmosphere Treatment Storage period
were held at 220 and 240 ◦ C, respectively. Volatile compounds were 19 weeks 31 weeks
identified by comparing retention indexes with those of standards
Air Untreated 2.8 Ba 2.9 Ba
and by enriching apple extract with authentic samples. The quan- CaCl2 3.4 Ab 5.2 Aa
tification was made using butylbenzene (assay > 99.5%, Fluka) as
SCA Untreated 4.2 Ba 3.9 Ba
the internal standard, run with each added standard aside from
CaCl2 4.8 Aa 4.9 Aa
the matrix to develop standard curves for each volatile analysed.
ULO Untreated 3.6 Ba 4.1 Ba
A GC–MS system (Agilent Technologies 6890N-5973N) was used
CaCl2 5.8 Aa 5.3 Aa
for compound confirmation, in which the same capillary column
was used as in the GC analyses. Mass spectra were obtained by Data represent means of three replicates. Means within the same column for a given
storage atmosphere followed by different capital letters are significantly different at
electron impact ionisation at 70 eV. Helium was used as the carrier
P ≤ 0.05 (LSD test). Means in the same row followed by different lower case letters
gas (42 cm s−1 ), according to the same temperature gradient pro- are significantly different at P ≤ 0.05 (LSD test).
gram as described above. Spectrometric data were recorded (MSD
Chemstation D.03.00.611) and compared with those from the NIST
NBS75A original library mass spectra. The concentration of each al., 2009). Previous work on ‘Golden Delicious’ apples, from which
volatile compound is expressed as mg kg−1 . ‘Golden Reinders’ originated, identified ethyl 2-methylbutanoate,
butyl acetate, 2-methylbutyl acetate, pentyl acetate, ethyl hex-
2.7. Extraction and assay of aroma volatile-related enzyme anoate and 2-methylbutyl butanoate as the main volatile esters
activities contributing to flavour at commercial harvest (López et al., 2000;
Mehinagic et al., 2006). These observations partially disagree with
Samples of both skin and flesh (outer cortex) tissue were taken the present data, and highlight flavour as a highly cultivar-specific
separately from six apples (2 apples/replicate × 3 replicates), frozen attribute which should be considered when evaluating overall qual-
in liquid nitrogen, freeze-dried, powdered and kept at −80 ◦ C until ity of fruit in response to postharvest handling. We were thus
processing. One hundred milligrams of lyophilised powdered tissue interested in assessing effects on this fruit quality attribute related
was used for each determination. Extraction and assay of pyruvate to treatment and storage conditions.
decarboxylase (PDC; EC 4.1.1.1), alcohol dehydrogenase (ADH; EC
1.1.1.1) and alcohol o-acyltransferase (AAT; EC 2.3.1.84) activities 3.1. Biosynthesis of flavour-related volatile compounds in
on crude enzyme extracts were performed as described elsewhere calcium-treated fruit after storage
(Lara et al., 2006). Total protein content in the enzyme extract was
determined with the Bradford method (1976), using BSA as a stan- Some other straight- and branched-chain esters, including ethyl
dard. In all cases, one activity unit (U) was defined as the variation butanoate, butyl acetate, butyl propanoate, pentyl acetate, butyl
in one unit of absorbance per minute. Results were expressed as butanoate, ethyl hexanoate, hexyl butanoate and 2-methylpropyl
specific activity (U mg protein−1 ). acetate, turned out to contribute actively to overall flavour of
‘Golden Reinders’ fruit after cold storage (Table 3 ). Important
2.8. Statistical analysis modifications in the emission of individual esters were observed
according to calcium treatment, although treatment effects were
A multifactorial design with storage period, storage atmosphere dependent upon storage length and atmosphere. After mid-term
and calcium treatment as factors was used to statistically analyse (19 weeks) storage, the emission of most straight-chain esters was
the results. All data were tested by analysis of variance (GLM- significantly enhanced by calcium treatment in air-stored samples
ANOVA procedure) with the SAS System 9.0 program package (SAS (Table 3A). Production of methyl butanoate and butyl hexanoate
Institute, Cary, NC, 2002), and means were separated by the Fisher’s arose above their OTH in these samples, thus increasing the num-
LSD test at P ≤ 0.05. ber of aroma-contributing compounds. Higher emission of most
detected branched-chain esters was also observed in calcium-
3. Results and discussion treated apples (Table 3B). These results suggest that postharvest
calcium dips have the potential to improve aroma quality of ‘Golden
Dipping of ‘Golden Reinders’ apples in 2% (w/v) CaCl2 resulted Reinders’ fruit stored in air for mid-term periods, and confirm simi-
in significantly higher concentrations of calcium in the flesh of lar previous findings for ‘Fuji Kiku-8’ apples (Ortiz et al., 2009). Since
fruit (Table 1), thus indicating that CaCl2 treatment was efficient ‘Fuji’ and ‘Golden’ fruit display considerably different postharvest
in incorporating calcium into fruit tissues. GC–MS analysis of the behaviour, these data suggest that treatment-related benefits on
volatile fraction isolated from intact fruit at harvest identified aroma quality might be a general, genotype-independent feature.
33 esters (8 acetates, 7 propanoates, 9 butanoates, 6 hexanoates, In contrast, when extending the storage period to 7 months (31
and 3 octanoates), 8 alcohols and 1 terpene (Table 2). Hexyl 2- weeks), total emission of straight-chain esters, including those con-
methylbutanoate, ethyl 2-methylbutanoate, hexyl butanoate, butyl tributing actively to overall flavour, was decreased significantly in
hexanoate and hexyl acetate showed productions higher than calcium-treated fruit after storage in air (Table 3A). Although the
50 mg kg−1 , and were quantitatively prominent, accounting for 55% same trend was observed for some branched-chain esters, no signif-
of total volatiles emitted (Table 2). On the basis of OAV, three of icant effects of treatment were observed for total emission of these
these esters (hexyl 2-methylbutanoate, ethyl 2-methylbutanoate compounds (Table 3B). However, it should be pointed out that,
and hexyl acetate) were considered to contribute actively to the although lower than in untreated fruit, ester emission in calcium-
overall flavour of ‘Golden Reinders’ apples, together with hexyl treated samples was significantly higher than in those stored under
propanoate, butyl 2-methylbutanoate and 2-methylbutyl acetate. CA.
Hexyl acetate and 2-methylbutyl acetate are deemed major con- Ester production was generally inhibited in CA- as compared
tributors to the characteristic apple flavour in a wide range of with air-stored samples, in agreement with previous reports on
cultivars (Fellman et al., 2000; Echeverría et al., 2008; Raffo et different apple cultivars (Streif and Bangerth, 1988; Mattheis et
Table 2
Emission of aroma volatile compounds (mg kg−1 ) by ‘Golden Reinders’ apples at harvest.
Volatile compound RIa OTHb Amountc log OAVd
Methyl acetate 854 8300(1) 15.2

Ethyl acetate 882 5000(2) 30.4
Ethanol 912 100000(2) 34.0
Tert-Butyl propanoate 928 19(1) 14.1
Propyl acetate 945 2000(3) <0.5
Methyl butanoate 955 5(4) 4.9
2-Methylpropyl acetate 976 4 (1) 0.6
1-Propanol 992 9000(2) 9.0
Ethyl butanoate 1002 1(2) 0.7
Propyl propanoate 1008 57(2) 0.9
Ethyl 2-methylbutanoate 1015 0.006(5) 128.4 4.33
Butyl acetate 1040 10(6) 3.7
2-Methyl-1-propanol 1054 250(7) 1.7
2-Methylbutyl acetate 1096 5(2) 15.1 0.48
1-Butanol 1119 500(2) 19.4
Butyl propanoate 1123 25(2) 1.6
Butyl 2-methylpropanoate 1131 80(8) 0.8
2-Methylpropyl butanoate 1138 7.3
Pentyl acetate 1161 5(2) 3.1
2-Methylbutyl propanoate 1180 19(1) 2.5
2-Methylbutyl 2-methylpropanoate 1190 5.0
2-Methyl-1-butanol 1199 250(9) 0.8
d-Limonene 1205 10(8) 0.25
Butyl butanoate 1218 100(8) 6.6
Butyl 2-methylbutanoate 1235 17(8) 24.0 1.15
Ethyl hexanoate 1241 1(5) 0.8
1-Pentanol 1262 4000(7) 1.3
Hexyl acetate 1292 2(6) 53.0 1.42
2-Methylbutyl 2-methylbutanoate 1324 11.9
Propyl hexanoate 1360 8.0
Hexyl propanoate 1379 8(7) 36.8 0.66
1-Hexanol 1392 150(10) 4.6
2-Methylpropyl hexanoate 1399 0.5
Butyl hexanoate 1473 250(10) 53.2
Hexyl butanoate 1477 250(5) 65.6
Hexyl 2-methylbutanoate 1488 6(8) 136.0 1.36
Ethyl octanoate 1502 92(5) 5.1
2-Ethyl-1-hexanol 1565 270000(11) 34.4
Pentyl hexanoate 1590 6.6
Hexyl hexanoate 1687 64000(12) 33.2
Butyl octanoate 1690 4.2
Hexyl octanoate 1840 <0.5
Total emission 785.3

a
Kovats retention index in cross-linked FFAP column.
b
Odour threshold in water (mg L−1 ) from references: (1) Schnabel et al. (1988); (2) Flath et al. (1967); (3) Takeoka et al. (1996); (4) Schieberle and Hofmann (1997); (5)
Takeoka et al. (1992); (6) van Gemert (2003); (7) Buttery (1993); (8) Takeoka et al. (1990); (9) Rychlik et al. (1998); (10) Herrmann (1991); (11) Fazzalari (1978); and (12)
Burdock (2002).
c
Means of four samples obtained each from 2 kg of apples after 4 h of collection.
d
Log10 of odour activity value = log10 (amount/OTH).
al., 2005; Lara et al., 2006; Echeverría et al., 2008). This inhibition Generally speaking, treatment effects on alcohol production
included all flavour-contributing esters, and was more pronounced were parallel to observations for volatile esters (Table 4). After mid-
for lower O2 concentrations during storage (Table 3). No significant term (19 weeks) storage under air, the emission of all detected
differences in total ester emission were found between untreated alcohols, with the exception of 2-ethyl-1-hexanol, was enhanced
and calcium-treated fruit after CA storage (Table 3), although cal- in calcium-treated fruit. Exogenous calcium resulted in higher pro-
cium treatment induced important modifications in the emission of duction of only five alcohols in SCA-stored samples, whereas no
specific compounds, particularly of flavour-contributing ones. After treatment effects were found for ULO-stored fruit. For long-term
mid-term (19 weeks) storage, the production of some esters (butyl storage, calcium treatment caused a decrease in the emission of
acetate, pentyl acetate, ethyl hexanoate, hexyl propanoate, 2- ethanol, 1-propanol, 1-butanol, 2-methyl-1-butanol and 1-hexanol
methylpropyl acetate, ethyl 2-methylbutanoate and 2-methylbutyl by fruit stored in air; however, production was higher in calcium-
acetate) was enhanced as a result of calcium treatment in SCA- treated than in CA-stored fruit. No calcium-related effects on
stored fruit, whereas for ULO-stored apples higher concentrations alcohol production were observed in ULO-stored samples (Table 4).
in treated fruit were found only for hexyl propanoate and ethyl 2- Alcohols have high OTH, and generally do not contribute to over-
methylbutanoate. Extending storage to 31 weeks further lessened all fruit flavour. Nevertheless, alcohol production is relevant for this
the effectiveness of calcium treatment regarding flavour-related attribute, as alcohols are immediate precursors for the biosynthesis
volatile production: among aroma-contributing compounds, only of volatile esters (Fellman et al., 2000; Beekwilder et al., 2004).
hexyl propanoate and hexyl 2-methylbutanoate were observed The linkage of the acyl moiety from an acyl-CoA to the appro-
to increase in treated fruit stored in SCA or ULO. Although ethyl priate alcohol is catalysed by AAT (Souleyre et al., 2005; Li et al.,
butanoate and butyl hexanoate were also enhanced, emission 2006; Pérez and Sanz, 2008), and previous work has shown that
remained below their OTH. ethylene is involved in volatile ester biosynthesis via modulation
Table 3
Emission (mg kg−1 ) of straight- (A) and branched-chain (B) esters by ‘Golden Reinders’ apples after 7 d at 20 ◦ C following cold storage.
Volatile compound Treatment Storage period
19 weeks 31 weeks
Air SCA ULO Air SCA ULO
A
Methyl acetate Untreated 8.4 Ab 12.1 Aa 9.5 Ab 11.2 Aa 8.6 Bb 9.2 Aab
CaCl2 7.7 Ab 11.5 Aa 7.5 Ab 12.0 Aa 11.3 Aa 10.5 Aa
Ethyl acetate Untreated 16.6 Bab 19.7 Aa 14.9 Ab 48.9 Aa 27.7 Ac 35.2 Ab
CaCl2 23.8 Aa 15.7 Ab 11.9 Ab 49.9 Aa 30.9 Ac 35.8 Ab
Propyl acetate Untreated 10.3 Ba 4.1 Bb 4.3 Bb 31.1 Aa 3.0 Ac 4.7 Ab

CaCl2 18.6 Aa 6.5 Ab 6.9 Ab 17.4 Ba 3.7 Ab 1.3 Bc
Methyl butanoate Untreated 4.3 Ba 3.3 Bb 3.1 Bb n.d. <0.5 n.d.

CaCl2 *7.1 Aa 4.2 Ac 5.5 Ab n.d. 0.6 0.8
Ethyl butanoate Untreated *1.1 Ba 0.6 Bb 0.5 Bb *3.1 Aa 0.5 Bb 0.5 Bb

CaCl2 *1.7 Aa 0.9 Ab 0.8 Ab *3.0 Aa 0.8 Ab 0.8 Ab
Propyl propanoate Untreated 1.6 Ba 0.5 Ab <0.5 4.4 Aa 0.6 Ab n.d.

CaCl2 2.9 Aa 0.5 Ac 1.5 b 2.0 Ba 0.7 Ab n.d.
Butyl acetate Untreated *425.8 Ba *84.7 Bb *25.5 Ac *1473.1 Aa *75.9 Ab *25.4 Ac

CaCl2 *821.3 Aa *121.3 Ab *24.8 Ac *854.7 Ba *81.7 Ab *19.6 Ac
Butyl propanoate Untreated *38.7 Ba 10.1 Ab 3.1 Ac *87.9 Aa 8.7 Ab 1.8 Ac

CaCl2 *63.8 Aa 11.9 Ab 3.0 Ac *44.9 Ba 10.7 Ab 2.8 Ac
Pentyl acetate Untreated *16.0 Ba *12.1 Bb 4.8 Ac *76.0 Aa *12.1 Ab 3.8 Ac

CaCl2 *60.1 Aa *16.2 Ab 3.4 Ac *53.3 Ba *14.8 Ab 3.2 Ac
Butyl butanoate Untreated *198.6 Ba 26.9 Ab 10.4 Ab *417.1 Aa 32.1 Ab 11.5 Ab

Ethyl hexanoate Untreated *1.5 Ba <0.5 n.d. *2.5 Aa 0.8 Ab n.d.

CaCl2 *2.6 Aa *1.4 b n.d. *2.3 Aa 0.9 Ab n.d.
Hexyl acetate Untreated *499.8 Ba *179.9 Ab *52.2 Ac *1255.3 Aa *155.8 Ab *80.8 Ac

Propyl hexanoate Untreated 11.1 Ba 4.5 Ab 0.9 Ac 17.3 Aa 4.7 Ab 3.3 Ac

CaCl2 17.7 Aa 5.1 Ab 1.4 Ac 8.7 Ba 5.6 Ab 1.1 Bc
Hexyl propanoate Untreated *80.7 Ba *28.9 Bb 0.9 Bc *166.4 Aa *44.4 Bb *12.7 Ac

Butyl hexanoate Untreated 226.4 Ba 58.4 Bb 39.4 Ab *395.4 Aa 80.0 Bb 49.3 Ac

Hexyl butanoate Untreated *411.9 Ba 101.6 Bb 58.2 Ab *838.1 Ba 170.2 Ab 61.8 Ac

CaCl2 *593.8 Aa 150.4 Ab 46.4 Ac *897.5 Aa 239.8 Ab 46.2 Ac
Ethyl octanoate Untreated 1.8 B n.d. n.d. 10.4 Aa <0.5 1.0 Ab

CaCl2 2.0 A <0.5 <0.5 2.1 Ba 2.0 a 1.0 Ab
Pentyl hexanoate Untreated 17.9 Ba 8.8 Bb 4.0 Ac 23.5 Ba 10.1 Bb 4.7 Ac

CaCl2 25.0 Aa 10.5 Ab 3.9 Ac 25.9 Aa 14.1 Ab 4.5 Ac
Hexyl hexanoate Untreated 171.2 Ba 87.0 Bb 39.8 Ac 217.5 Ba 109.6 Bb 14.1 Bc

Butyl octanoate Untreated 12.1 Ba 9.6 Bb 3.7 Ac 22.4 Aa 12.3 Ab 3.7 Ac

CaCl2 21.2 Aa 12.0 Ab 3.4 Ac 16.4 Ba 11.9 Ab 4.0 Ac
Hexyl octanoate Untreated 2.1 Ba 1.6 Bb n.d. 3.2 Aa 2.1 Bb n.d.

CaCl2 3.1 Aa 2.3 Ab n.d. 2.6 Ba 2.8 Aa n.d.
Total emission Untreated 2157.9 Ba 654.5 Ab 275.6 Ac 5104.8 Aa 758.9 Ab 323.5 Ac

B
Tert-butyl propanoate Untreated 7.0 Aa 5.2 Ab 0.9 Ac 8.2 Aa 6.1 Ab 5.5 Ab
CaCl2 4.1 Ba 5.3 Aa 1.5 Ab 7.7 Aa 6.0 Ab 1.3 Bc
2-Methylpropyl acetate Untreated *15.7 Ba *6.4 Bb 3.6 Ac *65.0 Aa *7.7 Ab *4.8 Ac

Ethyl 2-methylbutanoate Untreated *10.9 Aa *7.7 Bb *7.3 Bb *4.0 a <0.5 *2.1 b

CaCl2 *10.5 Ab *10.1 Ab *12.1 Aa n.d. n.d. n.d.
2-Methylbutyl acetate Untreated *153.5 Ba *152.9 Ba *57.5 Ab *178.4 Aa *116.2 Ab *83.1 Ac

CaCl2 *262.4 Aa *219.9 Ab *62.2 Ac *128.5 Ba *126.5 Aa *70.2 Ab
Butyl 2-methylpropanoate Untreated 2.0 Ba 0.5 Bb <0.5 4.1 Aa 1.2 Ab n.d.

Table 3 (Continued )
Volatile compound Treatment Storage period
19 weeks 31 weeks
CaCl2 3.5 Aa 1.4 Ab <0.5 3.4 Aa 0.9 Ab n.d.
2-Methylpropyl butanoate Untreated 1.9 Aa 1.9 Aa 1.9 Aa 4.1 Aa 2.9 Ab n.d.

CaCl2 1.9 Aa 1.0 Aab 0.6 Bb 3.0 Ba 2.7 Aa n.d.
2-Methylbutyl propanoate Untreated 2.9 Ba 1.5 Bb 0.9 Ac n.d. 1.2 Ba 1.1 Ba

CaCl2 3.6 Aa 2.2 Ab 1.0 Ac n.d. 1.8 Aa 1.8 Aa
2-Methylbutyl 2-methylpropanoate Untreated 0.5 Ba 0.5 a 0.9 Aa 1.0 Bb 1.9 Ba 0.9 Bb

CaCl2 2.2 Aa <0.5 0.6 Ab 2.4 Aa 2.8 Aa 1.9 Ab
Butyl 2-methylbutanoate Untreated *76.2 Ba *36.7 Ab 10.4 Ac *145.0 Aa *29.9 Ab 9.3 Ac

2-Methylbutyl 2-methylbutanoate Untreated 6.5 Ba 4.9 Bb 3.5 Ab 11.4 Aa 9.4 Ab 6.7 Ac

CaCl2 10.0 Aa 7.1 Aa 3.6 Ab 8.9 Ba 7.2 Bb 4.0 Bc
2-Methylpropyl hexanoate Untreated 0.6 B <0.5 <0.5 5.5 Aa 0.5 Ab 1.0 Ab

CaCl2 2.4 Aa 0.5 b <0.5 1.1 Ba 0.9 Aa 1.2 Aa
Hexyl 2-methylbutanoate Untreated *225.9 Ba *188.1 Ab *70.1 Ac *206.5 Aa *174.4 Bb *46.9 Ba

CaCl2 *295.4 Aa *197.4 Ab *73.2 Ac *223.5 Aa *255.9 Aa *72.7 Ab
Total emission Untreated 503.6 Ba 406.6 Ab 157.4 Ab 633.2 Aa 351.7 Ab 161.4 Ab

CaCl2 762.5 Aa 501.9 Ab 171.6 Ac 482.4 Aa 447.3 Aa 168.3 Ab
Data represent means of four replicates obtained each from 2 kg of apples after 4 h of collection. Means followed by different capital letters within a column for a given
compound are significantly different at P ≤ 0.05 (LSD test). Means in the same row for a given storage period showing different lower case letters are significantly different
at P ≤ 0.05 (LSD test). Values bearing an asterisk are associated to positive log (OAV).
of this enzyme activity (Yahyaoui et al., 2002; Defilippi et al., 2005). treatment on AAT activity were dependent on storage atmosphere:
However, important differences in the regulation of each member for air-stored fruit, activity levels were inhibited in the skin but
of the AAT gene family have been reported among apple culti- enhanced in the flesh. No calcium-related differences were found
vars (Souleyre et al., 2005; Li et al., 2006; Zhu et al., 2008). In this in the skin of fruit stored under CA, whereas activity levels were
work, AAT activity in the skin of apples stored for 19 weeks was inhibited in the flesh. These treatment-related modifications in AAT
inhibited by both calcium treatment and CA storage, while for the activity were apparently not concomitant with ethylene emission
flesh an inhibitory effect of CA storage was observed for untreated rates, which were inhibited by both calcium treatment and CA stor-
apples only (Table 5). After long-term storage, the effects of calcium age regardless of storage period (Table 6). At any rate, data show
Table 4
Emission of alcohol precursors (mg kg−1 ) for volatile ester biosynthesis by ‘Golden Reinders’ apples after 7 d at 20 ◦ C following cold storage.
Compound Treatment Storage period
19 weeks 31 weeks
Ethanol Untreated 10.9 Ba 11.3 Ba 9.2 Aa 29.0 Aa 16.2 Ab 17.3 Aab

CaCl2 19.6 Aa 19.7 Aa 10.7 Ab 21.7 Ba 16.3 Ab 18.4 Ab
1-Propanol Untreated 4.0 Ba 1.4 Bb 0.6 Ac 16.5 Aa 2.0 Ab 1.3 Ab

2-Methyl-1-propanol Untreated 6.3 Ba 2.1 Bb 1.0 Ac 16.9 Ba 3.8 Bb 0.9 Ac

1-Butanol Untreated 66.8 Ba 10.3 Bb 5.2 Ac 304.3 Aa 18.4 Bb 5.2 Ac

CaCl2 95.3 Ac 18.5 Ab 6.7 Ac 199.6 Ba 33.1 Ab 7.0 Ac
2-Methyl-1-butanol Untreated 10.3 Ba 8.1 Bb 4.2 Ab 16.0 Aa 10.2 Ab 4.7 Ac

CaCl2 20.1 Aa 12.4 Ab 4.5 Ac 12.4 Ba 10.3 Aa 3.4 Ac
1-Pentanol Untreated 2.5 Ba 0.5 Ab <0.5 5.5 Aa 0.6 Bb 0.9 Ab

CaCl2 2.6 Aa 0.5 Ab <0.5 5.6 Aa 2.4 Ab 1.2 Ac
1-Hexanol Untreated 36.1 Ba 16.4 Ab 8.9 Ab 111.5 Aa 28.7 Ab 7.8 Ac

CaCl2 53.9 Aa 18.0 Ab 9.4 Ab 86.1 Ba 30.3 Ab 7.9 Ac
2-Ethyl-1-hexanol Untreated 27.6 Aa 3.7 Ab 2.3 Ab 10.3 Aa 2.2 Bb 1.8 Ab

CaCl2 28.7 Aa 2.5 Ab 3.0 Ab 9.6 Aa 10.0 Aa 2.5 Ab
Total emission Untreated 164.5 Ba 53.8 Ab 31.8 Ab 510.0 Aa 82.1 Ab 40.0 Ab

CaCl2 235.0 Aa 77.4 Ab 36.7 Ab 366.7 Ba 109.9 Ab 43.0 Ac
Data represent means of four replicates obtained each from 2 kg of apples after 4 h of collection. Means followed by different capital letters within a column for a given
compound are significantly different at P ≤ 0.05 (LSD test). Means in the same row for a given storage period showing different lower case letters are significantly different
at P ≤ 0.05 (LSD test).
Table 5
Specific activities (U mg−1 protein) of volatile-related enzymes in the skin and flesh tissues of ‘Golden Reinders’ apples after 7 d at 20 ◦ C following cold storage.
Enzyme activity Treatment Storage period
19 weeks 31 weeks
Skin tissue
PDC Untreated 12.308 Ba 12.007 Ba 9.609 Ab 16.921 Aa 17.571 Ba 14.663 Bb
CaCl2 15.982 Ab 17.891 Aa 9.627 Ac 11.592 Bc 23.376 Aa 21.979 Ab
ADH Untreated 15.178 Ba 13.986 Bab 12.243 Ab 14.194 Ab 17.181 Ba 17.864 Aa

CaCl2 18.912 Ab 24.724 Aa 12.974 Ac 16.245 Ab 24.647 Aa 17.635 Ab
AAT Untreated 0.309 Aa 0.212 Ac 0.262 Ab 0.142 Aa 0.104 Ab 0.070 Ac

CaCl2 0.245 Ba 0.161 Bb 0.160 Bb 0.098 Ba 0.089 Aa 0.068 Aa
Flesh tissue
PDC Untreated 8.718 Ba 8.275 Ba 8.455 Aa 5.806 Aa 5.242 Aa 7.502 Aa
CaCl2 12.524 Aa 9.591 Ab 7.150 Bc 6.237 Ab 5.306 Ab 7.626 Aa
ADH Untreated 5.656 Ba 4.957 Ba 5.436 Ba 6.918 Aa 4.077 Ab 4.688 Bb

CaCl2 7.391 Aa 5.793 Ab 6.074 Ab 3.808 Bb 4.257 Ab 6.004 Aa
AAT Untreated 0.153 Aa 0.088 Ab 0.108 Ab 0.279 Bb 0.342 Aa 0.224 Ac

CaCl2 0.126 Aa 0.090 Aa 0.100 Aa 0.376 Aa 0.300 Bb 0.164 Bc
Data represent means of three replicates (2 fruit/replicate). Means followed by different capital letters within a column for a given enzyme activity are significantly different
at P ≤ 0.05 (LSD test). Means in the same row for a given storage period showing different lower case letters are significantly different at P ≤ 0.05 (LSD test).
that these changes apparently did not affect the ester-synthesising and increased skin resistance to gas diffusion (Rajapackse et al.,
capacity, as strong correlations were found between the emission 1992), and to increased internal CO2 levels (Hewett and Thompson,
of individual alcohols and that of the corresponding ester family 1992). Therefore, dipping of ‘Golden Reinders’ apples might have
(Fig. 1). These findings suggest that low AAT activity levels are diminished O2 levels and caused CO2 accumulation inside the fruit.
sufficient for the intrinsic requirements of ester production, while Indeed, increased PDC and ADH activity levels were observed in
accessibility to the necessary alcohol substrates may be a major CaCl2 -treated fruit after storage for 19 weeks under air or SCA
limiting factor (Knee and Hatfield, 1981; Berger and Drawert, 1984; (Table 5), which possibly led to improved substrate availability for
Lara et al., 2008; Zhu et al., 2008). AAT action during the shelf-life period. Accordingly, acetaldehyde
Aldehydes obtained through different metabolic pathways can content (Table 6) and the emission of most alcohols (Table 4) were
be reduced to alcohols by the action of ADH (Knee and Hatfield, enhanced in these samples. Increases in the production of these
1981). PDC uses pyruvic acid to produce acetaldehyde, which can be precursors paralleled those of most aroma-related esters, support-
subsequently processed by ADH to render ethanol, or by aldehyde ing the idea of precursor availability as a key factor determining
dehydrogenase (EC 1.2.1.5) to produce acetyl-CoA, the acylating the composition of the volatile fraction emitted by fruit. Extend-
agent for acetate ester formation. PDC and ADH are induced by ing storage to 31 weeks maintained calcium-related effects on PDC
low O2 levels in fruit tissues, and thus ethanol and acetaldehyde and ADH activities in the skin of SCA-, but not of air-stored fruit
can accumulate during fruit ripening under anaerobic conditions. (Table 5), and accordingly most volatile esters detected were emit-
Calcium dips have been demonstrated to modify the internal atmo- ted at lower concentrations in calcium-treated than in untreated
sphere of fruit due to decreased O2 diffusivity in flesh tissues fruit after storage in air (Table 3).
Table 6
Maturity and quality parameters of ‘Golden Reinders’ apples after 7 d at 20 ◦ C following cold storage.
Parameter Treatment Storage period
19 weeks 31 weeks
Ethylene production (mL kg−1 h−1 ) Untreated 297.1 Aa 118.0 Ab 112.1 Ab 334.2 Aa 123.7 Ab 115.2 Ab
CaCl2 230.7 Ba 89.3 Ab 82.4 Ab 279.4 Ba 98.9 Ab 87.5 Ab
Acetaldehyde content (mL L−1 ) Untreated 0.9 Ba 1.0 Ba 0.9 Ba 1.8 Aa 1.7 Aa 1.6 Aa
CaCl2 1.4 Aa 1.4 Aa 1.5 Aa 1.6 Aa 1.8 Aa 1.9 Aa
Firmness (N) Untreated 58.0 Ab 61.8 Ab 72.8 Aa 46.2 Bb 50.8 Bb 65.8 Aa

CaCl2 56.3 Ac 64.9 Ab 73.8 Aa 55.4 Ab 62.7 Aa 60.6 Aab
SSC (%) Untreated 15.6 14.8 15.2 15.1 15.4 15.5

CaCl2 14.9 14.5 15.3 14.4 15.5 14.7
TA (g L−1 ) Untreated 2.1 Bb 3.9 Aa 4.2 Aa 1.2 Bb 3.4 Aa 3.9 Aa

CaCl2 2.6 Ab 4.0 Aa 4.1 Aa 2.0 Ac 3.4 Ab 4.0 Aa
Hue Untreated 104.9 Bb 126.4 Aa 127.2 Aa 95.7 Bb 121.6 Aa 125.5 Aa

CaCl2 116.3 Ab 124.6 Aa 126.4 Aa 108.4 Ab 121.2 Aa 130.1 Aa
Data represent means of 3 (ethylene production) or 15 (acetaldehyde content and standard quality parameters) replicates. Means followed by different capital letters within
a column for a given parameter are significantly different at P ≤ 0.05 (LSD test). Means in the same row for a given storage period showing different lower case letters are
significantly different at P ≤ 0.05 (LSD test).
Fig. 1. Correlation between the emission (mg kg−1 ) of ethyl (A), propyl (B), butyl (C), hexyl (D), 2-methylpropyl (E) and 2-methylbutyl (F) esters, and the corresponding
alcohol precursor. Each point represents the mean of four replicates.
3.2. Standard quality parameters in calcium-treated fruit after that firmness of cold-stored ‘Golden Reinders’ apples was retained
storage at acceptable levels regardless of treatment or storage conditions
(Table 6). After storage for 19 weeks, no significant effects of cal-
Quality attributes such as fruit firmness, soluble solids content, cium application were observed on firmness of air-stored samples.
titratable acidity and skin colour are also important drivers of con- Calcium treatment was useful to improve fruit firmness after long-
sumer preference for apples (Hoehn et al., 2003; Harker et al., 2008), term storage under either air or SCA, while no treatment-related
and therefore the modifications in these quality parameters were effects were observed for ULO-stored fruit, which in turn dis-
also assessed together with flavour-related volatile emission, as the played firmness values above 60 N. TA values were also improved in
effects of any postharvest procedure on final quality of fruit should air-stored fruit in response to exogenous calcium, whereas no sig-
be evaluated as a whole. For ‘Golden Delicious’, for instance, it has nificant effects were observed on SSC (Table 6). Enhancement of TA
been estimated that minimum readings of 44 N for firmness, 12% by calcium treatment, together with improved firmness, may rep-
for SSC and 3.2 g L−1 for TA are required to attain acceptable eating resent an additional advantage for consumer acceptability (Harker
quality (Hoehn et al., 2003). For several apple cultivars, includ- et al., 2008). Therefore, eating quality of ‘Golden Reinders’ apples
ing ‘Golden Delicious’, it has also been found that acceptability stored in air is likely to have been enhanced in response to cal-
increases substantially as firmness rises up to 62 N, and that high cium. Skin colour was also retained better as indicated by higher
SSC and/or TA may result in further improvements in consumer hue values, representative of greener colour, in treated samples
acceptability of apples that are firm (Harker et al., 2008). If similar stored in air (Table 6); therefore, calcium treatment was also help-
minimum values for acceptability are considered for the ‘Golden ful to improve visual appearance, a main factor influencing apple
Reinders’ cultivar as for that from which it originated, results show purchasing patterns (Crassweller and Hollender, 1989). Satisfac-
tory levels of standard quality parameters were also found for Goldberg, I., 1984. Functional Foods, Designer Foods, Pharmafoods, Nutraceuticals.
CA-stored fruit (Table 6). Effects of CA storage on TA and SSC were Chapman & Hall, New York, USA.
Guadagni, D.R., Buttery, R.G., Harris, J., 1966. Odor intensities of hop oil constituents.
similar to those resulting from calcium treatment, although CA- J. Sci. Food Agric. 17, 142–144.
stored fruit had higher TA values. Higher TA and firmness values Harker, F.R., Kupferman, E.M., Marin, A.B., Gunson, F.A., Triggs, C.M., 2008. Eating
in CA-stored fruit suggest improved eating quality in compari- quality standards for apples based on consumer preferences. Postharvest Biol.
Technol. 50, 70–78.
son to samples stored in air. Nevertheless, detrimental effects of Herrmann, K., 1991. Die Aromastoffe des Obstes. Teil 1: Allgemeine Angaben zu den
storage under CA on the biosynthesis of aroma-related volatile Aromastoffen, ihren Schwellenwerten und ihrer Zusammensetzung. Erwerb-
compounds (Table 3) may compromise flavour quality in these sobstbau 33, 4–7.
Hewett, E.W., Thompson, C.J., 1992. Modification of internal carbon dioxide and
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to improve flavour quality of ‘Golden Reinders’ apples after mid- Hoehn, E., Gasser, F., Guggenbühl, B., Künsch, U., 2003. Efficacy of instrumental
measurements for determination of minimum requirements of firmness, sol-
term storage under air or, to a lesser extent, under SCA, through
uble solids, and acidity of several apple varieties in comparison to consumer
the enhancement of the emission of flavour-contributing volatile expectations. Postharvest Biol. Technol. 27, 27–37.
esters. Improved volatile compound production was probably the Holland, D., Larkov, O., Bar-Ya’akov, I., Bar, E., Zax, A., Brandeis, E., Ravid, U., Lewin-
consequence of increased PDC and ADH activities, with conse- sohn, E., 2005. Developmental and varietal differences in volatile ester formation
and acetyl-CoA: alcohol acetyl transferase activities in apple (Malus × domestica
quently better supply of immediate precursors for the biosynthesis Borkh.) fruit. J. Agric. Food Chem. 53, 7198–7203.
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Ke, D., Yahia, E.M., Mateos, M., Kader, A.A., 1994. Ethanolic fermentation of ‘Barlett’
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Lara, I., Graell, J., López, M.L., Echeverría, G., 2006. Multivariate analysis of modifi-
cations in biosynthesis of volatile compounds after CA storage of ‘Fuji’ apples.
Acknowledgements Postharvest Biol. Technol. 39, 19–28.
Lara, I., Ortiz, A., Echeverría, G., López, M.L., Graell, J., 2008. Development of aroma-
A. Ortiz is the recipient of a FPU grant from the Ministerio de synthesising capacity throughout fruit maturation of ‘Mondial Gala’ apples. J.
Hort. Sci. Biotechnol. 83, 253–259.
Ciencia e Innovación (MICINN) of Spain. This work was supported
López, M.L., Lavilla, M.T., Recasens, I., Graell, J., Vendrell, M., 2000. Changes in aroma
through the AGL2006-00345/ALI project, financed by the Ministe- quality of ‘Golden Delicious’ apples after storage at different oxygen and carbon
rio de Educación y Ciencia (MEC) of Spain. The authors are indebted dioxide concentrations. J. Sci. Food Agric. 80, 311–324.
to P. Sopeña and A. Latorre for technical assistance. Li, D., Xu, Y., Xu, G., Gu, L., Li, D., Shu, H., 2006. Molecular cloning and expression
of a gene encoding alcohol acyltransferase (MdAAT2) from apple (cv. Golden
Delicious). Phytochemistry 67, 658–667.
Mattheis, J.P., Fan, X., Argenta, L., 2005. Interactive responses of Gala apple fruit
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LWT - Food Science and Technology 42 (2009) 1520–1529
LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt
Overall quality of ‘Rich Lady’ peach fruit after air- or CA storage. The importance
of volatile emission
Abel Ortiz, Gemma Echeverrı́a, Jordi Graell, Isabel Lara*
Àrea de Post-Collita, XaRTA, UdL-IRTA, Alcalde Rovira Roure 191, 25198 Lleida, Spain
Article history: In this work, ‘Rich Lady’ peach fruit picked at three different dates were stored at 2 C under air or
Received 3 September 2008 controlled atmosphere (CA) conditions for 3 or 15 days with the purpose of assessing the effects of the
Received in revised form different factors considered on some variables (standard quality parameters and emission of volatile
20 March 2009
compounds) potentially having an impact on sensory acceptance after storage. Extending cold storage
Accepted 30 April 2009
under air resulted in lowered acceptance scores, which were improved by CA storage. Multivariate
analysis of results revealed that acceptance of ‘Rich Lady’ peach fruit was related closely to the perception
Keywords:
of the characteristic flavour, which in turn was related to soluble solids content and to the emission of
Aroma
Alcohol o-acyltransferase specific volatile compounds. Observed differences in alcohol o-acyltransferase (AAT) activity as affected
Air storage by factors considered in this work did not appear to be large enough to explain differences in ester
Controlled atmosphere production after storage. Data suggest that observed differences in the emission of volatile esters arose
Peach mainly from modifications in the activity of enzymes located upstream of AAT, causing changes in the
Volatile esters supply of precursors for ester biosynthesis in ‘Rich Lady’ peach fruit.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction 10–20% CO2 for 4 weeks were juicier and had better flavour after
storage than those kept in cold air (Burmeister & Harman, 1998).
Ripening-related events in climacteric fruit, including softening The understanding of the fundamental mechanisms that control
and volatile ester production, are coordinated by ethylene. changes in flavour is limited, and many biochemical pathways
Handling and commercialization of peach (Prunus persica (L.) determining this quality trait are still unknown (Song, 2007). These
Batsch.) fruit are limited by rapid softening and overall ripening, pathways are influenced by many pre- and post-harvest factors,
which results in short shelf life potential. If harvested before including harvest maturity and post-harvest handling and storage.
optimal maturity, firm enough to withstand handling and Intensive research has been conducted on flavour-related volatiles
marketing, peach fruit do not reach full flavour. Novel post-harvest emitted by peach fruit, and more than 100 compounds have been
technologies have often neglected this attribute too, as they have identified (reviewed in Aubert & Milhet, 2007). Important varia-
focused mainly on appearance and decay resistance of fruit, tions have been shown in the volatile profile of peaches as deter-
notwithstanding flavour is one of the most important characteris- mined by cultivar or maturity stage (see, for instance, Horvat et al.,
tics consumers use to judge quality of peaches (Bruhn, 1995). 1990; Lavilla, Recasens, López, & Puy, 2002; Visai & Vanoli, 1997).
Refrigerated storage of peaches and nectarines preserves fruit Similarly, the effects of storage temperature have also been the
firmness and delays the incidence of fungal infections, but this subject of a number of reports (Anderson, 1979; Robertson, Mer-
practice often leads to a range of chilling-induced disorders edith, Horvat, & Senter, 1990), and it has been shown that the
(reviewed in Lurie & Crisosto, 2005), which can be alleviated production of volatiles generally decreases during cold storage.
through storage under controlled atmospheres (CA), particularly However, to our best knowledge no previous research papers have
with high CO2 levels (Anderson, Parsons, & Smith, 1969; Roig, reported the effects of CA storage on the aroma volatile profile of
Vendrell, & Lara, 2003; Streif, Retamales, Cooper, & Kania, 1992). peaches.
Moreover, it has been reported that ‘Fantasia’ nectarines stored in Therefore, the objective of this work was to assess whether CA
storage could be a suitable means of preserving overall quality of
‘Rich Lady’ peach fruit during the post-storage period at 20 C, with
* Corresponding author. Tel.: þ34 973 702526; fax: þ34 973 238264. especial emphasis focused on the emission of volatile compounds.
E-mail address: lara@quimica.udl.cat (I. Lara). The combination of instrumental and sensory analysis should help
0023-6438/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2009.04.010
A. Ortiz et al. / LWT - Food Science and Technology 42 (2009) 1520–1529 1521
defining the role of particular volatile compounds or quality attri- six analysis sessions were conducted (three picking dates two
butes in the perception of flavour by consumers. The information storage periods). All participating judges were every-day stone fruit
thus obtained would broaden current understanding of changes in consumers from the UdL-IRTA campus, and were the same for all
this attribute during post-harvest handling and hence facilitate six sessions. Each piece was identified by a random three-digit
clues for the enhancement of the post-harvest preservation of code, and the order in which pieces were presented to each judge
peach fruit. was randomised. Mineral water was used as a palate cleanser
between samples. The judges were asked to rate overall fruit
2. Materials and methods acceptability according to a hedonic test (1: dislike very much; 9:
like very much). Sensory sourness, sensory sweetness, sensory
2.1. Plant material juiciness, and intensity of peach flavour were also evaluated with
a test in which the judges were requested to order the samples from
Peach (P. persica (L.) Batsch.) fruit of the melting flesh cultivar weaker to stronger perception of each attribute considered, and
‘Rich Lady’ were picked at a commercial orchard in Aitona (Segrià, data were parametrised as ÿ0.56 (weaker perception) or 0.56
NE Spain) at commercial maturity according to the usual standards (stronger perception) according to Anzaldúa-Morales (1994). The
in the producing area (diameter 70 mm; 100% red surface). Fruit samples could be retested as often as desired. All evaluations were
were picked at three different dates (29th June, 3rd July, 6th July conducted in individual booths under white illumination and at
2006; henceforth P1, P2 and P3, respectively) within one week in room temperature.
order to simulate the usual practice by the local growers. Standard
quality parameters of fruit at each picking date are shown (Table 1) 2.4. Analysis of volatile compounds
as a reference. After harvest, samples were stored at 2 C and 92%
relative humidity under air or CA (3 kPa O2: 10 kPa CO2) for 3 or 15 The extraction of volatile aroma compounds from a sample
days, and subsequently kept in air 1 day at 7 C to simulate (2 kg 4 replicates) of intact fruit was performed at ambient
refrigerated transport (henceforth, 3 þ 1 and 15 þ 1 fruit, respec- temperature according to the method of dynamic headspace. Each
tively). After cold storage, samples were placed at 20 C, and fruit sample was placed in a 8-l Pyrex glass container, and an air
analyses were carried out 0 and 3 days thereafter. stream (900 ml/min) was passed through for 4 h; the effluent was
then passed through an ORBO-32 adsorption tube filled with
2.2. Analysis of standard quality parameters 100 mg of activated charcoal (20/40 mesh), from which volatile
compounds were desorbed by agitation for 40 min with 0.5 ml of
Twenty fruit per each combination of factors (picking date diethyl ether. Identification and quantification of volatile
storage atmosphere storage period at 2 C shelf life period at compounds were achieved on a Hewlett Packard 5890 gas chro-
20 C) were analysed individually for flesh firmness, soluble solids matograph equipped with a flame ionisation detector and a cross-
content (SSC) and titratable acidity (TA). Flesh firmness was linked free fatty acid phase (FFAP; 50 m 0.2 mm i.d. 0.33 mm) as
measured on two opposite sides of each fruit with a penetrometer the capillary column, where a volume of 1 ml from the extract was
(Effegi, Milan, Italy) equipped with an 8-mm diameter plunger tip; injected in all the analyses. Helium was used as the carrier gas
results were expressed in N. SSC and TA were assessed in juice (42 cm/s), with a split ratio of 40:1. Both the injector and the
pressed from the whole fruit. SSC was determined using a digital detector were held at 240 C. The analysis was conducted according
hand refractometer (Atago, Tokyo, Japan), and results were to the following programme: 70 C (1 min); 70–142 C (3 C/min);
expressed as Brix. TA was measured by titration of 10 ml of juice 142–225 C (5 C/min); 225 C (15 min). Volatile compounds were
with 0.1 mol/l NaOH to pH 8.1; data are given as g malic acid/l. identified by comparing retention indices with those of standards
and by enriching peach extract with authentic samples. The
2.3. Sensory evaluation quantification was made using butylbenzene (assay > 99.5%) as the
internal standard. A GC–MS system (Hewlett Packard 5890) was
Fruit were analysed after 3 days at 20 C following cold storage. used for compound confirmation, onto the same capillary column
Twenty peaches per each combination of factors were used. Each as in the GC analyses. Mass spectra were obtained by electron
fruit was divided into four pieces, which were evaluated separately impact ionisation at 70 eV. Helium was used as the carrier gas
by four different judges included in a consumer panel comprised of (42 cm/s), according to the same temperature gradient program as
50 judges. Two pieces (one per storage atmosphere) were placed on described above. Spectrometric data were recorded (Hewlett
white plates and immediately presented to each panellist. A total of Packard 3398GC Chemstation) and compared with those from the
Table 1
Standard quality parameters of ‘Rich Lady’ peaches at harvest and 3 days after removal from storage.
At harvest P1 P2 P3
Firmness (N) 47.0 a 42.5 b 41.2 b
SSC ( Brix) 11.1 a 11.9 a 11.2 a
TA (g/l) 10.1 a 9.8 a 9.4 a

Brix/TA ratio 1.11 1.21 1.19
After storage P1 P2 P3
Perioda 3þ1 15 þ 1 3þ1 15 þ 1 3þ1 15 þ 1
Parameter Air CA Air CA Air CA Air CA Air CA Air CA

Firmness (N) 6.33 a 6.45 a <5 <5 1.08 b 6.05 a <5 <5 <5 <5 <5 <5
SSC ( Brix) 11.49 bc 11.71 b 10.63 d 11.55 bc 11.34 c 10.53 d 11.35 c 11.39 c 12.83 a 12.00 b 11.20 c 11.54 c
TA (g/l) 5.09 b 6.08 a 5.29 b 5.87 a 5.71 ab 5.92 a 4.98 cd 5.39 b 4.29 d 6.16 a 4.70 cd 5.10 b

Brix/TA ratio 2.26 1.93 2.01 1.97 1.99 1.78 2.28 2.11 2.99 1.95 2.38 2.26
Values represent means of twenty replicates. Means within the same row followed by different letters are significantly different at p 0.05 (LSD test).
a
3 þ 1: 3 days at 2 C þ 1 day at 7 C; 15 þ 1: 15 days at 2 C þ 1 day at 7 C.
1522 A. Ortiz et al. / LWT - Food Science and Technology 42 (2009) 1520–1529
NIST HP59943C original library mass spectra. Results were 3. Results and discussion
expressed as mg/kg.
Because of the large amount of information obtained, PCA was
used in order to aid its interpretation. Separate full-data PCA
2.5. Analysis of acetaldehyde concentration models were developed for each one of the three picking dates
considered, in which volatile-related enzyme activities, volatile
Juice from twenty fruit per each combination of factors was compounds emitted and standard instrumental quality parameters
frozen individually (ÿ20 C) prior to acetaldehyde analysis were used to characterise the samples. In all three cases, samples
according to Ke, Yahia, Mateos, & Kader (1994). Frozen juice was were found to group closely according to shelf life period (data not
thawed, and a 5-ml sample was incubated at 65 C for 1 h in shown), which was thus apparently the main factor accounting for
a hermetically closed tube. A 1-ml headspace gas sample was taken sample differentiation, consistent with the poor shelf life potential
with a syringe and injected into a Hewlett Packard 5890 gas of peach fruit. Since an objective of this work was to assess whether
chromatograph equipped with a flame ionisation detector, using CA storage could be a suitable means of preserving overall quality of
Carbowax (5%) on Carbopack (60/80, 2 m 2 mm i.d.) as the fruit during the post-storage period, we chose to focus on samples
stationary phase, and nitrogen (24 cm/s) as the carrier gas. Oven, held 3 days at 20 C after storage.
injector and detector temperatures were 80 C, 180 C and 220 C,
respectively. Acetaldehyde was identified and quantified by 3.1. Sensory quality of fruit after storage
comparison with an external standard; results were expressed
as ml/l. CA storage had no apparent influence on sensory acceptance of
3 þ 1 fruit in comparison with fruit stored in air (Fig. 1). Extending
cold storage under air to 15 days resulted in lowered scores, but
storage under CA significantly improved sensory acceptance of
2.6. Extraction and assay of volatile-related enzyme activities
15 þ 1 fruit as compared with storage in air. The sensory attributes
considered were then used to characterise the samples by means of
Lipoxygenase (LOX), hydroperoxide lyase (HPL), pyruvate
a PCA model (12 samples 5 variables), in order to visualise
decarboxylase (PDC), alcohol dehydrogenase (ADH) and alcohol
possible relationships to acceptability. 82% of total variability was
o-acyltransferase (AAT) activities were determined in skin and
explained by the two first principal components (PC) alone. The
flesh. Samples were taken separately from four peaches per each
corresponding loadings plot (Fig. 2) shows that acceptance was
combination of factors, frozen in liquid nitrogen, lyophilised and
apparently related mainly to perception of juiciness and of flavour
powdered. One hundred milligrams of dry tissue was used for each
by consumers. Perception of sweetness was also well correlated to
determination. Extraction and activity assay on crude enzyme
acceptance and inversely related to perception of sourness, in
extracts were performed as described by Lara et al. (2003) (LOX,
accordance with previous reports showing that increases in sucrose
PDC, ADH and AAT) and Vick (1991) (HPL). Total protein content in
concentration and in volatile components, and decreases in acidity
the enzyme extract was determined with the Bradford (1976)
were the changes mainly affecting the sensory acceptance ratings
method, using BSA as a standard. In all cases, one activity unit (U)
of ‘Harvester’ peaches (Meredith, Robertson, & Horvat, 1989).
was defined as the variation in one unit of absorbance per minute.
Therefore, we focused on a correlation analysis applied to
Each determination was done in triplicate, and results were
several variables susceptible of having some weight on perception
expressed as specific activity (U/mg protein).
of peach flavour. Partial least squares regression (PLSR) was used to
relate this attribute (Y variable) to a set of potentially explanatory
variables (X variables), including emission of volatile compounds as
2.7. Statistical and multivariate analyses
well as soluble solids content (SSC), titratable acidity (TA) and
firmness. These variables accounted together for 63% of variability
A multi-factorial design with picking date, storage atmosphere,
in flavour perception of fruit assessed by the consumers (Fig. 3).
storage period and shelf life period as factors was used for analysis
15 þ 1 fruit stored in air separated clearly from the rest of the
of variance of data. Means were separated by L.S.D. test at p 0.05.
samples along the PC1 axis (Fig. 3A), which explained alone 51% of
To provide a general visualisation of all the information contained
total variance. The loadings plot (Fig. 3B) shows that 15 þ 1 samples
in the data set obtained, principal component analysis (PCA) was
stored in CA, as well as 3 þ 1 fruit, were perceived as more flavoury
used. Partial least square regression (PLSR) was used as a predictive
by the participating assessors, possibly in relation to higher
method to relate a matrix of several dependent variables (Y) to a set
acceptance scores (Fig. 1).
of explanatory variables (X) in a single estimation procedure.
Perception of peach flavour was related to emission of some
Samples were labelled XYZ, where each digit takes values 1, 2 or 3 as
lactones, namely g-octalactone, d-decalactone and g-dodeca-
described in Table 2. Data were weighed by the inverse of the
lactone (Fig. 3B). Lactones, particularly g- and d-decalactones and
standard deviation of each variable to avoid dependence on
g- and d-dodecalactones, are character impact compounds in peach
measured units (Martens & Naes, 1989). Full cross-validation was
aroma (Aubert, Günata, Ambid, & Baumes, 2003; Lavilla et al., 2002;
run as a validation procedure.
Rizzolo, Eccher Zerbini, Grassi, Cambiagui, & Bianchi, 2006), often
in association with other volatiles, such as C6 aldehydes, aliphatic
Table 2 alcohols, and terpenes. Odour descriptors for decalactones and
Meaning of X-, Y- and Z-values for the sample generic labels. dodecalactones include ‘‘peach’’ or ‘‘peach-like’’ (Rizzolo et al.,
1 2 3
2006), and thus higher production of these compounds must have
an influence on perception of the characteristic peach flavour by
Xa P1 P2 P3
Yb Air 3:10 the consumer.
Zc 3 15 Flavour perception was also related to SSC (Fig. 3B), consistent
a
Picking date (P1, P2, P3: 29th June, 3rd July and 6th July 2006, respectively).
with the relationship found between sensory acceptance and
b
Storage atmosphere conditions (O2:CO2). perception of sweetness (Fig. 2). For some peach cultivars including
c
Storage period at 2 C (days) þ 1 day at 7 C þ 3 days at 20 C. ‘Rich Lady’, consumer preference favours high Brix/TA ratios
Fig. 1. Sensory acceptance of ‘Rich Lady’ peach fruit after 3 days at 20 C subsequent to cold storage (3 þ 1: 3 days at 2 C þ 1 day at 7 C; 15 þ 1: 15 days at 2 C þ 1 day at 7 C) in air
(-) or CA ( ). Means showing different letters for a given picking date are significantly different at P 0.05 (LSD test).
resulting from high SSC and low TA. Still, high Brix/TA ratios can 3.2. Biosynthesis of aroma volatile compounds after storage
also arise from excessively decreased acidity levels, which would
impair an adequate balance with SSC and thus be detrimental for Because perception of the characteristic aroma had been found
consumer acceptability, and indeed TA was rather low at the time in to be an important attribute when considering overall quality of
which sensory assessments were undertaken (Table 1) in compar- ‘Rich Lady’ peach fruit, special attention was focused on the emis-
ison with values at harvest. Contrarily, perception of flavour was sion of volatile compounds after storage. Up to 32 compounds (21
apparently not related to firmness. This latter result might have esters, six alcohols, four lactones and one terpene) were identified
arisen from the fact that analyses were conducted when fruit in the volatile fraction emitted by fruit (Table 3), some of which
softness was very perceptible, in some cases even not measurable were detectable only after storage (data not shown). Volatile
penetrometrically, and similar among storage conditions (Table 1). compounds and acetaldehyde were used to characterise samples 3
Results also show that a large part of the volatile compounds days after removal from cold storage by means of a PCA model (12
emitted by ‘Rich Lady’ fruit, including g-hexalactone, had no direct samples 33 variables). The two first PCs explained 66% of total
influence on flavour perception, while ethyl 2-methylbutanoate variability in emission of volatile compounds. Storage period
and butyl 2-methylbutanoate were observed to correlate inversely appeared to be the main factor for sample differentiation (Fig. 4A),
to this attribute (Fig. 3B). Interestingly, most volatile compounds illustrating the limited potential of peach fruit for post-harvest
identified were associated to 15 þ 1 fruit stored in air, which preservation. 3 þ 1 samples clustered together, with no apparent
received lower acceptance scores than those stored in CA (Fig. 1). separation among them, and clearly away from 15 þ 1 fruit. These
This observation agrees with previous reports on other fruit species results show that some time was necessary for CA conditions to
that CA-stored fruit received better scores than samples stored in induce alterations in volatile emission. In contrast to peaches stored
air, although more volatile compounds were produced by the latter for a shorter period, 15 þ 1 samples differentiated along the second
(Saftner, Abbott, & Conway, 2002), and suggests that an appropriate PC according to storage atmosphere, but the low percentage of total
balance of volatiles is more important than high production rates as variance explained by PC2 (18%) suggests a lesser weight of this
a driver of consumer acceptability. factor on sample differentiation. 15 þ 1 fruit were characterised by
Fig. 2. Loadings plot of PC1 vs. PC2 corresponding to a PCA model for sensory attributes assessed in ‘Rich Lady’ peach fruit after storage þ 3 days at 20 C (Juici, perception of
juiciness; Flv, perception of flavour; Sour, perception of sourness; Sweet, perception of sweetness).
Fig. 3. Scores (A) and loadings (B) plot of PC1 vs. PC2 corresponding to a PLSR model for flavour perception (Y variable) vs. emission of volatile compounds and standard quality
attributes (X variables) of ‘Rich Lady’ peach fruit after storage þ 3 days at 20 C (3 þ 1: 3 days at 2 C þ 1 day at 7 C; 15 þ 1: 15 days at 2 C þ 1 day at 7 C). Codes for samples are
defined in Table 2. Volatile compounds are labelled as indicated in Table 3 (Flv, perception of flavour; Firm, firmness; TA, titratable acidity; SSC, soluble solids content).
higher emission of most compounds identified in the volatile

fraction of ‘Rich Lady’ peaches, whereas 3 þ 1 samples were asso-
ciated to acetaldehyde, linalool and g-octalactone (Fig. 4B), sug- Table 3
gesting that the production of these volatile compounds declined Compounds identified in the volatile fraction emitted by ‘Rich Lady’ peach fruit.
during storage. Robertson et al. (1990) reported that linalool Compound Codea Compound Codea
concentrations in ‘Cresthaven’ peaches decreased throughout Methyl acetate ma 1-Hexanol hOH
storage at 0 C, which agrees with data reported herein. Similarly, Ethyl acetate ea 2-Methylpropyl hexanoate 2mprh
acetaldehyde has been observed to accumulate in ‘Springcrest’ Ethanol etOH Methyl octanoate mo
peach fruit treated with short-term (24 and 48 h) streams of 1 kPa Propyl acetate pra Butyl hexanoate bh
2-Methylpropyl acetate 2mpra Hexyl butanoate hb
O2 or 30 kPa CO2 (Bonghi, Ramina, Ruperti, Vidrih, & Tonutti, 1999),
Ethyl 2-methylbutanoate e2mb Hexyl 2-methylbutanoate h2mb
followed by a decline upon transfer to air. This is interesting, as Butyl acetate ba Ethyl octanoate eo
increases in both acetaldehyde and ethanol concentrations induced 3-Pentanol 3pOH 2-Ethyl-1-hexanol 2ehOH
by such treatments have been related to the appearance of off- 2-Methylbutyl acetate 2mba Pentyl hexanoate ph
Pentyl acetate pa Linalool lin
flavours (Tonutti, Bonghi, Ramina, & Vidrih, 1998). In contrast,
2-Methylbutyl 2mb2mpr Hexyl hexanoate hh
15 þ 1 peaches stored under CA were characterised by higher 2-methylpropanoate
emissions of d-decalactone and g-dodecalactone, possibly in rela- 3-Methyl-1-butanol 3mbOH Butyl octanoate bo
tion to improved sensory acceptance in comparison with air-stored Butyl 2-methylbutanoate b2mb g-Hexalactone hlac
fruit (Fig. 1). Although different ratios of esters and monoterpenes 1-Pentanol pOH g-Octalactone olac
Hexyl acetate ha d-Decalactone dlac
to lactones have been reported for a number of peach cultivars,
Hexyl propanoate hpr g-Dodecalactone ddlac
these compounds are considered to make the major contribution to
a
Codes used for multivariate analysis.
the ‘‘peachy’’ aroma (Horvat et al., 1990).
Fig. 4. Scores (A) and loadings (B) plot of PC1 vs. PC2 corresponding to a PCA model for emission of volatile compounds of ‘Rich Lady’ peach fruit after storage þ 3 days at 20 C
(3 þ 1: 3 days at 2 C þ 1 day at 7 C; 15 þ 1: 15 days at 2 C þ 1 day at 7 C). Codes for samples are defined in Table 2. Volatile compounds are labelled as indicated in Table 3 (AA,
acetaldehyde).
The metabolic pathways for volatile production in fruit are not hexanoate, butyl hexanoate, butyl octanoate, hexyl acetate, hexyl
fully understood, but both fatty acid- and branched-chain amino butanoate and 2-methylbutyl acetate. The plot of predicted vs.
acid-derived intermediates may serve as precursors for the measured AAT activity in the flesh (Fig. 5B) shows that samples
biosynthesis of aroma compounds (Sanz, Olı́as, & Pérez, 1997). AAT distributed preferentially according to picking date, P2 fruit
catalyzes the final linkage of an acyl moiety and an alcohol to form showing the highest levels of AAT activity, which agrees with the
esters and is thus directly responsible for the production of volatile observation that the emission of volatile esters after storage was
esters by fruit tissues, but other enzymes such as LOX, HPL, PDC and highest for P2 fruit (Fig. 4). The lowest values for AAT activity were
ADH are also involved in the pathways to supply the required found for P3 peaches, while the activity in the flesh tissue of P1
precursors. Specifically, LOX is assumed to contribute to the samples was dependent upon storage atmosphere. These results
breakdown of long-chain fatty acids to C6 aldehydes, subsequently suggest that a too advanced maturity stage at harvest can also
converted to alcohols by ADH. compromise the capacity of fruit for adequately regenerating
A PLSR model was developed with AAT activity in both skin and volatile compounds after storage. In all cases, AAT activity increased
flesh tissues (X variables), and the emission of non-cyclic and cyclic along the post-storage period at 20 C, indicating some regenera-
esters (Y variables). AAT activity accounted for up to 44% of total tion or enhancement of the capacity for ester biosynthesis after
variability in ester production (Fig. 5A). Interestingly, all three cold storage of fruit (Table 4). However, similar levels of AAT activity
lactones contributing to the perception of peach flavour (Fig. 3B) were observed upon removal from storage regardless of storage
were among the compounds seemingly most dependent on AAT atmosphere and period. For P3 peaches uniquely, longer storage
activity levels, particularly in the flesh tissue, together with pentyl time caused significantly lessened AAT activity in fruit flesh after 3
Fig. 5. (A) X-loading weights and Y-loadings plot of PC1 vs. PC2 corresponding to a PLSR model for emission of esters and lactones (Y variables) vs. alcohol o-acyltransferase activity
(X variable) in skin (AATs) and flesh (AATf) of ‘Rich Lady’ peach fruit after storage þ 3 days at 20 C. (B) Plot of predicted vs. measured alcohol o-acyltransferase activity in the flesh of
‘Rich Lady’ peach fruit after storage þ 3 days at 20 C. Codes for samples are defined in Table 2. Volatile compounds are labelled as indicated in Table 3.
days at 20 C, which was restored in CA- as compared with air- Beekwilder et al., 2006; Souleyre, Greenwood, Friel, Karunair-
stored peaches. etnam, & Newcomb, 2005; Yayaoui et al., 2002), suggesting that the
Broad substrate preferences have been reported for the product ultimate preference of the corresponding enzyme for alcohol
of all AAT genes isolated to date from fruit (Aharoni et al., 2000; precursors is dependent on their concentration and availability,
Table 4
Alcohol o-acyltransferase (AAT) and alcohol dehydrogenase (ADH) specific activities (U mg/protein) in the flesh tissue of ‘Rich Lady’ peach fruit after storage.
AAT activity ADH activity

Storage perioda 3þ1 15 þ 1 3þ1 15 þ 1
Shelf life periodb 0 3 0 3 0 3 0 3

P1 Air 0.312 Ba 0.452 Aa 0.310 Ba 0.424 Aa 8.361 Ba 17.723 Aa 4.274 Bb 5.849 Ba
CA 0.282 Ba 0.404 Aa 0.273 Ba 0.384 Aa 5.268 Aa 7.654 Ab 10.623 Aa 5.198 Aa
P2 Air 0.295 Ba 0.468 Aa 0.329 Ba 0.424 Aa 6.760 ABb 5.787 Ba 2.881 Bb 11.416 Aa
CA 0.324 Ba 0.412 Aa 0.329 Ba 0.440 Aa 20.740 Aa 5.515 Ba 8.064 Ba 6.637 Bb
P3 Air 0.236 Ca 0.386 Aa 0.247 Ca 0.311 Bb 22.303 Aa 7.784 Ba 16.286 ABa 6.072 Ba
CA 0.230 Ba 0.350 Aa 0.258 Ba 0.421 Aa 13.969 Aa 6.735 Aa 5.069 Aa 3.216 Aa
Values represent means of three replicates. Means within the same row for a given enzyme activity followed by different capital letters are significantly different at p 0.05
(LSD test). Means within the same column for a given picking date followed by different small letters are significantly different at p 0.05 (LSD test).
a
3 þ 1: 3 days at 2 C þ 1 day at 7 C; 15 þ 1: 15 days at 2 C þ 1 day at 7 C.
b
Days at 20 C following cold storage.
which thus seems to be a major limiting factor determining the storage period, 15 þ 1 fruit having higher levels of ethanol and thus
final aroma profile of fruit. This is consistent with data reported higher production of the corresponding esters. Although excess
herein, since the observed variations in AAT activity (Table 4) did concentrations of ethanol may be related to the appearance of off-
not appear to be large enough to explain differential ester flavours in peach (Karakurt, Huber, & Sherman, 2000; Tonutti et al.,
production after storage (Fig. 4). Hence a PLSR model was devel- 1998), it has also been observed that high ethanol levels induced by
oped to test the importance of precursors for volatile ester short-term hypoxic treatments decline rapidly upon transfer to air
production. 57% of variability in the emission of non-cyclic and (Bonghi et al., 1999). In this work, increased emission of ethyl esters
cyclic esters was dependent on supply of substrates (Fig. 6). in 15 þ 1 fruit suggest that esterification of ethanol might have
Samples separated along PC1 according to storage period (Fig. 6A), prevented the accumulation of high ethanol contents in the
15 þ 1 fruit having higher levels of precursors available and thus samples, thus avoiding the development of off-flavours and maybe
higher emission of esters. In addition, some differentiation within contributing to higher acceptance scores for fruit stored in CA
15 þ 1 samples was found among picking dates. P2 fruit were (Fig. 1). However, data also show that the effect of CA storage on
characterised by higher levels of ethanol and 1-hexanol, which ADH activity and thus on ethanol accumulation was strongly
were the variables having most weight on differentiation along PC1 dependent on maturity stage at harvest (Table 4). CA-induced
(regression coefficients ¼ 0.60 and 0.49, respectively) (Fig. 6B). As increases in ADH activity were found uniquely for P2 fruit, and only
an example, the biplot of a PLSR model developed for ethanol (X upon removal from storage regardless of period. Interestingly, P2
variable) vs. emission of ethyl esters (Y variable) is shown (Fig. 7). fruit were also those displaying higher production of non-cyclic and
88% of variation in the emission of these esters was dependent cyclic esters (Fig. 6). These observations are also symptomatic of
upon supply of ethanol. Samples separated along PC1 according to metabolic differences among fruit seemingly at very similar
Fig. 6. Scores (A) and loadings (B) plot of PC1 vs. PC2 corresponding to a PLSR model for emission of esters and lactones (Y variables) vs. availability of alcohols and acetaldehyde (X
variables) in ‘Rich Lady’ peach fruit after storage þ 3 days at 20 C. Codes for samples are defined in Table 2. Volatile compounds are labelled as indicated in Table 3 (AA,
acetaldehyde).
Fig. 7. Biplot of PC1 vs. PC2 corresponding to a PLSR model for emission of ethyl esters (Y variables) vs. availability of ethanol (X variable) in ‘Rich Lady’ peach fruit after storage þ 3
days at 20 C. Codes for samples are defined in Table 2. Volatile compounds are labelled as indicated in Table 3.
maturity stage at harvest as indicated by usual instrumental quality References

parameters (Table 1), and illustrate the difficulty of establishing
suitable maturity indices for peach. Aharoni, A., Keizer, L. C., Bouwmeester, H. J., Sun, Z. K., Álvarez-Huerta, M., Verho-
even, H. A., et al. (2000). Identification of the SAAT gene involved in strawberry
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Food Chemistry 123 (2010) 698–704
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Volatile ester-synthesising capacity in ‘Tardibelle’ peach fruit in response

to controlled atmosphere and 1-MCP treatment
Abel Ortiz a, Jordi Graell b, M. Luisa López b, Gemma Echeverría c, Isabel Lara a,*
a
b
c
IRTA Lleida, Unitat de Postcollita-XaRTA, Rovira Roure 191, 25198 Lleida, Spain
Article history: Peach fruit is highly perishable, which drastically restricts storage potential and marketing possibilities.
Received 7 October 2009 Although aroma is a very important attribute for sensory quality of peach, post-harvest procedures,
Received in revised form 24 March 2010 aimed at extending commercial availability of fruit, have focused preferentially on other quality aspects.
Accepted 5 May 2010
In this work, we were interested in assessing the effect of 1-methylcyclopropene (1-MCP) treatment and
controlled atmosphere storage on the post-storage production of volatile esters, important aroma-con-
tributing compounds, by fruit of the late season cultivar ‘Tardibelle’. Results indicate that the supply of
Keywords:
alcohol and acyl-CoA precursors was altered as a consequence of treatments considered, leading to sig-
Aroma
Controlled atmosphere
nificant changes in the emission of some volatile esters, particularly of the straight-chain type. Some
1-Methylcyclopropene enzyme activities involved in the production of volatile esters from fatty acids were partially inhibited
Peach in 1-MCP-treated fruit, suggesting that they are under ethylene regulation, although tissue-specific dif-
Volatile esters ferences were also observed. Lipoxygenase and hydroperoxide lyase activities were particularly relevant
for these modifications.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction peach fruit is particularly prone (Lurie & Crisosto, 2005). Different
post-harvest treatments have been studied with the purpose of
Peach (Prunus persica L. Batsch) is a climacteric stone fruit spe- limiting the incidence of chilling-related disorders during cold
cies displaying excellent organoleptic and nutraceutical properties storage and commercial life of peach, among which cold storage
(Tomás-Barberán et al., 2001). The area of Lleida (NE Spain) is the under controlled atmosphere (CA) has been demonstrated to be
main producer of peach in Spain, which, in turn, is the second pro- particularly effective (Murray, Lucangeli, Polenta, & Budde, 2007;
ducer of this fruit crop in the European Union. The increasing Roig, Vendrell, & Lara, 2003; Streif, Retamales, Cooper, & Kania,
peach growing in Spain means that an ever larger part of the pro- 1992), especially under high CO2 concentrations. Furthermore,
duce will have to be stored for longer periods in order to regulate storage of ‘Rich Lady’ fruit for 15 days, under 3% O2 + 10% CO2 at
commercial availability. Unfortunately, peach fruit is characterised 2 °C, improved juiciness, sweetness, perception of peach flavour,
by high perishability, owing to rapid firmness loss during ripening, emission of aroma volatile compounds and sensory acceptance in
which favours the incidence of rots and physiological disorders and comparison with fruit stored in cold air (Ortiz, Echeverría, López,
drastically restricts storage potential and marketing possibilities Graell, & Lara, 2009).
(Robertson, Meredith, Horvat, & Senter, 1990). Harvest at a slightly Another option for the extension of shelf life of fruit, which
unripe stage may allow better withstanding of post-harvest han- would avoid chilling injury-related disorders, is to antagonise eth-
dling, but these fruit generally do not adequately develop their ylene action, responsible for the coordination of ripening-related
organoleptic attributes, and consumer acceptance is significantly events in climacteric fruit. 1-Methylcyclopropene (1-MCP), which
decreased. binds to ethylene receptors with 10 times more affinity than
Refrigerated storage, one of the main tools used to decrease res- ethylene itself (Blankenship & Dole, 2003), is one such ethylene-
piration rates and to delay post-harvest spoilage of fruits, has as a antagonising compound, and a useful tool for both commercial
major associated drawback the appearance of a number of physio- and research purposes. 1-MCP treatment of peach and nectarine
logical disorders, generically known as ‘‘chilling injury”, to which (P. persica L. Batsch var. nectarina) fruit has been shown to
maintain flesh firmness and acidity, but it has been also reported
* Corresponding author. Tel.: +34 973 702526; fax: +34 973 238264. to decrease juiciness and to increase the incidence of some physi-
E-mail address: lara@quimica.udl.cat (I. Lara). ological disorders (Dong, Zhou, Sonego, Lers, & Lurie, 2001; Fan,
0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.05.037
A. Ortiz et al. / Food Chemistry 123 (2010) 698–704 699
Argenta, & Mattheis, 2002; Mathooko, Tsunashima, Owino, Kubo, & malic acid lÿ1. For determination of acetaldehyde content, a sample
Inaba, 2001). Other undesirable side effects have been found as of juice (5 ml) from each fruit was introduced in a 10-ml test tube,
well: 1-MCP-treated ’Big Top’ nectarines had altered fruit odour which was closed with a rubber cap and incubated at 65 °C for 1 h
patterns (Rizzolo, Eccher Zerbini, Grassi, Cambiaghi, & Bianchi, according to previous work (Ke, Yahia, Mateos, & Kader, 1994). A
2006), in accordance with reports for other fruit species, such as headspace gas sample (1 ml) was taken with a syringe and injected
apricots (Prunus armeniaca L.) (Fan, Argenta, & Mattheis, 2000), into a Hewlett Packard 5890 series II gas chromatograph, equipped
melon (Cucumis melo L.) (Flores et al., 2002), banana (Musa sp. with a column containing Carbowax (5%) on Carbopack (60/80,
[AAA group, Cavendish subgroup]) (Golding, Shearer, McGlasson, 2 m 2 mm i.d.) as the stationary phase, and a flame ionisation
& Wyllie, 1999), or apple (Malus domestica Borkh.) (Li et al., detector. Nitrogen was used as the carrier gas (24 cm sÿ1), and
2006). operating conditions were as follows: oven temperature 80 °C,
Thus, any positive effects on storage potential or quality attri- injector temperature 180 °C, detector temperature 220 °C. Acetal-
butes derived from a given post-harvest procedure need to be eval- dehyde was identified and quantified by comparison with an exter-
uated as a whole. Flavour is particularly relevant in this regard, as nal standard, and results were expressed as ll lÿ1.
it has been reported that sensory acceptance of peach fruit is
strongly associated with perception of the characteristic flavour 2.3. Analysis of volatile compounds
(Ortiz et al., 2009), in turn related to the emission of particular vol-
atiles. The effects of post-harvest handling on the biosynthesis of The extraction of volatile aroma compounds from a sample
flavour-related volatile compounds are therefore of particular (2 kg 4 replicates) of intact fruit was performed by the method
interest for the final quality of peach fruit. Hence, the purpose of of dynamic headspace. Each fruit sample was placed in a 8-l Pyrex
this work was to assess the influence of CA storage and 1-MCP glass container, and an air stream (900 ml minÿ1) was passed
treatment on volatile production by ‘Tardibelle’ peach fruit. Late through for 4 h; the effluent was then passed through an adsorp-
harvest date, when commercial availability of peach fruit is consid- tion tube (ORBO-32™; SUPELCO, Bellefonte, PA) filled with
erably lower than in previous months, makes this cultivar a good 100 mg of activated charcoal (20/40 mesh), from which volatile
subject for the implementation of post-harvest technologies, compounds were desorbed by agitation for 40 min with 0.5 ml of
allowing for an extension of storage potential. diethyl ether. Identification and quantification of volatile com-
pounds were achieved on a Hewlett Packard 5890 series II gas
2. Materials and methods chromatograph equipped with a flame ionisation detector and a
cross-linked free fatty acid phase (FFAP; 50 m 0.2 mm i.d.
2.1. Plant material and post-harvest handling 0.33 lm) as the capillary column, where a volume of 1 ll from
the extract was injected in all the analyses. Helium was used as
Late season peach (P. persica L. Batsch cv. Tardibelle) fruit were the carrier gas (42 cm sÿ1), with a split ratio of 40:1. Both the
picked (18th September, 2006) at a commercial orchard in Torre- injector and the detector were held at 240 °C. The analysis was
lameu (Segrià, NE Spain), according to the usual maturity stan- conducted according to the following programme: 70 °C (1 min);
dards in the producing area (diameter P 70 mm; 100% red 70–142 °C (3 °C minÿ1); 142–225 °C (5 °C minÿ1); 225 °C (15 min).
surface). Immediately after harvest, fruit were divided into four Volatile compounds were identified by comparing retention indi-
lots, two of which were placed in a sealed plastic container and ex- ces with those of standards and by enriching peach extract with
posed to 1 ll lÿ1 of 1-MCP (SmartFresh™; Agrofresh Inc.) at 1 °C for authentic samples. The quantification was done using butylben-
24 h. The rest of the samples were meanwhile kept at 1 °C. Both zene (assay >99.5%, Fluka) as the internal standard. A GC–MS
controls and treated fruit were stored, subsequently, for 21 days system (Agilent Technologies 6890N–5973N) was used for com-
at 0 °C and 92% relative humidity under either air or CA (3 kPa pound confirmation, in which the same capillary column was used
O2:10 kPa CO2), and then placed at 20 °C to simulate commercial as in the GC analyses. Mass spectra were obtained by electron
shelf life. Analyses were carried out 0 and 7 days after removal impact ionisation at 70 eV. Helium was used as the carrier gas
from cold storage. (42 cm sÿ1), according to the same temperature gradient pro-
gramme as described above. Spectrometric data were recorded
2.2. Analysis of maturity and standard quality parameters (MSD Chemstation D.03.00.611) and compared with those from
the NIST NBS75A original library mass-spectra. Results were
Ethylene production was measured from three individual fruits expressed as lg kgÿ1.
per treatment, kept in respiration jars and aerated continuously
with humidified air at a rate of 5 l hÿ1. Gas samples (1 ml) of the 2.4. Extraction and assay of aroma volatile-related enzyme activities
effluent air were taken with a syringe and injected into a gas chro-
matograph (Agilent Technologies 6890N) equipped with a flame Lipoxygenase (LOX; EC 1.13.11.12), hydroperoxide lyase (HPL;
ionisation detector and an alumina column (1.5 m 3 mm). Anal- EC 4.2.1.-), pyruvate decarboxylase (PDC; EC 4.1.1.1), alcohol dehy-
yses were conducted isothermally at 100 °C, with N2 as the carrier drogenase (ADH; EC 1.1.1.1) and alcohol o-acyltransferase (AAT; EC
gas, in the presence of air and H2 (45, 400, and 45 ml minÿ1, 2.3.1.84) activities were determined. Samples of both skin and
respectively). The injector and detector were held at 120 and flesh tissue were taken separately from six peaches per treatment
180 °C, correspondingly, and results were expressed as ll kgÿ1 hÿ1. (2 fruit/replicate 3 replicates), frozen in liquid nitrogen, lyophi-
Standard quality parameters of fruit were measured individually at lised and powdered. One hundred milligrammes of dry tissue were
harvest, and after storage, on 15 fruit. Flesh firmness was measured used for each determination. Extraction and assay of LOX, PDC,
on two opposite sides of each fruit with a hand-held penetrometer ADH and AAT activities on crude enzyme extracts were performed
(Effegi, Milan, Italy) equipped with a 8-mm diameter plunger tip; as described elsewhere (Lara et al., 2003). HPL activity was ex-
results were expressed in N. Soluble solids content (SSC) and titrat- tracted and assayed as reported previously (Vick, 1991). Total pro-
able acidity (TA) were assessed in juice pressed from the whole tein content in the enzyme extract was determined by the Bradford
fruit. SSC was determined using a digital hand refractometer (Atag- method (Bradford, 1976), using BSA as a standard. In all cases, one
o, Tokyo, Japan), and results were expressed as °Brix. TA was mea- activity unit (U) was defined as the variation in one unit of absor-
sured by titration of 10 ml of juice with 0.1 N NaOH to pH 8.1 with bance per minute. Results were expressed as specific activity
1% (v/v) phenolphthaleine as the indicator, and data are given as g (U mg proteinÿ1).
700 A. Ortiz et al. / Food Chemistry 123 (2010) 698–704
2.5. Statistical analysis Weksler, Zutahi, Lurie, & Kosto, 2004). Ethylene treatment of
preclimacteric peach fruit failed to induce the accumulation of
A multi-factorial design, with storage atmosphere, and 1-MCP ACC synthase transcripts (Callahan, Fishel, & Dunn, 1993), thus
treatment as factors, was used to statistically analyse results. All questioning a possible ethylene modulation of Pp-ACS1 gene
data were tested by analysis of variance (GLM-ANOVA), and means expression.
were separated by LSD test at P 6 0.05. Significant changes in some standard quality parameters were
found in response to the post-harvest procedures considered here-
in (Table 2). Weight loss immediately after cold storage was signif-
3. Results and discussion icantly lower in CA-stored fruit, regardless of 1-MCP treatment
while, after one week at 20 °C, this effect was found only for un-
3.1. Ethylene production and standard quality after cold storage treated fruit. The combination of 1-MCP and CA storage did not im-
prove retention of firmness, SSC or TA in comparison to CA storage
Ethylene production one week after removal from storage was alone. The SSC/TA ratio, 7 days after removal from storage in air,
partially inhibited in CA-stored fruit while, in 1-MCP-treated sam- was lower for 1-MCP-treated samples, reflecting better preserva-
ples, it was simply delayed by a few days (Table 1). The latter tion of acidity. However, the major effect found for 1-MCP on stan-
observation is in accordance with a previous report (Mathooko dard quality was to slow down the softening process: higher
et al., 2001), in which a single dose of 1-MCP was shown to have firmness retention was observed for treated fruit, both 0 and
little effect on ethylene biosynthesis by ‘Hakuho’ peaches. Ethylene 7 days subsequent to cold storage in air. These positive effects on
production was lower in 1-MCP-treated fruit during the first days firmness retention would allow the extension of marketing possi-
at 20 °C subsequent to cold storage, which is in accordance with bilities, as rapid firmness loss is the main factor limiting the com-
this suggested requirement. It has been hypothesised (Mathooko mercial life of stone fruit (Murray et al., 2007). Yet sensory quality
et al., 2001) that new ethylene receptors could be synthesised of peach fruit, and thus consumer satisfaction, have been shown to
within a short time, so that these fruit would need continuous or be largely associated with flavour perception rather than with
intermittent exposure to 1-MCP for continuous suppression of firmness (Ortiz et al., 2009). Therefore, we chose to focus on the
the expression of these genes. Similarly, no significant differences biosynthesis of flavour-related volatile compounds in order to gain
in ethylene production or in gene expression and activity of ACC a deeper insight of the final quality of treated fruit.
synthase and ACC oxidase were found between untreated and 1-
MCP-treated peaches and nectarines (Dong et al., 2001; Liguori,
3.2. Production of aroma-related volatile compounds after cold storage
Table 1 Twenty-eight compounds were identified in the volatile frac-

Ethylene production (ll kgÿ1 hÿ1) by ‘Tardibelle’ peaches at harvest and after storage
tion emitted by ‘Tardibelle’ peach fruit at harvest, including 15
at 0 °C for 21 days.
straight-chain esters, 7 branched-chain esters, 1 lactone, 4 alco-
At harvest 2.00 hols, and 1 aldehyde (Table 3). Important differences in the emis-
After storage Air CA sions of these compounds were found after cold storage as a
Days after storage Untreated 1-MCP Untreated 1-MCP function of 1-MCP treatment and atmosphere composition. How-
ever, not all detected volatile compounds were equally affected.
0 0.83 a 1.19 a 0.49 a 0.35 a
1 15.2 a 10.1 ab 6.92 b 10.2 ab The production of straight-chain esters after one week at 20 °C
2 20.5 a 11.3 b 3.70 c 7.72 bc was partially decreased in CA-stored fruit. Production levels were
4 7.85 a 8.40 a 3.31 b 7.55 a also lower in 1-MCP-treated than in untreated samples, regardless
5 6.45 b 19.3 a 3.91 b 8.00 b of storage atmosphere (Table 4). In contrast, the emission of
7 6.12 b 13.5 a 3.40 b 7.69 b
branched-chain esters was apparently unaffected by either treat-
Values represent means of three replicates. Means showing different letters within ment, although significantly inhibited levels were found for CA-
a row are significantly different at P 6 0.05 (lsd test). stored fruit immediately after removal from storage.
Table 2
Maturity and quality parameters of ‘Tardibelle’ peaches at harvest and after storage at 0 °C for 21 days.
Parameter Ha SLb Storage atmosphere

Air CA
Untreated 1-MCP Untreated 1-MCP
Weight (g) 352.3 0 306.2 b 287.2 b 339.1 a 341.3 a
LSD = 24.6 7 320.3 ab 297.0 b 343.8 a 316.3 b
Firmness (N) 58.2 0 51.7 c 68.9 a 61.5 b 56.0 c
LSD = 5.9 7 <5 12.5 a 6.3 b <5
SSC (°Brix) 11.1 0 9.9 c 11.1 ab 11.7 a 10.5 bc
LSD = 0.9 7 10.3 a 10.5 a 10.5 a 10.8 a
TA (g lÿ1) 7.9 0 6.3 ab 6.4 ab 6.6 a 6.0 b
LSD = 0.5 7 3.8 b 4.5 a 4.4 a 4.6 a
SSC/TA ratio 1.4 0 1.6 1.7 1.8 1.8
7 2.7 2.3 2.4 2.3
Acetaldehyde (ll lÿ1) 0.56 0 1.14 b 0.71 c 1.76 a 1.07 b
LSD = 0.34 7 6.90 a 6.11 bc 5.93 c 6.31 b
Values represent means of 15 replicates. Means showing different letters within a row are significantly different at P 6 0.05 (lsd test).
a
At harvest.
b
Shelf life (days).
Table 3 Table 5
Emission of aroma volatile compounds (lg kgÿ1) by ‘Tardibelle’ peach fruit at Methyl, ethyl, hexyl and acetate esters (lg kgÿ1) emitted by untreated and 1-MCP-
commercial harvest. treated ‘Tardibelle’ peach fruit after storage at 0 °C for 21 days.
Compound KIa Amountb SLa Storage atmosphere

Methyl acetate 834 2.69 ± 1.42 Air CA
Ethyl acetate 901 13.0 ± 5.76
Ethanol 936 7.98 ± 2.51
Propyl acetate 986 7.19 ± 1.22 Methyl esters 0 3.01 a 3.42 a 1.24 a 0.86 a
2-Methylpropyl acetate 1022 <0.5 LSD = 16.53 7 79.4 a 38.9 b 54.7 b 18.0 c
Ethyl 2-methylbutanoate 1061 2.93 ± 1.06
Ethyl esters 0 15.6 c 82.0 a 46.6 b 20.6 c
Butyl acetate 1085 2.02 ± 0.64
LSD = 18.25 7 93.0 a 15.1 c 34.3 b 15.5 c
2-Methylbutyl acetate 1134 2.63 ± 1.60
Pentyl acetate 1186 2.50 ± 1.38 Hexyl esters 0 6.14 a <0.5 1.15 b 0.80 b
2-Methylbutyl 2-methylpropanoate 1205 <0.5 LSD = 2.10 7 9.83 a 4.20 b 1.50 c 3.13 bc
2-Ethylhexanal 1208 <0.5 Acetate esters 0 42.9 b 97.5 a 53.7 b 30.2 b
2-Methyl-1-butanol 1212 <0.5 LSD = 25.81 7 182 a 61.7 c 95.2 b 39.7 c
Butyl-2-methylbutanoate 1243 <0.5
Ethyl hexanoate 1246 0.71 ± 0.43 Values represent means of four replicates (2 kg of intact fruit each) after 4 h of
1-Pentanol 1256 n.d. collection. Means showing different letters within a row are significantly different
Hexyl acetate 1286 1.50 ± 0.80 at P 6 0.05 (lsd test).
a
2-Methylbutyl 2-methylbutanoate 1293 1.50 ± 0.80 Shelf life (days).
Trans-2-hexenyl acetate 1348 n.d.
Hexyl propanoate 1351 <0.5
1-Hexanol 1361 <0.5
Methyl octanoate 1405 <0.5
a direct precursor for the synthesis of ethyl esters, by the specific
Butyl hexanoate 1426 1.34 ± 0.83 action of ADH. Therefore, augmented production of ethyl esters
Hexyl butanoate 1429 2.12 ± 0.44 could have arisen from increased ADH activity. Indeed, higher lev-
Hexyl 2-methylbutanoate 1440 2.03 ± 1.63 els of ADH activity were found in the skin, but not in the flesh, of
Ethyl octanoate 1448 <0.5
these fruit (Table 7), which possibly contributed to the upsurge
2-Ethyl-1-hexanol 1497 2.22 ± 1.03
Pentyl hexanoate 1525 <0.5 in ethanol production and thus in the emission of ethyl esters.
Linalool 1557 n.d. 1-MCP treatment also clearly inhibited the production of
Hexyl hexanoate 1625 <0.5 methyl esters after one week at 20 °C and of hexyl esters in fruit
Butyl octanoate 1628 <0.5 stored in air (Table 5), but no effect was found for butyl esters (data
c-Hexalactone 1768 <0.5
d-Decalactone 2284 n.d.
not shown). CA storage was likewise detrimental for the produc-
tion of methyl and hexyl esters during the post-storage period at
a
Kovats retention index in a cross-linked FFAP column. 20 °C (Table 5). The inhibition of the emission of methyl esters
b
Values are the means of four replicates (2 kg of intact fruit each) after 4 h of
by 1-MCP treatment is interesting in the light of results showing
collection ± standard deviation (n.d.: not detected at harvest, but found after
storage). higher flesh firmness in treated fruit (Table 2). Naturally occurring
methanol in plants is produced largely during the degradation of
pectin in the cell walls (Fall & Benson, 1996), one of the biochem-
Ethyl and acetate esters were particularly affected by 1-MCP ical processes underlying fruit softening. Methyl groups esterified
treatment, although a sharp increase in the production of these es- to cell wall pectin are hydrolysed by apoplastic pectinmethylester-
ters was found for treated fruit immediately after removal from ase (PME; EC 3.1.1.11) and PME over-expression in plants has been
cold air (Table 5), concomitant with a remarkable upsurge in emit- reported to result in overproduction of methanol (Hasunuma,
ted ethanol (Table 6), which was reflected in total emission of Fukusaki, & Kobayashi, 2003). The methanol thus produced is likely
straight-chain esters (Table 4). These notable increases in the emis-
sion of acetate and ethyl esters might have arisen from higher acet-
aldehyde availability; however, acetaldehyde content was Table 6
significantly lower in these samples (Table 2). This observation Alcohol production (lg kgÿ1) by untreated and 1-MCP-treated ‘Tardibelle’ peach fruit
might indicate that acetaldehyde was being diverted to the pro- after storage at 0 °C for 21 days.
duction of acetate esters, as plant tissues can metabolise acetalde- Compound SLa Storage atmosphere
hyde to acetate and acetyl-CoA (Kreuzwieser, Scheerer, &
Air CA
Rennenberg, 1999). Acetaldehyde can also be reduced to ethanol,
Ethanol 0 4.93 bc 187 a 5.73 b 1.72 c
LSD = 3.70 7 13.9 a 7.17 b 12.1 a 7.96 b
Table 4
Straight- and branched-chain esters (lg kgÿ1) emitted by untreated and 1-MCP- 2-Methyl-1-butanol 0 n.d. <0.5 n.d. <0.5
treated ‘Tardibelle’ peach fruit after storage at 0 °C for 21 days. LSD = 4.13 7 1.44 b <0.5 6.39 a 6.15 a
1-Pentanol 0 n.d. n.d. n.d. <0.5
SLa Storage atmosphere
7 n.d. n.d. n.d. n.d.
Air CA
1-Hexanol 0 0.67 b 1.99 a <0.5 n.d.
Untreated 1-MCP Untreated 1-MCP LSD = 1.19 7 <0.5 n.d. 9.83 a 9.75 a
Straight-chain esters 0 45.4 bc 98.2 a 53.1 b 31.0 c 2-Ethyl-1-hexanol 0 2.04 a 1.51 ab 1.02 b 1.02 b
LSD = 19.08 7 189 a 60.4 c 95.3 b 39.2 d LSD = 0.61 7 2.43 a 1.44 bc 0.94 c 1.64 b
Branched-chain esters 0 51.1 a 56.3 a 8.42 b 21.0 b Linalool 0 <0.5 n.d. 0.58 <0.5
LSD = 16.21 7 17.8 a 16.0 a 14.7 a 11.9 a LSD = 1.21 7 1.64 c <0.5 4.84 a 3.50 b
Values represent means of four replicates (2 kg of intact fruit each) after 4 h of Values represent means of four replicates (2 kg of intact fruit each) after 4 h of
collection. Means showing different letters within a row are significantly different collection (n.d.: not detected). Means showing different letters within a row are
at P 6 0.05 (lsd test). significantly different at P 6 0.05 (lsd test).
a a
Shelf life (days). Shelf life (days).
Table 7 AAT catalyses the esterification reaction between an alcohol

Flavour-related enzyme activities (U mg proteinÿ1) in untreated and 1-MCP-treated and an acyl-CoA, and is thus directly responsible for the generation
‘Tardibelle’ peach fruit after storage at 0 °C for 21 days.
of volatile esters (Beekwilder et al., 2004; Fellman, Miller, Mattin-
Enzyme SLa Storage atmosphere son, & Mattheis, 2000; Yahyaoui et al., 2002). When AAT activity
Air CA levels were examined, no apparent relationship to the emissions
of volatile esters was found. Furthermore, AAT gene expression
has been reported to be ethylene-dependent in apple (Defilippi,
Skin tissue
LOX 0 3.37 a 1.48 b 2.50 ab 1.35 b
Kader, & Dandekar, 2005), while results do not support an ethylene
LSD = 1.781 7 7.81 a 5.73 b 1.64 c 3.04 c modulation of AAT activity in ‘Tardibelle’ peach fruit (Table 7). De-
HPL 0 89.2 c 56.3 d 182 a 150 b
creased AAT activity in the flesh of 1-MCP-treated fruit was ob-
LSD = 26.874 7 72.9 b 40.8 c 161 a 172 a served, uniquely, one week after removal from storage under CA.
PDC 0 9.16 c 14.4 b 26.5 a 15.2 b
1-MCP-related inhibition of activity levels for this enzyme was also
LSD = 2.210 7 14.0 c 12.4 c 16.4 b 31.0 a found in the skin of fruit stored in air upon removal from storage,
ADH 0 10.8 c 17.3 ab 19.9 a 15.9 b
but activity recovered rapidly during the subsequent period at
LSD = 3.562 7 18.0 b 14.9 b 7.92 c 25.9 a 20 °C. For the rest of the samples, 1-MCP treatment either had no
AAT 0 0.189 a 0.141 b 0.161 ab 0.136 b
significant effect on enzyme activity, or actually increased it. These
LSD = 0.039 7 0.267 c 0.340 b 0.256 c 0.572 a results strongly suggested the relevance of substrate supply for the
biosynthesis of volatile esters, and thus of other control points in
Flesh tissue the metabolic pathway. This is in accordance with previous work
LOX 0 7.48 b 3.22 c 16.9 a 4.15 bc
on pear (Pyrus comunis L.) (Lara et al., 2003) and apple (Lara, Ech-
LSD = 3.563 7 9.61 a 1.26 b 9.10 a 8.46 a
everría, Graell, & López, 2007), for which we have consistently ob-
HPL 0 80.8 a 75.8 a 54.9 b 46.6 b
served that an adequate supply of precursors is a key factor for
LSD = 19.115 7 25.1 c 82.3 a 50.6 b 33.9 bc
volatile ester production, provided sufficient AAT activity is pres-
PDC 0 12.6 a 10.5 b 10.1 b 9.60 b
ent. Moreover, biochemical characterisation of MpAAT1 has shown
LSD = 1.610 7 16.9 a 13.1 b 9.32 c 7.89 c
that binding of the alcohol substrate is rate-limiting for the AAT-
ADH 0 11.1 a 5.77 c 11.1 a 9.49 b
catalysed reaction (Souleyre, Greenwood, Friel, Karunairetnam, &
LSD = 0.833 7 5.24 b 5.97 b 12.2 a 11.5 a
Newcomb, 2005). So, although being statistically significant, differ-
AAT 0 0.323 ab 0.289 b 0.184 c 0.346 a
ences found in AAT activity might be not really of consequence for
LSD = 0.048 7 0.206 c 0.320 b 0.438 a 0.138 d
ester production by fruit.
Values represent means of four replicates (2 kg of intact fruit each) after 4 h of Therefore, the observed changes in the production of straight-
collection. Means showing different letters within a row are significantly different
chain esters might have arisen from modifications in the regulation
at P 6 0.05 (lsd test).
a
Shelf life (days). and functional properties of precursor-providing enzyme activities.
Specifically, the availability of alcohols is believed to be a bottle-
neck for ester production (Beekwilder et al., 2004), as substrate
to be emitted mainly to the atmosphere (Nemecek-Marshall, Mac- specificity of AATs isolated from fruit tissues to date is reportedly
Donald, Franzen, Wojciechowski, & Fall, 1995). Therefore, the ben- wide (Olías, Pérez, & Sanz, 2002; Souleyre et al., 2005; Wyllie &
eficial effects of post-harvest procedures on firmness preservation Fellman, 2000). In this work, 1-MCP treatment and CA storage
might have associated detrimental consequences for the produc- led to significant changes in the emission of alcohols by ‘Tardibelle’
tion of methyl esters. peach fruit, particularly after 7 days at 20 °C. No common trend
Currently, 1-MCP is not authorised by the Spanish legislation for was found for any of the detected alcohols, reflecting the different
commercial use in stone fruit. Therefore, no sensory analyses could metabolic origins of these compounds. For some alcohols (2-
be undertaken in this work. However, the observed changes in the methyl-1-butanol, 1-hexanol and linalool), higher production was
emission of specific volatile compounds must have had conse- found one week after storage for CA-stored samples than for those
quences for fruit flavour. 1-MCP has indeed been reported to stored in air (Table 6), concomitantly with lower or similar ester
change the odour pattern in nectarines by enhancing the ‘‘fresh” production (Tables 4 and 5). In the case of ethanol, no storage
note and reducing the ‘‘overmature” note (Rizzolo et al., 2006). atmosphere-related differences were found after shelf life, while
For instance, the odour threshold for hexyl acetate, a major fla- the emission of ethyl esters was significantly reduced (Table 5).
vour-contributing volatile ester conferring ‘‘fruity, sweet” odour Both observations suggest that ester-forming capacity was par-
notes (Herrmann, 1991) is reportedly 2 lg kgÿ1. CA storage tially blocked after CA storage. However, in untreated fruit, no sig-
strongly suppressed the emission of this compound to values well nificant differences in AAT activity in the skin tissue were found
below its detection limit, whereas 1-MCP treatment decreased it to between air- and CA-stored samples, whereas activity levels in
half the level found in untreated fruit (data not shown). Similarly, the flesh one week after removal from storage were significantly
methyl esters are known to contribute sweet and fruity notes to higher in CA- than in air-stored fruit (Table 7). This observation
the aroma of different fruits (Supriyadi et al., 2003). might be indicating that other factors, in addition to activity levels
and alcohol availability, also contribute to the control of volatile
3.3. Aroma volatile-related enzyme activities after cold storage ester production. For example, depletion of respiration rates during
CA storage, arising from lowered O2 concentrations, is related to
The alterations found in the emission of specific volatile fami- diminished production of volatile compounds (Bangerth & Streif,
lies in response to the post-harvest procedures applied must have 1987; Streif & Bangerth, 1988), as lower respiration rates are asso-
resulted from readjustments of the metabolic pathways involved ciated with an insufficient supply of energy-carrying molecules,
in response to the factors considered. On account of the quantita- such as adenine and/or pyridine nucleotides. Similarly, partial ar-
tive relevance of straight-chain esters in the volatile fraction emit- rest of oxidative reactions under hypoxic conditions might have
ted by fruit, we focused on modifications of the activities of some led to a decrease in pyruvate dehydrogenase (PDH; EC 1.2.4.1)
enzymes related to the production of volatile compounds from activity, which contributes to the oxidative decarboxylation of
fatty acids, considered to be major precursors of this type of esters pyruvate and other oxo-acids into acyl-CoAs (Arjunan et al.,
(Schwab, Davidovich-Rikanati, & Lewinsohn, 2008). 2002). The consequently reduced synthesis of acyl-CoA substrates
might also have accounted for lower volatile ester production after atile compounds, such as hexyl acetate or the aldehyde precursor
CA storage. On the other hand, PDC is an allosteric enzyme (Hüb- hexanal, were preferentially produced in the skin of apple fruit
ner, Weidhase, & Schellenberger, 1978), central to respiratory (Ferreira, Perestrelo, Caldeira, & Câmara, 2009).
metabolism. Reduced respiration rates might affect the modulation In conclusion, the supply of alcohol and acyl-CoA precursors
of the different subunits, and the overall activity of the enzyme. was altered as a consequence of the treatments considered, leading
Accordingly, PDC activity levels during the shelf life period were af- to significant changes in the emission of some volatile esters, par-
fected by CA storage: whereas activity in the skin tissue was en- ticularly of the straight-chain type. Some involved enzyme activi-
hanced after storage under hypoxia, activity levels in the flesh ties were partially inhibited in 1-MCP-treated fruit, suggesting
were partially inhibited in CA-stored samples (Table 7). Acetalde- that they are under ethylene regulation, although tissue-specific
hyde, the product of PDC action on pyruvate, accumulated in CA- differences were also observed. In addition to enzyme activity lev-
in comparison to air-stored fruit, but was more readily metabo- els and precursor availability, observations for CA-stored fruit sug-
lised upon transfer to air (Table 2), maybe in relation to higher gest that shortage of energy-carrying compounds, due to depleted
ADH activity levels (Table 7). respiration rates, or substrate preferences of the AAT isoforms
1-MCP also led to significant modifications in alcohol produc- present, might also have a role in the modulation of the biosynthe-
tion during the post-storage period at 20 °C. The most affected sis of flavour-contributing volatile esters. Therefore, in spite of po-
alcohols were ethanol, 2-methyl-1-butanol, 1-hexanol and the ter- sitive effects of both treatments considered herein on key standard
pene alcohol linalool (Table 6), even though the corresponding es- quality attributes, such as firmness or TA, flavour quality of treated
ter family was not always in accordance with alcohol availability. fruit is likely to have been compromised, since a strong association
For instance, although the production of 2-methyl-1-butanol was between sensory acceptance and flavour perception has previously
clearly decreased by 1-MCP treatment in fruit stored in air (Ta- been reported for peach fruit (Ortiz et al., 2009). However, sensory
ble 6), no effects of treatment were observed on the emission of evaluations would be needed to confirm this conclusion.
2-methylbutyl esters (data not shown). Therefore, substrate prefer-
ences of the AAT isoforms present in the tissues must also have had Acknowledgements
a role in determining the specific esters produced in each case.
The biosynthesis of straight-chain esters is largely dependent A. Ortiz is the recipient of a FPU grant from the Ministerio de
on an adequate supply of lipid-derived precursors, and therefore Ciencia e Innovación (MICINN) of Spain. This work was supported
LOX activity, which catalyses the hydroperoxidation of polyunsat- through the ISAFRUIT project, funded by the European Commission
urated fatty acids (Porta & Rocha-Sosa, 2002), is central to this pro- under the Thematic Priority 5 – Food Quality and Safety of the 6th
cess. Over-expression of LOX genes has been demonstrated to lead Framework Programme of RTD (Contract No. FP6-FOOD-CT-2006-
to enhanced delivery of straight-chain alcohols, such as 1-octanol 016279). The views and opinions expressed in this publication
(Beekwilder et al., 2004), whereas the production of terpene alco- are purely those of the writers and may not, in any circumstances,
hols, such as geraniol, required the introduction of a geraniol syn- be regarded as stating an official position of the European Commis-
thase (Iijima et al., 2004). Apple fruit, in which ethylene production sion. The authors are indebted to A. Latorre and P. Sopeña for tech-
had been depleted by means of genetic modification, showed sig- nical assistance. E. Dupille (Agrofresh Inc.) is also acknowledged for
nificant changes in the hexanal/(2E)-hexenal ratio, suggesting that the supply of 1-MCP and for technical advice.
ethylene might be involved in the regulation of either LOX or HPL
activities (Dandekar et al., 2004). Our results are in accordance
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Lurie, S., & Crisosto, C. H. (2005). Chilling injury in peach and nectarine. Postharvest bananas (Musa sapientum L.). Journal of Agricultural and Food Chemistry, 48(8),
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0

Cell Wall Modifications during On-Tree Development and Maturation
of ‘Golden Reinders’ Apples
A. Ortiz, G. Echeverría, J. Graell and I. Lara
Àrea de Post-Collita, XaRTA, UdL-IRTA, Rovira Roure 191
25198 Lleida
Spain
Keywords: Malus domestica, hemicellulose, pectin, polygalacturonase, pectate lyase,

softening
Abstract
The objective of this study was to examine possible relationships between
softening, cell wall composition, and activities of pectate lyase (PL),
polygalacturonase (PG) and endo-1,4- -glucanase (EGase) throughout on-tree
maturation of ‘Golden Reinders’ apple fruit. Fruit were picked weekly during two
months prior to and at commercial harvest. Non-covalently bound pectins, a main
constituent of the middle lamella, were found to decrease concurrently with
firmness, possibly in relation to higher PL and PG activities at the beginning of the
experimental period and at commercial harvest, respectively. In contrast, softening
was not related to the yield of the hemicellulose-containing fraction, in accordance
with the absence of significant changes in EGase activity throughout the
experimental period.
INTRODUCTION
Fruit firmness at harvest is an important maturity and quality parameter
determining not only storage potential, but also sensory quality of fruit. Textural
attributes such as crispness, hardiness and juiciness are very important traits for
consumer’s acceptability of apple fruit (Mignani and Bassi, 2005).
Softening arises largely from modifications in cell wall structure and
polysaccharide composition, as a result of the coordinated action of several related
enzyme activities. Polygalacturonase (PG), pectate lyase (PL) and endo-1,4- -glucanase
(EGase) have been proposed as principal enzymes acting on the linkages between cell
wall polymers. These modifications include not only solubilisation and depolymerisation
of the polysaccharides contained in the wall, but also rearrangements of their associations
(Redgwell et al., 1997). Although a number of studies have reported the activity of
several cell wall-modifying enzyme activities during growth and development of apples,
only few works have focused on possible relationships between modifications on cell wall
architecture and related enzyme activities during fruit maturation (Goulao et al., 2007).
Thus, this work was carried out with the aim of establishing possible relationships, if any,
between cell wall composition and the above-stated cell wall-modifying enzyme activities
in relation to firmness loss during on-tree development of ‘Golden Reinders’ apple fruit.
Plant Material
Apple (Malus domestica Borkh. ‘Golden Reinders’) fruit, selected for
uniformity of size and absence of defects, were picked weekly from 6-year-old trees at the
IRTA-Experimental Station in Mollerussa (NE Spain). The sampling period was from
10 July to 4 September 2007, corresponding to 90 and 146 days after full bloom (dafb),
respectively. Commercial harvest at the producing area took place at 139 dafb, when fruit
size averaged 86.6 mm.
Flesh Firmness Measurement
Firmness was measured on two opposite sides of 15 fruits per sampling date, with
Proc. 6th International Postharvest Symposium

Eds.: M. Erkan and U. Aksoy 1031
Acta Hort. 877, ISHS 2010
a hand-held Effegi penetrometer equipped with an 11.1-mm diameter tip. Results are
given as N.
Enzyme Extraction and Assay

For the extraction and analysis of enzyme activities, three replicates of pulp tissue
(150 g each), were frozen in liquid nitrogen, freeze-dried, powdered and kept at -80ºC until
processing. A 10% (w/v) pulp homogenate was prepared by homogenizing 100 mg of
freeze-dried pulp tissue in an extraction buffer prepared according to Lohani et al. (2004).
Total protein content in the crude extract was determined with the Bradford (1976)
method, using BSA as a standard. Results were expressed as specific activity (activity
units mg protein-1).
PG activity was determined according to Pathak and Sanwall (1998), with
galacturonic acid (GalUA) as a standard. One unit (U) of PG activity was defined as the
liberation of 1 µmol of GalUA min-1.
PL activity was assayed according to Moran et al. (1968) as modified by Lohani et
al. (2004). An increase in A235, due to PL-mediated release of GalUA, was measured over
time. One unit of PL activity was defined as the increase in one unit of A235 min-1. For
EGase activity, the assay mixture consisted of carboxymethyl cellulose (CMC), water and
enzyme extract. The amount of reducing sugar released was determined using the DNS
(3,5-dinitrosalicylic acid) method (Miller, 1959), with glucose as a standard. One unit of
EGase activity was defined as the release of 1 µmol of glucose min-1.
Extraction and Fractionation of Cell Wall Materials

Cell wall materials (CWM) were extracted in triplicate from fruit flesh according
to Redgwell et al. (1992), with some modifications (Lara et al., 2004). Lyophilised tissue
(3 g) was homogenised in phenol:acetic acid:water (2:1:1, w/v/v) (PAW). After
centrifugation of the homogenate, the pellet was washed in water. The PAW and water
wash supernatants were combined and defined as the PAW-soluble fraction (PAWsf),
dialysed, lyophilised and weighed. The pellet was washed in acetone, filtered, lyophilised
and weighed to determine yield of CWM (g 100 g FW-1).
As previously described (Selvendran and O’Neill, 1987), CWM from each sample
was fractionated sequentially with water, cyclohexane-trans-1,2-diamine tetra-acetate
(CDTA), Na2CO3, and KOH, in order to fractionate soluble, non covalently-bound pectin,
covalently-bound pectin and matrix glycans (hemicelluloses), respectively. Each fraction
was filtered, dialysed, lyophilised and weighed. Yields were expressed as g 100 g-1
CWM.
Statistical Analysis
Results were treated for multiple comparisons by analysis of variance (GLM-
ANOVA), followed by the least significant difference (LSD) test at P<0.05. ANOVA was
performed according the SAS/STAT 9.1 procedures (SAS Institute Inc., 2004).
Unscrambler vers. 6.11a software (CAMO ASA, 1997) was used for Principal
Component Analysis (PCA) of data. Full cross-validation was run as a validation
procedure. For multivariate analysis, samples were coded H1 to H9, corresponding to
fruit picked between 90 and 146 dafb, respectively.
RESULTS AND DISCUSSION

From the first to the last sampling date, fruit size increased from 70 to 89 mm
(data not shown) and flesh firmness decreased 25 N (Fig. 1). However, it should be noted
that average firmness value of fruit picked 118 dafb (three weeks prior to commercial
harvest) was not significantly different to that corresponding to apples picked around
commercial harvest date. Firmness loss was not progressive throughout the sampling
period, but rather three different firmness stages were revealed by statistical treatment of
data (H1-H2; H3-H4; H5-H9).
To examine the modifications in cell wall polymers which underlie cell wall
1032
structural changes, CWM was isolated from the samples and sequentially extracted to
produce fractions enriched in particular wall components. Taking yields of CWM, PAWsf
and different CWM fractions as variables, a PCA model was developed with the purpose
of providing a global overview of the samples. Principal components 1 (PC1) and 2 (PC2)
accounted for 50 and 19% of total variability. In this model, ‘Golden Reinders’ apples
showed a sequential distribution over PC1, leftwards from early to advanced maturity
stage of fruit (Fig. 2A), thus indicating differences in cell wall composition between
samples.
Fruit picked at early sampling dates were characterised by higher amounts of
CWM and non-covalently bound pectin (CDTA-soluble), which in turn appeared well
related to higher firmness levels (Fig. 2B). This observation suggested softening of
‘Golden Reinders’ apples during on-tree maturation as being partly dependent on
decreased CWM yields, which might be in part the consequence of the solubilisation of
non-covalently bound pectin, similarly to results of previous report (Yoshioka et al.,
1992). In contrast, yields of covalently-bound pectin (NaCO3-soluble) appeared not to
play a key role in firmness loss during on-tree maturation, although previous work has
suggested this fraction to play a central role for the preservation of fruit firmness after
cold-storage of apples (Ortiz et al., 2010). Also, decreases in the amount of CWM during
maturation of apples were paralleled by PAWsf yields, indicative of polymers solubilised
in vivo. Higher yields of PAWsf, as well as of the water-soluble CWM, were associated to
samples corresponding to commercial harvest (Fig. 2B). Increases in yields corresponding
to these fractions have been reportedly associated to softening in apple (Siddiqui et al.,
1996).
Fruit cell walls contain many enzymes capable of modifying pectin and matrix
glycans, including both pectolytic and non-pectolytic (hemicellulose-degrading) enzymes,
such as PG and PL, and EGase, respectively. However, some species including apple
have been reported to lack detectable endo-PG activity or to exhibit very low expression
of PG (Siddiqui et al., 1996; Wakasa et al., 2006). Thus, PG activity in apple could be
considered as being mainly of the exo-type, with less dramatic consequences on pectin
depolymerisation than that corresponding to the endo-type. Consistent with this
supposition, our results suggests that PG might not play a main role in fruit softening
during on-tree maturation of ‘Golden Reinders’ apples, since no parallel patterns were
observed for softening and this enzyme activity in this study (Fig. 1 and Table 1). In
general, PG activity during fruit maturation remained constant for most of the
experimental period, and although a significant increase in this activity was detected after
commercial harvest, no effects on fruit firmness were observed. Higher PL activities,
causing pectin depolymerisation via a -elimination reaction, were detected at early
maturity stages, preceding a major firmness loss and thus suggesting a synergistic action
with PG. Even though no significant pectin depolymerisation is thought to occur in apple
fruit (Siddiqui et al., 1996), modifications of cell walls as a result of PL activity might be
critical in firmness loss, thus being worth further research.
Apparently, modifications in the amount of hemicelluloses, as indicated by yields
of the KOHsf fraction, were not related to firmness loss during maturation of ‘Golden
Reinders’ apples (Fig. 2B), in accordance with the absence of significant changes in
EGase activity during the experimental period (Table 1). These results agree with
previous reports (Abeles and Biles, 1991), in which the importance of EGase activity was
reported as negligible during advanced maturity stages and ripening of apples.
In summary, our results suggest that firmness loss during on-tree maturation of
‘Golden Reinders’ apples arose mainly from the solubilisation of non-covalently bound
pectin, a main constituent of the middle lamella, and partially as a result of a synergistic
action between PG and PL enzyme activities. Apparently, softening of apples during
maturation was not related to the yield of the hemicellulose-containing fraction, probably
in relation with the absence of significant changes in EGase activity throughout the
experimental period.
1033
ACKNOWLEDGEMENTS
A. Ortiz is the recipient of an FPU grant from the Ministerio de Ciencia e
Innovación (MICINN) of Spain. This work was supported through the AGL2006-
00345/ALI project, financed by the Ministerio de Educación y Ciencia (MEC) of Spain.
The authors are indebted to P. Sopeña for technical assistance.
Literature Cited
Abeles, F.B. and Biles, C.L. 1991. Cellulase activity in developing apple fruit. Sci.
Hortic. 47:77-87.
Bradford, M. 1976. A rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.
72:248-254.
CAMO ASA. 1997. Unscrambler Users Guide, version 6.11ª. Program package for
multivariate calibration. Trondheim, Norway: CAMO ASA.
Goulao, L.F., Santos, J., de Sousa, I. and Oliveira, C.M. 2007. Patterns of enzymatic
activity of cell wall-modifying enzymes during growth and ripening of apples.
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Lara, I., García, P. and Vendrell, M. 2004. Modifications in cell wall composition after
cold storage of calcium-treated strawberry (Fragaria ananassa Duch.) fruit.
Lohani, S., Trivedi, P.K. and Nath, P. 2004. Changes in activities of cell wall hydrolases
during ethylene-induced ripening in banana: effect of 1-MCP, ABA and IAA.
Postarvest Biol. Technol. 31:119-126.
Mignani, I. and Bassi, D. 2005. The effect of calcium treatments on aspects of cell wall
metabolism in apple cv. ‘Braeburn’. Acta Hort. 682:191-198.
Miller, G.L. 1959. Use of dinitrosalycilic acid reagent for determination of reducing
sugar. Anal. Chem. 31:426-428.
Moran, F., Nasuno, S. and Starr, M.P. 1998. Extracellular and intracellular
polygalacturonic acid trans-eliminase of Erwinia carotovora. Arch. Biochem.
Biophys. 123:298-306.
Ortiz, A., Echeverría, G., Graell, J. and Lara, I. Cell wall-modifying enzyme activities
after controlled atmosphere storage of calcium-treated ‘Fuji’ apples. Acta Hort.
858:213-216.
Pathak, N. and Sanwall, G.G. 1998. Multiple forms of polygalacturonase from banana
fruits. Phytochemistry 48:249-255.
Redgwell, R.J., Melton, L.D. and Brasch, D.J. 1992. Cell wall dissolution in ripening
kiwifruit (Actinidia deliciosa). Plant Physiol. 98:71-81.
Redgwell, R.J., Fischer, M., Kendal, E. and MacRae, E.A. 1997. Galactose loss and fruit
ripening: high-molecular-weight arabinogalactans in the pectic polysaccharides of
fruit cell walls. Planta 203:174-181.
SAS Institute, Inc. 2004. SAS/STAT© 9.1 User’s Guide. Cary, NC: SAS Institute Inc.
Selvendran, R.R. and O’Neill, M.A. 1987. Isolation and analysis of cell walls from plant
material. p.25-153. In: D. Glick (ed.), Methods of Biochemical Analysis, vol. 32. John
Wiley Interscience, New York.
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storage of apples: cell wall composition and fruit softening. J. Hortic. Sci. 71:613-620.
Wakasa, Y., Kudo, H., Ishikawa, R., Akada, S., Senda, M., Niizeki, M. and Harada, T.
2006. Low expression of an endopolygalacturonase gene in apple fruit with long-term
storage potential. Postharvest Biol. Technol. 39:193-198.
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1034
Tables
Table 1. Cell wall-modifying enzyme activities in pulp tissue of ‘Golden Reinders’ apples
during on-tree maturationa.
Enzyme activity (U·mg protein-1)

Dafbb
Polygalacturonase Pectate lyase Endo-1,4- -glucanase
90 17.84 d 1.59 b 7.70
97 21.33 bc 1.96 a 8.56
104 21.96 b 1.52 b 9.11
111 21.70 b 1.40 bc 11.03
118 22.50 b 1.08 d 9.82
125 22.21 b 1.16 d 9.25
132 21.07 bc 0.98 d 9.87
139 18.70 cd 0.71 e 10.12
146 26.10 a 1.17 cd 8.10
a
Values represents means of three repetitions. Means in the same column showing different letters are
significantly different at p!0.05 (LSD test).
b
Days after full bloom.
Figures
110
a
100 a
90
b b
80 bc
Firmness (N)
d d cd
d
70
60
50
40
85 95 105 115 125 135 145
dafb (days)
Fig.1. Firmness evolution during on-tree maturation of ‘Golden Reinders’ apples. Values
represent means of 15 apples. Different letters denote significant differences
between sampling dates at p!0.05 (LSD test) (dafb: days after full bloom).
1035
A
Fig. 2. Scores (A) and loadings (B) plot of PC1 vs. PC2 corresponding to a PCA model
for firmness and yields of cell wall fractions at different sampling dates of ‘Golden
Reinders’ apples (sf: soluble fraction).
1036

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Postharvest Biology and Technology 58 (2010) 88–92
Postharvest Biology and Technology

journal homepage: www.elsevier.com/locate/postharvbio
Cell wall disassembly during the melting phase of softening in

‘Snow Queen’ nectarines
Abel Ortiz a , Graham B. Seymour b , Gregory A. Tucker b , Isabel Lara a,∗
a
b
School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK
Article history: Nectarine samples of the melting flesh ‘Snow Queen’ cultivar were harvested periodically around the
Received 6 April 2010 commercial harvest date. A sharp decline in the ratio of insoluble to soluble cell wall materials preceded
Accepted 22 May 2010 the melting-like decrease in fruit firmness, apparently arising from depolymerisation of polysaccharides
bound tightly to the cell wall. Results suggest that part of the arabinose-rich side-chains removed from
Keywords: the pectic polymers remained linked transiently to the chelator-soluble fraction of the cell wall. Sugar
Cell wall
analyses also suggest that cell wall disassembly was aided by previous elimination of galactan side-chains,
Firmness
which may have facilitated pectin solubilisation. Activity patterns of the cell wall-modifying enzymes
Fruit softening
Melting flesh nectarine
considered were very similar, the highest levels being found immediately prior to commercial harvest,
Prunus persica followed by some increase again in over-ripe fruit. No apparent relationship with the melting phase of
fruit softening was observed, which suggests the presence of different isoforms contributing to the total
activity levels measured.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction depolymerisation of the polysaccharides composing the cell wall,

as well as rearrangements of their associations, result in extensive
Peaches (Prunus persica L. Batsch) and nectarines (P. persica L. disassembly (Redgwell et al., 1997). Cell wall disassembly appears
Batsch var. nectarina) show noticeable differences regarding the to be regulated by many factors, including developmentally- and
softening process among the large number of existing cultivars. environmentally-controlled expression of multiple genes encoding
These fruit can be divided into melting flesh (MF) and non-melting cell wall-modifying proteins, whose cooperative action is required
flesh (NMF) types according to softening behaviour. Softening dur- to adequately orchestrate the softening process (Bennett and
ing maturation and ripening in MF-type peaches and nectarines Labavitch, 2008).
displays two phases: early in ripening, softening rates undergo a Storage potential and postharvest handling possibilities of nec-
slow decline, followed by sharp firmness loss. In comparison, NMF tarine fruit are limited by high softening rates, and therefore
fruit do not show the extensive softening phase during the latter this ripening-related physiological event has obvious commercial
stages of ripening, and thus remain firmer when fully ripe (Pressey relevance, especially for MF-type cultivars, which are particu-
and Avants, 1978; Fishman et al., 1993). Most fresh market nec- larly prone to pests, rots and mechanical damage. Information
tarines belong to the MF-type, and have tender, juicy (melting) flesh on the biochemical mechanisms underlying the softening pro-
when ready to eat. cess may be important for the development of strategies allowing
Firmness loss of nectarine fruit begins early in ripening and the delay of postharvest deterioration of fruit. Although exten-
occurs at the same time as changes in the ultrastructure of the cell sive work on fruit ripening has been done using tomato (Solanum
wall, which are considered to play a major role in ripening-related lycopersicum L.) as a plant model, profound differences have been
fruit softening. Fruit cell walls consist of rigid, inextensible cel- reported between fruit regarding the extent and enzyme regu-
lulose microfibrils held together by interpenetrating coextensive lation of the modifications of cell wall polysaccharides during
networks of matrix glycans (hemicelluloses), pectins and struc- ripening-related softening (Gross and Sams, 1984; Goulao and
tural glycoproteins (Carpita and Gibeaut, 1993). Solubilisation and Oliveira, 2008). In this work, we examined the temporal pat-
terns of changes in cell wall composition and the activity of a
range of cell wall-associated enzymes during on-tree ripening of
∗ Corresponding author. Tel.: +34 973 702526; fax: +34 973 238264. ‘Snow Queen’ fruit, a white-fleshed, melting flesh-type nectarine
E-mail address: lara@quimica.udl.cat (I. Lara). cultivar.
0925-5214/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.postharvbio.2010.05.013
filtration through Whatman grade 4 paper filters, lyophilised and

weighed to determine yield of CWM, expressed as % (w/w) FW.
For further fractionation, CWM (100 mg) from each replicate were
extracted sequentially with water, 0.05 M cyclohexane-trans-1,2-
diamine tetra-acetate (CDTA), 0.05 M Na2 CO3 , and 4 M KOH as
described previously (Selvendran and O’Neill, 1987), in order
to fractionate water-soluble pectin, non-covalently-bound pectin,
covalently-bound pectin and matrix glycans (hemicelluloses),
respectively. Each fraction was filtered through Miracloth, exten-
sively dialysed (mol wt. cut-off 7000) for two days against Milli-Q
water at 4 ◦ C, lyophilised and weighed. Yields were expressed as %
(w/w) CWM.
2.3. Analysis of cell wall fractions
CWM, CDTA- and Na2 CO3 -soluble fractions were hydrolysed

with sulphuric acid for further analysis. For hydrolysis, 1 mL of 12 M
H2 SO4 was added to 30–35 mg of fractions to be analysed. After
pre-hydrolysis for 1 h at 37 ◦ C, the suspension was diluted with
11 mL distilled water and heated at 100 ◦ C for 2 h. Uronic acid con-
tent in the hydrolysate was measured by the m-hydroxydiphenyl
method (Blumenkrantz and Asboe-Hansen, 1973) using galactur-
onic acid as a standard. Neutral sugar composition in CWM, CDTAsf
and Na2 CO3sf was determined by gas chromatography using a
flame ionisation detector (GC-FID). Briefly, sugars from an aliquot
of the hydrolysed CWM and pectin-rich fractions were reduced
Fig. 1. Firmness (A) and yield of insoluble () and PAW-soluble () cell wall mate- with sodium borohydride and derivatised to their alditol acetates
rials (B) in flesh of ‘Snow Queen’ nectarines at each sampling date. Points represent with anhydrous acetic acid and 1-methylimidazole. To this pur-
means of 15 (firmness) or three (cell wall materials) replicates. Means bearing dif-
pose, 3 mL hydrolysed sample, 0.5 mL internal standard (1 g L−1
ferent letters for a given fraction are significantly different at P ≤ 0.05 (LSD test).
allose) and 1.2 mL freshly prepared sodium borohydride solution
(50 mg mL−1 in 3 M ammonium hydroxide) were mixed and incu-
2. Materials and methods bated for 1 h at 40 ◦ C. After incubation, 0.5 mL glacial acetic acid
was added to each tube and tempered at 20 ◦ C in order to stabilise
2.1. Plant material and firmness measurements the samples. Subsequently, 0.5 mL aliquots were transferred to a
glass tube and mixed with 0.5 mL cold 1-methylimidazole and 5 mL
Samples of white-fleshed nectarines (P. persica L. Batsch var. nec- anhydrous acetic acid. After 10 min at room temperature, 0.8 mL
tarina cv. ‘Snow Queen’), selected for uniformity of size and absence absolute ethanol was added and the tube contents were mixed prior
of defects, were harvested at a commercial orchard located in Mas- to incubation for 5 min at room temperature. Then, 5 mL distilled
salcoreig (Segrià, NE Spain) at intervals of three or four days around water were added to each tube, and after further 5 min incubation,
the commercial harvest date, which in the producing area took 0.5 mL bromophenol blue solution (0.04%, w/v) was also added.
place between 15th and 20th June. Samples were coded H1–H8, After mixing, 10 mL 7.5 M KOH were added to each tube. The upper
corresponding to successive picking dates. At each sampling date, layer was transferred to vials and allowed to evaporate overnight
firmness (N) was measured on two opposite sides of 15 fruit, using at room temperature before GC analysis. Once evaporated, 100 mL
an Effegi penetrometer equipped with an 8-mm diameter convex of ethyl acetate were added to each vial and aliquots of 1 mL were
tip. According to firmness values (Fig. 1A), the physiological stage injected in splitless mode. Helium was used as carrier gas at a flow
of samples ranged from mature-unripe (H1) to over-ripe (H8). rate of 5 mL min−1 , in the presence of air (450 mL min−1 ) and H2
(45 mL min−1 ). The GC (Perkin Elmer Autosystem, Beaconsfield, UK)
2.2. Extraction and fractionation of cell wall materials was fitted with a capillary column (SGE, Milton Keynes, UK) with
70% cyanopropyl polysilphenylene-siloxane as the stationary phase
Samples of fruit flesh (2 fruit/replicate × 3 replicates) were taken (BPX-70, 25 m × 0.32 mm i.d. × 0.25 mm). The oven program was set
at each sampling date, frozen in liquid nitrogen, freeze-dried, and at 190 ◦ C (1 min), and the temperature was raised to 220 ◦ C at a
powdered. Weight loss after lyophilisation was consistently around rate of 2.5 ◦ C min−1 . The injector and detector were held at 260 and
85%. Cell wall materials (CWM) were extracted in triplicate from 280 ◦ C, respectively. Individual sugars were identified by compari-
lyophilised tissue (3 g) according to Redgwell et al. (1992), with son of their retention times with those of authentic standards, and
some modifications as explained elsewhere (Lara et al., 2004). Sam- quantified using allose as an internal standard.
ples were homogenised in 20 mL phenol:acetic acid:water (2:1:1,
w/v/v) (PAW) for 20 min. After centrifugation of the homogenate at 2.4. Extraction and assay of cell wall-modifying enzyme activities
4000 × g and 4 ◦ C for 45 min, the pellet was resuspended in 10 mL
water and centrifuged again. The PAW and water wash super- For the extraction of polygalacturonase (exo-PG; EC 3.2.1.67 and
natants were combined and intensively dialysed (mol wt. cut-off endo-PG; EC 3.2.1.15), pectinmethylesterase (PME; EC 3.1.1.11),
7000) for two days against Milli-Q water at 4 ◦ C. The dialysate was pectate lyase (PL; EC 4.2.2.2) and endo-1,4-b-d-glucanase (EGase;
centrifuged at 4000 × g and 4 ◦ C for 45 min to sediment out the EC 3.2.1.4) activities, a 10% (w/v) pulp homogenate was prepared
precipitate formed during the dialysis. The supernatant (hence- by homogenising 100 mg of freeze-dried pulp tissue in an extrac-
forth, PAW-soluble fraction; PAWsf ) was recovered, lyophilised and tion buffer prepared according to Lohani et al. (2004). PG activity
weighed. The pellet obtained after PAW extraction and water wash was determined on apple pectin (d.e. 70–75%) as described previ-
was subsequently washed twice in acetone, recovered by vacuum- ously (Pathak and Sanwall, 1998), with galacturonic acid (GalUA) as
a standard. One unit (U) of PG activity was defined as the liberation Table 1
Yield (% CWM) of fractions isolated from cell wall materials of ‘Snow Queen’ fruit at
of 1 mmol of GalUA min−1 . PME activity was measured according
each sampling date.
to Hagerman and Austin (1986). For the assay, the reaction mixture
contained enzyme extract, apple pectin and bromothymol blue pre- Watersf CDTAsf Na2 CO3sf KOHsf
pared as described previously (Alonso et al., 1997). One unit (U) of H1 June 10 3.220 de 16.787 a 24.727 a 13.025 a
PME activity was defined as the decrease of one unit of A620 min−1 . H2 June 13 3.785 de 18.271 a 26.495 a 11.636 b
PL activity was assayed with apple pectin as the substrate accord- H3 June 16 3.174 e 21.589 a 27.585 a 11.630 b
H4 June 19 3.627 de 21.961 a 19.461 b 3.235 f
ing to Moran et al. (1968) as modified by Lohani et al. (2004). One
H5 June 23 5.591 bc 22.595 a 16.053 c 3.801 ef
unit (U) of PL activity was defined as the increase of one unit of H6 June 27 4.791 cd 21.622 a 14.042 cd 5.160 d
A235 min−1 . For the assessment of EGase activity, the DNS method H7 July 1 7.609 a 18.717 a 11.596 d 4.739 de
(Miller, 1959), with carboxymethylcellulose as the assay substrate, H8 July 4 6.726 ab 19.535 a 7.244 e 7.056 c
was used to determine the amount of reducing sugars released, Values represent means of three replicates. Means followed by different letters
with glucose as a standard. One unit (U) of EGase activity was within the same column are significantly different at P ≤ 0.05 (LSD test).
defined as the release of 1 mmol of glucose min−1 .
For the extraction of b-galactosidase (b-Gal; EC 3.2.1.23) and a portion of these polysaccharides become water-soluble (Dawson
a-l-arabinofuranosidase (AFase; EC 3.2.1.55) activities, a 10% (w/v) et al., 1992; Brummell and Harpster, 2001), suggesting that most
pulp homogenate was prepared by homogenising 100 mg of freeze- of these polymers might remain linked by ionic bonds to other
dried pulp tissue in an extraction buffer prepared according to insoluble molecules in the wall.
previous work (Vicente et al., 2005). b-Gal and AFase activity assays In this work, the content of solubilised polymers increased as
were undertaken in the crude extract as described in Vicente et shown by enhanced yields of materials soluble in PAW (Fig. 1)
al. (2005) and Wei et al. (2010), respectively. One unit (U) of b- and water (Table 1), and indicating substantial changes in cell wall
Gal was defined as the liberation of 1 mmol of p-nitrophenol min−1 polysaccharides. This was confirmed by analysis of uronic acid con-
from p-nitrophenyl-b-d-galactopyranoside. One unit (U) of AFase tent in both the CDTA- and the Na2 CO3 -soluble fractions (Table 2),
was defined as the release of 1 nmol of p-nitrophenol min−1 from showing a significant decline during the melting phase, particularly
p-nitrophenyl-a-l-arabinofuranoside. in the Na2 CO3sf . Sugar analysis also indicated significant variations
Total protein content in the crude extracts was determined with in the content of neutral sugars in CWM during softening of ‘Snow
the Bradford (1976) method, using BSA as a standard. All analy- Queen’ fruit (Fig. 2), although these variations did not show the
ses were done in triplicate, and results were expressed as specific same patterns: while galactosyl and glucosyl residues declined sig-
activity (U mg protein−1 ). nificantly throughout the experimental time, a transient increase
in arabinosyl residues was found during the melting phase. No sig-
2.5. Statistical analysis nificant changes were found for xylosyl residues, whereas no clear
trend could be observed for mannosyl residues. Modifications in
Results were treated for multiple comparisons by analysis of neutral sugar contents in both pectin-containing fractions were
variance (GLM-ANOVA), followed by the least significant difference not totally parallel to those in the total CWM. The amounts of ara-
(LSD) Fisher’s test at P ≤ 0.05 with the SAS software package (SAS binosyl, galactosyl, glucosyl and mannosyl residues decreased in
Institute, Cary, NC, USA, 1988). the Na2 CO3 -soluble fraction while increasing in the fraction solu-
ble in CDTA (Table 2), with the only exception of xylosyl residues,
for which the opposite was observed. The transient increase in the
3. Results and discussion content of arabinosyl residues in the total CWM (Fig. 2) agreed
with a similar one in the CDTAsf (Table 2). These results sug-
The softening pattern during on-tree ripening of ‘Snow Queen’
nectarines was representative of that corresponding to a melting
Table 2
flesh-type cultivar. Firmness loss was moderate until commercial
Neutral sugar composition and uronic acid content (%, w/w) of the pectin-enriched
harvest date, followed by a sharp 50-N drop in only one week fractions obtained from insoluble cell wall materials of ‘Snow Queen’ fruit at each
(Fig. 1A) to values lower than 10 N, rendering fruit unsuitable for sampling date.
commercialisation. The melting-type drop in firmness was pre-
Fraction Composition (%, w/w)
ceded by a similar decrease in the ratio between insoluble and
PAW-soluble cell wall materials (Fig. 1B), in accordance with obser- Ara Xyl Man Gal Glc Uronic acids
vations for other nectarine cultivars (Dawson et al., 1992). Since CDTAsf
the PAW-soluble fraction is assumed to be comprised of the cell H1 0.32 e 0.28 a n.d. 0.30 d 0.24 d 34.31 a
wall materials solubilised in vivo, declining ratios are indicative H2 0.39 e 0.28 a n.d. 0.29 d 0.26 cd 25.99 b
H3 0.49 e 0.23 b n.d. 0.28 d 0.36 cd 19.15 c
that increased solubilisation of cell wall polymers was an important H4 2.00 d 0.14 c 0.07 c 0.78 c 0.55 c 17.74 c
factor for tissue softening. H5 3.10 a 0.15 c 0.13 b 0.98 b 1.01 b 17.63 c
Insoluble cell wall materials were further fractionated and anal- H6 2.98 a 0.14 c 0.12 b 1.00 b 1.16 b 15.46 c
ysed in order to help dissect the possible relationships between H7 2.72 b 0.13 cd 0.27 a 1.05 ab 3.81 a 8.76 d
H8 2.42 c 0.11 d 0.28 a 1.10 a 3.56 a 7.97 d
compositional and structural changes in the cell wall and flesh
Na2 CO3sf
firmness of fruit. The decline in CWM contributing to decreased H1 26.04 a 0.51 c 0.33 a 13.26 a 5.03 a 65.05 a
CWM:PAWsf ratios arose apparently from reduced yields of the H2 25.89 a 0.51 c 0.33 a 13.19 ab 4.78 a 65.01 a
CWM fractions soluble in Na2 CO3 (Na2 CO3sf ) and KOH (KOHsf ), H3 22.37 b 0.47 c 0.32 a 12.46 b 4.63 a 67.24 a
enriched in tightly-bound pectins and in matrix glycans, respec- H4 23.05 b 0.47 c 0.25 b 9.47 c 2.57 c 25.44 b
H5 23.07 b 0.59 c 0.19 cd 8.98 cd 2.62 c 24.88 b
tively (Table 1). In contrast, no significant changes in the yields H6 21.19 bc 0.85 b 0.20 c 8.40 de 2.33 c 24.94 b
of pectins bound non-covalently to the cell wall (CDTAsf ) were H7 19.99 c 1.37 a 0.18 d 8.09 e 3.50 b 24.57 b
observed at the different harvest dates, suggesting that this cell wall H8 19.79 c 1.50 a 0.16 e 6.92 f 3.64 b 24.74 b
fraction is not a key factor determining firmness of ‘Snow Queen’ Values represent means of three replicates (n.d., non-detected). Means followed by
fruit. It has been reported that pectins become depolymerised and different letters within the same column for a given cell wall fraction are significantly
solubilised during ripening of peaches and nectarines, but that only different at P ≤ 0.05 (LSD test).
Fig. 3. PME and PG activities in the flesh of ‘Snow Queen’ nectarines at each sampling
Fig. 2. Neutral sugar composition (%, w/w) of the insoluble cell wall materials date. Points represent means of three replicates. Means bearing different letters for
obtained from ‘Snow Queen’ fruit at each sampling date. Points represent means a given enzyme activity are significantly different at P ≤ 0.05 (LSD test).
of three replicates. Means bearing different letters for a given enzyme activity are
significantly different at P ≤ 0.05 (LSD test).
to the first hints of loss of uronic acids, observed three days later
from the Na2 CO3sf (Table 2).
gest some remobilisation of insoluble cell wall materials from the For some fruit including peach, pectin solubilisation has been
Na2 CO3 - to the CDTA-soluble fraction during the melting phase of reported to precede depolymerisation, which is initiated at mid-
fruit softening, consistent with the idea that a large part of the or late-softening (Brummell et al., 2004; Brummell, 2006). For
polysaccharides solubilised during ripening of peaches and nec- instance, it has been suggested that the highly branched structure
tarines remain linked to the cell wall by ionic bonds (Dawson et of a large part of cell wall pectins may limit susceptibility to attack
al., 1992; Brummell and Harpster, 2001) and in agreement with by pectolytic enzymes (Ahmed and Labavitch, 1980). Specifically,
observations that CDTAsf yields did not show significant variations galactosyl- and arabinosyl-containing side-chains are thought to
(Table 1) in spite of a significant decrease in uronic acids (Table 2). control pore size in the cell wall, thus hindering the accessibility to
At any rate, an important increase in the Ara:Gal ratio was found in pectolytic hydrolases and therefore protecting cell wall polysaccha-
the total CWM as well as in both pectin-enriched cell wall fractions rides from extensive depolymerisation (Brummell and Harpster,
(Table 3), which corresponded with the drop in the ratio between 2001; Brummell, 2006). In this work, the highest b-Gal activity was
insoluble and PAW-soluble cell wall materials (Fig. 1B) and with measured in H1 fruit (Table 4) and a loss of galactose from CWM
the decrease in the uronic acid content in both pectin-containing was apparent immediately afterwards (Fig. 2) together with a sig-
fractions (Table 2). nificant decline in fruit firmness (Fig. 1A), which is in accordance
Depolymerisation of polyuronides from the pectin network, with the previous idea. However, the pattern of changes in b-Gal
which may help pectin solubilisation, can be driven by the coor- activity is intriguing, as activity levels increased again in over-ripe,
dinate action of a large number of enzymes able to degrade or to very soft fruit (H5–H8 stages) (Table 4), for which steady galactose
modify cross-links between cell wall polysaccharides. Yet notice- loss from both from CWM (Fig. 2) and the Na2 CO3sf (Table 2) was
able differences in the pattern of gene expression and activity of still detectable. Similar trends were observed for the rest of the
these potentially cell wall-modifying enzymes have been reported pectolytic enzyme activities considered herein (PG, PME PL and
among different fruit species and even among cultivars of a given AFase), with no apparent coincidence with the melting phase of
species (Goulao and Oliveira, 2008). Significant differences in PG fruit softening. This may be indicative of a real situation (namely,
(Fig. 3) and PL (Table 4) activities, which can mediate pectin degra- the irretrievable onset of events leading to melting-like softening),
dation, were found during ripening-related softening of ‘Snow or it may be rather reflecting the presence of different isoforms with
Queen’ nectarines. In both cases, activity levels were highest prior divergent expression patterns along fruit softening, which might
to commercial harvest and declined noticeably thereafter, although have masked and distorted the measurement of the activity cor-
some increase was observed in over-ripe fruit. PG and PL activ- responding to the specific melting-related isozyme. For example,
ity levels were parallel to those found for PME (Fig. 3), consistent previous work (Smith et al., 2002) on tomato fruit suppressed for
with the view that previous de-esterification of methyl groups in TBG4 transcription showed reduced exo-galactanase activity and
polyuronides is a requisite for PG and PL action (Pressey and Avants, some transformed lines remained firmer than untransformed fruit,
1982; Bennett and Labavitch, 2008). These peaks in PG, PL and PME suggesting a key role for this isogene in tomato fruit softening.
activities, also observed for AFase (Table 4), did not match chrono- The drop in the CWM:PAWsf ratios (Fig. 1B) which preceded the
logically the melting phase of softening, as they were found prior melting phase of firmness loss was also well correlated with the
Table 3 Table 4
Ara:Gal ratio in the insoluble cell wall materials and in pectin-enriched fractions Some cell wall-modifying enzyme activities (U mg protein−1 ) in the flesh of ‘Snow
obtained from ‘Snow Queen’ fruit at each sampling date. Queen’ fruit at each sampling date.
CWM CDTAsf Na2 CO3sf PL b-Gal AFase EGase
H1 June 10 0.58 d 1.07 f 1.96 d H1 June 10 0.66 b 0.55 a 45.20 b 1.00 b

H2 June 13 0.72 c 1.34 e 1.96 d H2 June 13 0.87 a 0.29 b 62.46 a 1.77 a
H3 June 16 0.79 c 1.75 d 1.79 d H3 June 16 0.35 d 0.14 c 52.62 b 0.57 de
H4 June 19 1.31 b 2.56 b 2.34 c H4 June 19 0.46 cd 0.07 d 29.32 d 0.50 e
H5 June 23 1.52 a 3.16 a 2.57 b H5 June 23 0.42 cd 0.14 c 30.81 d 0.42 e
H6 June 27 1.35 b 2.98 a 2.52 b H6 June 27 0.37 d 0.18 c 23.60 e 0.50 e
H7 July 1 1.42 ab 2.59 b 2.47 b H7 July 1 0.45 cd 0.19 c 35.54 cd 0.77 c
H8 July 4 1.43 ab 2.20 c 2.86 a H8 July 4 0.52 bc 0.18 c 40.25 bc 0.70 cd
Values represent means of three replicates. Means followed by different letters Values represent means of three replicates. Means followed by different letters
within the same column are significantly different at P ≤ 0.05 (LSD test). within the same column are significantly different at P ≤ 0.05 (LSD test).
yields of the KOHsf (Table 1), which is an indicative of the hemicel- Bonghi, C., Ferrarese, L., Ruperti, B., Tonutti, P., Ramina, A., 1998. Endo-b-1,4-
lulose fraction and reportedly is comprised mainly of xyloglucan glucanases are involved in peach fruit growth and ripening, and regulated by
ethylene. Physiol. Plant 102, 346–352.
polymers (Wakabayashi, 2000). EGase activity can contribute par- Bradford, M., 1976. A rapid and sensitive method for the quantification of micro-
tially to xyloglucan breakdown. Similarly to the observations for gram quantities of protein utilizing the principle of protein-dye binding. Anal.
pectolytic enzymes, the highest EGase activity levels were mea- Biochem. 72, 248–254.
Brummell, D.A., Harpster, M.H., 2001. Cell wall metabolism in fruit softening and
sured for H2 fruit, with some slight increase again in over-ripe quality and its manipulation in transgenic plants. Plant Mol. Biol. 47, 311–340.
samples (Table 4). This pattern was not totally in accordance with Brummell, D.A., Dal Cin, V., Crisosto, C.H., Labavitch, J.M., 2004. Cell wall metabolism
KOHsf yields: although a significant decline was found already at during maturation, ripening and senescence of peach fruit. J. Exp. Bot. 55,
2029–2039.
the H2 stage (Table 1), suggesting a role for this enzyme in the first Brummell, D.A., 2006. Cell wall disassembly in ripening fruit. Funct. Plant Biol. 33,
phases of softening of ‘Snow Queen’ fruit, yields increased again 103–119.
during melting and over-ripening, the significance of which event Carpita, N.C., Gibeaut, D.M., 1993. Structural models of primary cell walls in flow-
ering plants: consistency of molecular structure with the physical properties of
is unclear. EGase activity and gene expression have been reported
the walls during growth. Plant J. 3, 1–30.
to increase during ripening of peaches (Bonghi et al., 1998), but Darley, C.P., Forrester, A.M., McQueen-Mason, S.J., 2001. The molecular basis of plant
in other cases the role of this enzyme family in peach softening cell wall extension. Plant Mol. Biol. 47, 179–195.
has been de-emphasised (Brummell et al., 2004) and associated Dawson, D.M., Melton, L.D., Watkins, C.B., 1992. Cell wall changes in nectarines
(Prunus persica). Solubilization and depolimerization of pectic and neutral poly-
preferentially to cell wall extensibility (Darley et al., 2001). mers during ripening and in mealy fruit. Plant Physiol. 100, 1203–1210.
In summary, our results suggest that melting-like firmness Fishman, M.L., Levaj, B., Gillespie, D., Scorza, R., 1993. Changes in the physico-
loss during on-tree ripening of ‘Snow Queen’ nectarines was chemical properties of peach fruit pectin during on-tree ripening and storage. J.
Am. Soc. Hortic. Sci. 118, 343–349.
preceded by removal of galactan side-chains, which may have facil- Goulao, L.F., Oliveira, C.M., 2008. Cell wall modifications during fruit ripening: when
itated pectin solubilisation prior to depolymerisation. Data are also a fruit is not the fruit. Trends Food Sci. Technol. 19, 4–25.
indicative that pectin solubilisation took place preferentially from Gross, K.C., Sams, C.E., 1984. Changes in cell wall neutral sugar composition during
fruit ripening: a species survey. Phytochemistry 23, 2457–2461.
the fraction bound tightly to the cell wall, and that some of the solu- Hagerman, A.E., Austin, P.J., 1986. Continuous spectrophotometric assay for plant
bilised polysaccharides, particularly the arabinose-rich side-chains, pectin methylesterase. J. Agric. Food Chem. 34, 440–444.
were reallocated transiently in the CDTAsf , suggesting the partici- Lara, I., García, P., Vendrell, M., 2004. Modifications in cell wall composition after
cold storage of calcium-treated strawberry (Fragaria × ananassa Duch.) fruit.
pation in the process of proteins involved in the rearrangement of Postharvest Biol. Technol. 34, 331–339.
the associations between different cell wall polymers. Lohani, S., Trivedi, P.K., Nath, P., 2004. Changes in activities of cell wall hydrolases
Postharvest Biol. Technol. 31, 119–126.
Acknowledgements Miller, G.L., 1959. Use of dinitrosalycilic acid reagent for determination of reducing
sugar. Anal. Chem. 31, 426–428.
Moran, F., Nasuno, S., Starr, M.P., 1968. Extracellular and intracellular polygalactur-
A. Ortiz is the recipient of a FPU grant from the Ministerio de onic acid trans-eliminase of Erwinia carotovora. Arch. Biochem. Biophys. 123,
Ciencia e Innovación (MICINN) of Spain. This work was supported 298–306.
by research contract 2005SGR00630, funded by the Generalitat de Pathak, N., Sanwall, G.G., 1998. Multiple forms of polygalacturonase from banana
fruits. Phytochemistry 48, 249–255.
Catalunya, and by Project AGL2006-00345/ALI, funded by MICINN.
Pressey, R., Avants, J.K., 1978. Difference in polygalacturonase composition of cling-
A part of the experimental work reported herein was undertaken stone and freestone peaches. J. Food Sci. 43, 1415–1417.
while A. Ortiz was a visiting scholar in G. Seymour and G. Tucker’s Pressey, R., Avants, J.K., 1982. Solubilization of cell walls by tomato polygalactur-
laboratories at the School of Biosciences, University of Nottingham, onases: effects of pectinesterases. J. Food Biochem. 6, 57–74.
Redgwell, R.J., Melton, L.D., Brasch, D.J., 1992. Cell wall dissolution in ripening
funded by MICINN. Partial financial support through the ISAFRUIT kiwifruit (Actinidia deliciosa). Plant Physiol. 98, 71–81.
project (contract no. FP6-FOOD–CT-2006-016279) is also gratefully Redgwell, R.J., MacRae, E., Hallet, I., Fisher, M., Perry, J., Harker, R., 1997. In vivo and
acknowledged. The authors are indebted to P. Sopeña and G. West in vitro swelling of cell walls during fruit ripening. Planta 203, 162–175.
SAS Institute Inc., 1988. SAS/STAT Guide for Personal Computers, 6th ed. SAS Institute
for technical assistance. Fruit samples were kindly provided by A. Inc., Cary, NC.
Latorre. Selvendran, R.R., O’Neill, M.A., 1987. Isolation and analysis of cell walls from plant
material. In: Glick, D. (Ed.), Methods of Biochemical Analysis, vol. 32. John Wiley
Interscience, NY, USA, pp. 25–153.
References Smith, D.L., Abbott, J.A., Gross, K.C., 2002. Down-regulation of tomato b-
galactosidase 4 results in decreased fruit softening. Plant Physiol. 129,
Ahmed, A., Labavitch, J.M., 1980. Cell wall metabolism in ripening fruit. I. Cell wall 1755–1762.
changes in ripening ‘Bartlett’ pears. Plant Physiol. 65, 1009–1013. Vicente, A.R., Costa, M.L., Martínez, G.A., Chaves, A.R., Civello, P.M., 2005. Effect
Alonso, J., Howell, N., Canet, W., 1997. Purification and characterization of two of heat treatments on cell wall degradation and softening in strawberry fruit.
pectinmethylesterase from persimmon (Diospyros kaki). J. Sci. Food Agric. 75, Postharvest Biol. Technol. 38, 213–222.
352–358. Wakabayashi, K., 2000. Changes in cell wall polysaccharides during fruit ripening. J.
Bennett, A.B., Labavitch, J.M., 2008. Ethylene and ripening-regulated expression and Plant Res. 113, 231–237.
function of fruit cell wall modifying proteins. Plant Sci. 175, 130–136. Wei, J., Fengwang, M., Shi, S., Qi, X., Zhu, Z., Yuan, J., 2010. Changes and posthar-
Blumenkrantz, N., Asboe-Hansen, G., 1973. New method for quantitative determi- vest regulation of activity and gene expression of enzymes related to cell wall
nation of uronic acids. Anal. Biochem. 54, 484–489. degradation in ripening apple fruit. Postharvest Biol. Technol. 56, 147–154.

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FOCH 10943 No. of Pages 9, Model 5G
19 April 2011
Food Chemistry xxx (2011) xxx–xxx

1
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
2 Cell wall-modifying enzymes and firmness loss in ripening ‘Golden Reinders’ apples:
3 A comparison between calcium dips and ulo storage
4 Abel Ortiz a, Jordi Graell b, Isabel Lara Ayala a,⇑
5 a
6 b
7
8
1 6
2 0
11 Article history: Calcium treatment and storage under ultra-low oxygen (ULO) conditions are common post-harvest prac- 27
12 Received 3 December 2010 tices aimed at delaying ripening-related softening of apple (Malus domestica Borkh.) fruit, but the bio- 28
13 Received in revised form 18 February 2011 chemical mechanisms underlying these effects have not been determined conclusively to date. In this 29
14 Accepted 6 April 2011
study, commercially mature ‘Golden Reinders’ apples were dipped in 2% calcium chloride prior to storage 30
15 Available online xxxx
at 1 °C and 92% RH under either regular air or ultra-low oxygen (ULO; 1kPa O2:2kPa CO2) for 19 or 31
31 weeks, and kept thereafter at 20 °C for 0, 7 or 14 days in order to simulate the usual marketing time. 32
16 Keywords:
Cell wall composition and cell wall-modifying enzyme activities were determined in relation to fruit 33
17 Apple
a-L-Arabinofuranosidase firmness. ULO-storage and calcium dips were effective for firmness preservation, seemingly due to 34
19 b-Galactosidase decreased pectin solubilisation. b-Galactosidase, a-L-arabinofuranosidase and pectate lyase activities 35
20 Calcium dips were correlated positively with firmness loss of ‘Golden Reinders’ fruit after storage. 36
21 Cell wall Ó 2011 Published by Elsevier Ltd. 37
22 Firmness
23 Pectate lyase
24 ULO storage
25
38
39
40 1. Introduction controlled atmosphere (CA) storage, mainly under ultra-low oxy- 61
gen (ULO) concentrations, has been widely adopted as the technol- 62
41 Standard quality specifications for commercialisation of apple ogy of choice for apple storage. Unfortunately, CA storage often 63
42 (Malus domestica Borkh.) fruit rely essentially on visual parame- leads to partial suppression of flavour-contributing volatile com- 64
43 ters such as size and surface colour. Yet these parameters are not pounds (Lara, Echeverría, Graell, & López, 2007; Ortiz, Echeverría, 65
44 sufficient to fit consumer’s expectations, as apple preference is dri- Graell, & Lara, 2010), which is a major drawback of CA technology 66
45 ven mainly by texture and flavour (Harker, Kupferman, Marin, often causing detrimental effects on the eating quality of produce. 67
46 Gunson, & Triggs, 2008; Jaeger, Andani, Wakeling, & MacFie, Ripening-related softening of fruit is generally associated to the 68
47 1998). Since firmness is associated to juicy and crispy texture, fir- disassembly of middle lamella and primary cell walls (Brummell & 69
48 mer apples are generally more appreciated. Contrarily, soft apples Harpster, 2001; Goulao & Oliveira, 2008), which are composed of 70
49 can develop mealiness, a texture attribute causing a starch-like rigid cellulose microfibrils held in concert by networks of matrix 71
50 sensation in the mouth. However, while a substantial increase in glycans (hemicelluloses) and pectins, with varying levels of struc- 72
51 apple acceptability was reported, as firmness rose up to 62 N (Har- tural proteins and phenolics (Caffall & Mohnen, 2009). During rip- 73
52 ker et al., 2008), only small improvements in consumer acceptance ening, these polysaccharides are extensively modified, mostly by 74
53 resulted from further increases in firmness, suggesting that the the action of a large number of cell wall-localised proteins, result- 75
54 enhancement of consumer acceptance in apples that are firm ing in solubilisation, depolymerisation and rearrangements of their 76
55 may rely on high levels of other attributes such as SSC and/or TA. associations, which eventually affect cell wall strength and lead to 77
56 In addition to its role on sensory quality, firmness is also impor- fruit softening (Brummell et al., 2001; Goulao, Cosgrove, & Oliveira, 78
57 tant for storage potential. Firmer fruit are more resistant to physi- 2008). The real contribution of these modifying proteins to the 79
58 cal damage and infections during handling and storage, which is an softening process remains unclear despite numerous experiments 80
59 economically relevant issue. Consequently, most post-harvest on genetic modification of individual cell wall-related enzymes 81
60 strategies have focused on delaying extensive fruit softening, and (Vicente, Saladié, Rose, & Labavitch, 2007). The degree of methyla- 82
tion of pectin is also a major factor determining pectin properties 83
and textural attributes of fruit (Fraeye et al., 2009). During fruit 84
⇑ Corresponding author. Tel.: +34 973 702526; fax: +34 973 702924. development and ripening, pectinmethylesterase (PME, EC 85
E-mail address: lara@quimica.udl.cat (I. Lara Ayala). 3.1.1.11)-catalysed pectin demethylation may delay firmness loss, 86
0308-8146/$ - see front matter Ó 2011 Published by Elsevier Ltd.

doi:10.1016/j.foodchem.2011.04.016
Please cite this article in press as: Ortiz, A., et al. Cell wall-modifying enzymes and firmness loss in ripening ‘Golden Reinders’ apples: A comparison be-
tween calcium dips and ulo storage. Food Chemistry (2011), doi:10.1016/j.foodchem.2011.04.016
19 April 2011
2 A. Ortiz et al. / Food Chemistry xxx (2011) xxx–xxx
87 as demethylated carboxyl groups can cross-link with divalent cat- per treatment using a hand-held Effegi penetrometer equipped 145
88 ions such as calcium, thus reinforcing the cell wall network and with an 11.1 mm-diameter probe with a convex tip. Results are gi- 146
89 reducing its porosity (Brummell et al., 2001; Voragen, Coenen, Ver- ven as N. 147
90 hoef, & Schols, 2009).
91 Calcium applications thus have the potential to delay softening 2.4. Extraction, fractionation and uronic acid analysis of cell wall 148
92 and to extend shelf life of apples, and therefore have an influence materials 149
93 on texture, the other major attribute determining consumer accep-
94 tance of apples. They are also more economical and simple than CA Samples of flesh tissue were taken from six apples per treat- 150
95 technology, and have been shown recently to have beneficial ef- ment (2 fruit/replicate 3 replicates), frozen in liquid nitrogen, 151
96 fects on aroma biosynthesis by ‘Golden Reinders’ apples (Ortiz freeze-dried, and powdered. Weight loss after lyophilisation was 152
97 et al., 2010). Although these are interesting findings from a techno- consistently around 82%. Cell wall materials (CWM) were extracted 153
98 logical perspective, the optimisation of post-harvest handling re- from lyophilised tissue (3 g) according to Redgwell, Melton, and 154
99 quires a broader overview of changes induced by the procedures Brasch (1992). Samples were homogenised in 20 ml phenol:acetic 155
100 applied in each case. While calcium applications and CA storage acid:water (2:1:1, w/v/v) (PAW) for 20 min. After centrifugation 156
101 have been shown to maintain apple firmness (reviewed in John- at 4000g and 4 °C for 45 min, the pellet was resuspended in 157
102 ston, Hewett, & Hertog, 2002), treatment effects on cell wall com- 10 ml water and centrifuged again. The PAW and water wash 158
103 position and related enzyme activities have received much less supernatants were combined and intensively dialysed (mol wt. 159
104 attention, and no conclusive results have been reported to date. cut-off 7000) for 2 days against Milli-Q water at 4 °C. The dialysate 160
105 The impact of post-harvest calcium dips on cell wall metabolism was centrifuged at 4000g and 4 °C for 45 min to sediment out the 161
106 of air- and CA-stored ‘Golden Reinders’ apples were assessed here- precipitate formed during the dialysis. The supernatant (hence- 162
107 in. A two-week post-storage period at 20 °C was chosen to simulate forth, PAW-soluble fraction; PAWsf) was recovered, lyophilised 163
108 the usual marketing time for these fruit. and weighed. The pellet obtained after PAW extraction and water 164
wash was subsequently washed twice in acetone, recovered by 165
vacuum-filtration through Whatman grade 4 paper filters, lyophi- 166
109 2. Materials and methods
lised and weighed to determine yield of CWM, expressed as % (w/ 167
w) FW. For further fractionation, CWM (100 mg) from each repli- 168
110 2.1. Plant material, calcium treatment and storage conditions
cate were extracted sequentially with water, 0.05 M cyclohex- 169
ane-trans-1,2-diamine tetra-acetate (CDTA), 0.05 M Na2CO3, and 170
111 Apple (Malus domestica Borkh., cv. Golden Reinders) fruit
4 M KOH as described previously (Selvendran & O’Neill, 1987), in 171
112 were harvested in 2007 at commercial maturity (139 days after full
order to fractionate water-soluble pectin, loosely-bound pectin, 172
113 bloom), from 7 year-old trees grafted on M-9 EMLA rootstocks at
covalently-bound pectin and matrix glycans (hemicelluloses), 173
114 the IRTA-Experimental Station in Mollerussa, in the area of Lleida
respectively. Each fraction was dialysed (mol wt. cut-off 7000) 174
115 (NE Spain). Ethylene production at harvest was 1.4 ll kgÿ1 hÿ1;
for 2 days against Milli-Q water at 4 °C, filtered through Miracloth, 175
116 firmness and starch index averaged 72.3 N and 5.3, respectively.
lyophilised and weighed. Yields are expressed as % (w/w) CWM. 176
117 Immediately after harvest, fruit were randomly divided into four
For uronic acid content determination, 30–35 mg of the CDTA- 177
118 lots, two of which were dipped in a CaCl2 solution (2%, w/v, in
and Na2CO3-soluble fractions were pre-hydrolysed in 1 ml of 178
119 deionised water) at ambient temperature for 5 min. Subsequently,
12 M H2SO4 for 1 h at 37 °C, prior to dilution in 11 ml distilled 179
120 CaCl2-treated and untreated apples were stored at 1 °C and 92% RH
water and further hydrolysis at 100 °C for 2 h. Uronic acid content 180
121 under either air or ultra-low oxygen (ULO) atmosphere (1kPa
in the hydrolysate was measured by the m-hydroxydiphenyl meth- 181
122 O2:2kPa CO2). O2 and CO2 concentrations were monitored continu-
od (Blumenkrantz & Asboe-Hansen, 1973), using galacturonic acid 182
123 ously, and corrected automatically using N2 from a tank and by
as a standard, and results were expressed as % (w/w). 183
124 scrubbing off excess CO2 with a charcoal system. A humidifier
125 was used to maintain RH to constant levels. Samples were taken
2.5. Extraction and assay of cell wall-modifying enzyme activities 184
126 after 19 or 31 weeks of storage, and placed at 20 °C for 0, 7 or
127 14 days in order to simulate commercial shelf life and final firm-
For the extraction of polygalacturonase (exo-PG; EC 3.2.1.67 185
128 ness of fruit reaching potential consumers.
and endo-PG; EC 3.2.1.15), pectinmethylesterase (PME; EC 186
3.1.1.11), pectate lyase (PL; EC 4.2.2.2) and endo-1,4-b-D-glucanase 187
129 2.2. Determination of calcium content (EGase; EC 3.2.1.4), 100 mg of freeze-dried flesh tissue was homog- 188
enised (10%, w/v) in extraction buffer prepared according to Loh- 189
130 Seven days after removal from cold storage, samples of flesh tis- ani, Trivedi, and Nath (2004). PG activity was determined on 190
131 sue were taken (3 replicates 2 apples/replicate), frozen in liquid apple pectin (d.e. 70–75%) as described previously (Pathak & San- 191
132 nitrogen, freeze-dried, powdered, and kept at ÿ80 °C until process- wall, 1998), with galacturonic acid (GalUA) as a standard. One unit 192
133 ing. One gramme of lyophilised powdered tissue was ashed in a (U) of PG activity was defined as the liberation of 1 lmol of GalUA 193
134 muffle furnace at 500 °C for 2 h. Ashes were digested thereafter minÿ1. PME activity was measured as described by Hagerman and 194
135 with 4 ml HCl:water (1:1, v/v) and heated at 70 °C until complete Austin (1986), with the reaction mixture containing crude enzyme 195
136 sample dehydration. Dried material was then resuspended in extract, pectin and bromothymol blue prepared as described by 196
137 2 ml HCl:water (1:1, v/v) for 15 min, filtered through ‘Whatman Alonso, Howell, and Canet (1997). One unit (U) of PME activity 197
138 40 Ashless’ paper filters, and the filtrate diluted to 50 ml in distilled was defined as the decrease of one unit of A620 minÿ1. PL activity 198
139 water. Samples were then analysed by inductively coupled plasma was assayed according to Moran, Nasuno, and Starr (1968) as mod- 199
140 emission spectroscopy (ICP-OES) in a ‘Horiba Jobin Yvon ACTIVA’ ified by Lohani et al. (2004). One unit (U) of PL activity was defined 200
141 spectrometer, and results expressed as mg 100 g FWÿ1. as the increase of one unit of A235 minÿ1. For the assessment of 201
EGase activity, the DNS method (Miller, 1959), with carboxymeth- 202
142 2.3. Determination of flesh firmness ylcellulose [1.3% (w/v) in 20 mM Tris–HCl, pH 7.0] as the assay 203
substrate, was used to determine the amount of reducing sugars 204
143 Skin tissue from two opposite sides was removed, and flesh released, using glucose as a standard. One unit (U) of EGase activity 205
144 firmness measurements were carried out individually on 15 fruit was defined as the release of 1 lmol of glucose minÿ1. 206
19 April 2011
A. Ortiz et al. / Food Chemistry xxx (2011) xxx–xxx 3
207 For the extraction of b-galactosidase (b-Gal; EC 3.2.1.23) and 3. Results and discussion 264
208 b-xylosidase (b-Xyl; EC 3.2.1.37) activities, a 10% (w/v) flesh
209 homogenate was prepared by homogenising 100 mg of freeze- 3.1. Softening and cell wall characterisation during post-storage 265
210 dried flesh tissue in an extraction buffer prepared according to ripening of ‘Golden Reinders’ apples 266
211 Vicente, Costa, Martínez, Chaves, and Civello (2005), and activity
212 assays were undertaken in the crude extract as described therein. Calcium concentration in the cortical tissue was higher in trea- 267
213 One unit (U) of b-Gal or b-Xyl activity was defined as the liberation ted than in untreated fruit (Table 1), which confirms the effective- 268
214 of 1 lmol of p-nitrophenol minÿ1 from p-nitrophenyl-b-D-galacto- ness of the dipping procedure for calcium application. Ethylene 269
215 pyranoside or 1 nmol of p-nitrophenyl-b-D-xylopyranoside, respec-
216 tively. For the extraction of a-L-arabinofuranosidase (AFase; EC
Table 1
217 3.2.1.55) activity, the crude extract was obtained after mixing Calcium content (mg 100 g FWÿ1) in the flesh of ‘Golden Reinders’ apples after 7 days
218 100 mg of freeze-dried flesh tissue with 1 ml of extraction buffer at 20 °C following cold storage at 1 °C.
219 [0.1 M sodium acetate (pH 5.2), 100 mM NaCl, 2% (v/v) b-mercap-
Storage atmosphere Treatment Storage period
220 toethanol, and 1% (w/v) PVPP], and the activity was measured
19 Weeks 31 Weeks
221 according to Wei et al., 2010. One unit (U) of AFase was defined
222 as the liberation of 1 nmol of p-nitrophenol minÿ1 from p-nitro- Air Untreated 2.8 b 2.9 b
223 phenyl-a-L-arabinofuranoside. All assays were done in triplicate, CaCl2 3.4 a 5.2 a
ULO Untreated 3.6 b 4.1 b
224 and total protein content in the extracts was determined with CaCl2 5.8 a 5.3 a
225 the Bradford method (1976), using BSA as a standard, and results
226 were expressed as specific activity (U mg proteinÿ1). Data represent means of three replicates. Means within the same column for a given
storage atmosphere followed by different letters are significantly different at
P 6 0.05 (LSD = 0.5).
227 2.6. Analysis of ethylene production
228 The rate of ethylene production was determined (3 repli-

229 cates 2 apples/replicate) by placing fruit in 3 l respiration flasks,
230 continuously aerated with humidified air at a flow rate of around
231 1.5 l hÿ1. Samples of the effluent air were taken with a 1 ml syr-
232 inge, and injected into a gas chromatograph (Agilent Technologies
233 6890 N) equipped with a flame ionisation detector and an alumina
234 column (1.5 m 3 mm). Gas analyses were conducted isother-
235 mally at 100 °C. N2 carrier gas, air and H2 flows were 45, 400 and
236 45 ml minÿ1, respectively. The injector and detector were held at
237 120 °C and 180 °C, correspondingly. Results were expressed as ll
238 ethylene kgÿ1 hÿ1.
239 2.7. Microscopy observations
240 Small pieces (1 mm3) of fruit cortex were fixed in 2% (w/v) p-

241 formaldehyde and 2.5% (v/v) glutaraldehyde in 0.1 M Na-phos-
242 phate buffer, pH 7.2, at 4 °C for 5 h. Samples were then washed
243 three times in the same buffer for 10 min at 4 °C, post-fixed with
244 2% osmium tetroxide for 2 h at 4 °C, and rinsed again in Na-phos-
245 phate buffer, pH 7.2. Tissues were dehydrated in a graded series
246 of ethanol solutions (50–100%, v/v), embedded in LR White resin
247 and polymerised at 60 °C for 48 h. For transmission–electron-
248 microscopy (TEM) observations, ultra-thin (60–90 nm thick) sec-
249 tions were cut and mounted onto copper grids, counter-stained
250 with uranyl acetate and lead citrate for 30 and 10 min, respec-
Fig. 1. Ethylene production by ‘Golden Reinders’ apples after storage at 1 °C for 19
251 tively, and viewed in a Phillips (EM 301) electron microscope at
(A) or 31 (B) weeks. Values represent means of three replicates. Vertical bar
252 80 kV. indicates LSD0.05.
253 2.8. Statistical analysis Table 2

Flesh firmness (N) of ‘Golden Reinders’ apples after storage at 1 °C.
254 Results were treated for multiple comparisons by analysis of
Atmosphere Treatment Storage period + days at 20 °C
255 variance (GLM-ANOVA), followed by the least significant difference
19 Weeks 31 Weeks
256 (LSD) Fisher’s test at P 6 0.05 with the SAS programme package
257 (SAS Institute, Cary, NC, USA, 1988). For these analyses, a multi-fac- 0d 7d 14 d 0d 7d 14 d
258 torial design was used, with calcium treatment, storage atmo- Air Untreated 61.9 b 58.0 b 47.9 c 50.4 c 46.2 c 42.4 d
259 sphere, storage period and shelf life period as the factors. Partial CaCl2 62.1 b 56.3 b 55.5 b 56.3 b 55.4 b 51.8 c
260 least squares regression (PLSR) was used as a predictive method ULO Untreated 69.6 a 72.8 a 62.9 a 70.1 a 65.8 a 57.7 b
CaCl2 71.4 a 73.8 a 66.4 a 70.5 a 66.3 a 63.6 a
261 to relate a matrix of dependent variables (Y) to a set of explanatory
262 variables (X). Unscrambler version 6.11a software (CAMO ASA, Values represent means of fifteen replicates. Means showing different letters within
263 1997) was used for developing these models. a column are significantly different at P 6 0.05 (LSD = 5.7).
19 April 2011
270 production was inhibited in calcium-treated samples during the Whitaker, & Sams, 1998). The inhibition of ethylene production 277
271 first week of shelf life subsequent to air storage (Fig. 1). Applied was even more accentuated in ULO-stored fruit, but in general no 278
272 calcium has been reported to inhibit ethylene biosynthesis through additive effects with calcium were observed. Accordingly, ULO- 279
273 its role in the preservation of cell membranes, thus delaying ACO- stored fruit remained firmer throughout the shelf life period con- 280
274 catalysed conversion of ACC into ethylene (Lara & Vendrell, 1998). sidered regardless of calcium treatment, with the only exception 281
275 Indeed calcium treatments have been shown to maintain cell of long term-stored fruit (Table 2), for which an additive effect of 282
276 membrane integrity in apple fruit (Picchioni, Watada, Conway, calcium dips was observed for samples stored under ULO after 283
Table 3
Yield of insoluble CWM (% FW) and of CWM fractions (% CWM) isolated from ‘Golden Reinders’ fruit after storage at 1 °C.
Fraction Atmosphere Treatment Storage period + days at 20 °C

19 Weeks 31 Weeks
0d 7d 14 d 0d 7d 14 d
CWM Air Untreated 2.45 b 2.23 b 2.14 b 2.43 b 2.10 b 1.73 b
(LSD = 0.11) CaCl2 2.50 b 2.47 a 2.25 a 2.62 a 2.43 a 1.99 a
ULO Untreated 2.55 ab 2.31 b 2.29 a 2.44 b 2.03 b 2.02 a
CaCl2 2.63 a 2.54 a 2.29 a 2.50 b 2.07 b 2.09 a
Wsf Air Untreated 2.36 a 3.75 a 4.95 a 4.54 a 3.25 ab 5.63 a
(LSD = 1.05) CaCl2 2.16 ab 3.75 a 4.03 ab 4.23 a 4.17 a 4.39 b
ULO Untreated 1.92 b 2.52 b 3.29 b 2.78 b 2.86 b 4.95 ab
CaCl2 1.18 b 1.56 b 2.16 b 2.26 b 2.90 b 4.37 b
CDTAsf Air Untreated 35.41 b 31.35 b 28.51 b 30.39 c 25.16 d 18.32 d
(LSD = 2.87) CaCl2 40.40 a 37.80 a 33.59 a 35.72 b 32.75 b 24.30 c
ULO Untreated 30.17 c 30.05 b 27.75 b 28.87 c 29.11 c 27.23 b
CaCl2 30.79 c 31.36 b 27.84 b 40.52 a 38.74 a 38.99 a
Na2CO3sf Air Untreated 18.03 b 17.65 b 16.67 c 19.17 b 19.72 b 16.60 b
(LSD = 1.43) CaCl2 21.90 a 18.80 b 18.50 b 19.33 b 19.59 b 17.42 ab
ULO Untreated 20.50 a 20.24 a 19.25 ab 19.06 b 18.40 b 16.75 b
CaCl2 21.45 a 21.42 a 20.48 a 20.43 a 20.10 a 18.20 a
KOHsf Air Untreated 9.74 a 7.90 a 4.02 b 5.07 a 5.23 a 5.95 a
(LSD = 1.62) CaCl2 9.83 a 9.40 a 6.07 a 5.35 a 6.10 a 5.97 a
ULO Untreated 7.33 b 4.67 b 3.74 b 4.80 a 4.51 a 3.40 b
CaCl2 6.61 b 4.31 b 3.92 b 5.04 a 5.07 a 3.89 b
Values represent means of three replicates. Means showing different letters within a column for a given fraction are significantly different at P 6 0.05 (LSD test).
Fig. 2. Transmission–electron-microscopy images of the cortex of untreated (A and C) and calcium-treated (B and D) ‘Golden Reinders’ apples after 31 weeks at 1 °C under air
(top) or ULO (bottom) + 7 days at 20 °C (ml, middle lamella). Bars = 1 lm.
19 April 2011
284 14 days at 20 °C. Calcium-treated fruit stored in air also remained Higher yields of Na2CO3sf, the fraction enriched in covalently- 306
285 firmer, although this effect was observed only at advanced ripening bound pectin, were found in fruit stored under ULO for 19 weeks 307
286 stages. (Table 3) regardless of calcium treatment, in parallel to higher 308
287 Since ripening-related softening occurs mainly as a result of firmness values (Table 2), indicating that this attribute might be 309
288 cell wall disruption (Brummell et al., 2001; Goulao et al., 2008), strongly dependent on the content of these polysaccharides. At 310
289 CWM were extracted from fruit cortex and fractionated sequen- the end of the shelf life period considered, the preservation of this 311
290 tially. Significant differences in the amount of CWM isolated were pectin fraction in air-stored samples was also aided by calcium 312
291 observed in response to calcium treatment or storage atmosphere applications, which additionally led to higher content of chelator- 313
292 (Table 3). For longer (14 days) shelf life periods, calcium applica- soluble pectins (Table 3), possibly sustaining firmness retention 314
293 tions and ULO storage caused higher CWM yields, both alone and (Table 2). The effects of calcium dips on CDTAsf yields were partic- 315
294 in combination (Table 3), possibly in relation to better firmness ularly noticeable after long-term (31 weeks) storage, when higher 316
295 retention in these samples (Table 2). The combined treatment contents of this fraction in treated samples were found for both air- 317
296 also led to lower solubilisation of cell wall materials after 14 days and ULO-stored fruit (Table 3). The chelator-soluble fraction is con- 318
297 at 20 °C as indicated by PAWsf yields (data not shown). Yet nei- sidered to contain the middle lamella pectin. This is consistent 319
298 ther CWM nor PAWsf yields were totally in accordance with the with TEM images (Fig. 2) showing better preservation of middle la- 320
299 differences in firmness observed during the post-storage period mella in calcium-treated fruit, and suggesting improved cell-to-cell 321
300 (Table 2). Previous works (Murayama, Katsumata, Horiuchi, & adhesion as a result of calcium deposition which may have contrib- 322
301 Fukushima, 2002; Brummell, Dal Cin, Crisosto, & Labavitch, uted to higher firmness values. For long-term storage, higher Na2- 323
302 2004; Peña & Carpita, 2004) have pointed out that modifications CO3sf yields were observed uniquely for the combined treatment. 324
303 in composition and linkages between polysaccharides, rather than Firmness levels of ‘Golden Reinders’ fruit were thus associated 325
304 the total amount of CWM, may be critical traits influencing fruit apparently with changes in pectin-containing fractions, rather than 326
305 softening. with total CWM amounts. 327
Fig. 3. Loadings plots of PC1 versus PC2 corresponding to a PLSR model for yields of CWM fractions and firmness (Y variables) versus cell wall modifying enzyme activities (X
variables) in ‘Golden Reinders’ apples after cold storage at 1 °C for 19 (A) or 31 (B) weeks.
19 April 2011
328 3.2. Cell wall-modifying enzyme activities during post-storage ripening

329 of ‘Golden Reinders’ apples
330 Research on ripening-related cell wall metabolism has generally

331 failed to establish satisfactory relationships between firmness loss
332 and individual related enzyme activities, which illustrates the
333 complexity of cell wall disassembly events. Seven different cell
334 wall-modifying enzyme activities (five pectolytic, two non-pecto-
335 lytic), potentially involved in cell wall disassembly, were analysed
336 in this work. Data were used to develop PLSR models of firmness
337 and cell wall fractions (Y variables), with enzyme activities as the
338 explanatory variables. The first two principal components of these
339 models explained 71% and 67% of total variability in the Y dataset
340 after storage for 19 (Fig. 3A) or 31 (Fig. 3B) weeks, respectively.
341 This was considered satisfactory taking into account the many
342 variables introduced and the intrinsic variability of fruit studies.
343 Both plots show that yields of PAWsf and Wsf, representative of sol-
344 ubilised polymers, were strongly associated to the activities of the
345 glycosidic enzymes b-Gal, AFase and b-Xyl, in turn inversely re-
346 lated to firmness (Fig. 3). This is interesting, as the loss of neutral
347 sugars from the side-chains attached to rhamnosyl residues in
348 the rhamnogalacturonan backbone of pectins is reportedly one of
349 the earliest cell wall modifications during fruit ripening (Redgwell,
350 Fischer, Kendall, & MacRae, 1997; Brummell et al., 2004; Peña Fig. 4. a-L-arabinofuranosidase activity in ‘Golden Reinders’ apples after storage at
351 et al., 2004). The elimination of these side-chain might promote 1 °C for 19 (A) or 31 (B) weeks. Values represent means of three replicates. Means
352 pectin solubilisation, as they connect covalently the rhamnogalac- showing different letters for a given period are significantly different at P 6 0.05
(LSD test).
353 turonan backbone to other cell wall materials (Hwang, Pyun, &
354 Kokini, 1993; Caffall et al., 2009; Jarvis, 2009), and presumably
355 protect polyuronides from enzymatic degradation through the inhibiting effect was detectable only for the combined treatment 362
356 control of cell wall porosity and thus of enzyme access to sub- (Table 4). Both ULO storage and applied calcium, alone and in com- 363
357 strates (Brummell et al., 2001). bination, reduced b-Gal activity during the first week following 364
358 b-Gal and AFase may remove galactosyl and arabinosyl residues long-term storage, whereas for a longer period at 20 °C only the 365
359 from rhamnogalacturonan side-chains. Decreased b-Gal activity combined treatment was effective. Significant inhibitory effects 366
360 was observed for ULO-stored samples upon removal from mid- of both post-harvest procedures considered were also observed 367
361 term storage, but during the subsequent shelf life at 20 °C this on AFase activity (Fig. 4). Partial inhibition of b-Gal and AFase 368
Table 4
Specific activities (U mg proteinÿ1) of some pectolytic and non-pectolytic cell wall-modifying enzymes in ‘Golden Reinders’ apple fruit after storage at 1 °C.
Enzyme Atmosphere Treatment Storage period + days at 20 °C

19 Weeks 31 Weeks
0d 7d 14 d 0d 7d 14 d
Pectolytic
PME Air Untreated 89.08 ab 83.66 b 63.80 b 63.57 b 87.97 b 69.99 b
(LSD = 19.74) CaCl2 70.66 b 72.80 b 40.63 c 60.12 b 69.35 b 75.05 b
ULO Untreated 87.92 ab 143.50 a 202.72 a 108.44 a 130.03 a 141.43 a
CaCl2 102.11 a 128.57 a 191.41 a 96.14 a 124.47 a 124.98 a
PG Air Untreated 17.83 a 19.32 a 18.94 a 17.85 a 22.79 a 21.72 a
(LSD = 2.03) CaCl2 13.33 b 13.89 b 12.73 b 13.18 b 12.87 c 12.88 b
ULO Untreated 18.04 a 20.58 a 18.99 a 18.97 a 18.82 b 23.72 a
CaCl2 14.62 b 15.37 b 14.19 b 13.15 b 12.77 c 13.15 b
PL Air Untreated 1.02 a 1.25 a 1.34 a 0.68 a 1.26 a 1.54 a
(LSD = 0.18) CaCl2 1.05 a 1.14 ab 1.15 b 0.59 a 1.15 a 1.21 b
ULO Untreated 0.99 ab 1.08 b 1.27 ab 0.37 b 0.89 b 1.27 b
CaCl2 0.82 b 0.81 c 0.95 c 0.37 b 0.87 b 1.07 c
b-Gal Air Untreated 0.65 a 0.66 a 0.78 a 0.37 a 0.64 a 0.66 a
(LSD = 0.16) CaCl2 0.69 a 0.69 a 0.71 a 0.15 b 0.33 b 0.66 a
ULO Untreated 0.30 b 0.72 a 0.72 a 0.17 b 0.42 b 0.54 a
CaCl2 0.35 b 0.36 b 0.54 b 0.16 b 0.28 b 0.36 b
Non-pectolytic
b-Xyl Air Untreated 36.24 a 44.09 a 36.41 a 29.92 a 45.43 a 46.91 a
(LSD = 3.71) CaCl2 35.18 a 40.26 b 28.94 b 31.99 a 44.63 a 45.92 a
ULO Untreated 34.53 a 31.95 c 29.36 b 19.70 b 26.80 b 26.17 b
CaCl2 29.17 b 29.28 c 28.26 b 19.25 b 27.82 b 27.85 b
EGase Air Untreated 8.45 b 6.86 b 5.17 b 10.68 b 6.48 c 4.66 c
(LSD = 2.29) CaCl2 6.38 b 3.85 c 3.97 b 10.51 b 8.30 c 5.95 c
ULO Untreated 12.99 a 11.30 a 11.27 a 13.00 a 15.95 a 16.00 a
CaCl2 10.87 a 9.75 a 9.82 a 10.17 b 12.23 b 12.80 b
Values represent means of three replicates. Means showing different letters within a column for a given enzyme activity are significantly different at P 6 0.05 (LSD test).
19 April 2011
Table 5
Uronic acid content (% w/w) in pectin-containing fractions isolated from ‘Golden Reinders’ fruit after storage at 1 °C.
Fraction Atmosphere Treatment Storage period + days at 20 °C

19 Weeks 31 Weeks
0d 7d 14 d 0d 7d 14 d
CDTAsf Air Untreated 10.28 b 9.15 b 9.72 b 10.27 a 8.27 c 8.75 c
(LSD = 1.39) CaCl2 13.01 a 12.97 a 13.23 a 10.07 a 10.61 b 14.75 a
ULO Untreated 6.29 d 10.14 b 10.62 b 7.24 b 9.32 bc 9.85 c
CaCl2 8.79 c 12.74 a 12.95 a 9.42 a 13.29 a 13.13 b
Na2CO3sf Air Untreated 65.65 b 63.36 b 61.23 c 64.52 b 61.01 c 58.43 c
(LSD = 1.94) CaCl2 66.67 b 64.37 b 64.58 b 65.45 b 63.00 b 63.35 b
ULO Untreated 69.94 a 69.96 a 67.00 a 68.66 a 66.82 a 65.09 a
CaCl2 71.46 a 69.42 a 67.55 a 68.31 a 67.87 a 66.28 a
Values represent means of three replicates. Means showing different letters within a column for a given fraction are significantly different at P 6 0.05 (LSD test).
369 activities in treated fruit might have delayed the cleavage of ram- decreased PL levels uniquely after 14 days at 20 °C, in agreement 419
370 ifications attached to the pectin backbone and contributed to the with firmness values (Table 2). Banana (Musa acuminata Colla) fruit 420
371 preservation of the covalently-bound pectin fraction. This idea is PL is reportedly ethylene-dependent (Domínguez-Puigjaner, Llop, 421
372 also supported by the observation of inverse relationships between Vendrell, & Prat, 1997; Lohani et al., 2004), and apple MdPL1 clusters 422
373 these enzyme activities and Na2CO3sf yields (Fig. 3), and in agree- with homologous genes known to accumulate during banana ripen- 423
374 ment with recent reports that these enzyme activities and the ing (Goulao et al., 2008). If similar regulation mechanisms are as- 424
375 associated release of neutral sugars from the cell wall are a key sumed as for banana PLs, partial suppression of PL activity might 425
376 indicator of storability in different apple cultivars including ‘Fuji’, have arisen from decreased ethylene production rates (Fig. 1). Uron- 426
377 ‘Golden Delicious’ and ‘Royal Gala’ (Siddiqui, Streif, & Bangerth, ic acid content in the Na2CO3sf (Table 5) closely paralleled fruit firm- 427
378 2004; Goulao, Santos, de Sousa, & Oliveira, 2007; Wei et al., ness (Table 2), and higher contents were found in samples showing 428
379 2010). Moreover, b-Gal in apple has been reported to be multi- lower PL activity levels (Table 5), suggesting a relevant role of this 429
380 functional, with associated b-D-fucosidase and a-L-arabinopyra- enzyme in the solubilisation of cell wall pectins. PL activity has been 430
381 nosidase activities acting synergistically (Dick, Opoku-Gyamfua, also shown to associate to firmness loss in fruit species displaying 431
382 & deMarco, 1990). Gene expression and activity of b-Gal and AFase different softening behaviour than apple (Domínguez-Puigjaner 432
383 are thought to be ethylene-dependent (Wei et al., 2010), which et al., 1997; Marín-Rodríguez, Orchard, & Seymour, 2002). For exam- 433
384 agrees with decreased ethylene production observed for treated ple, PL-suppressed ripe strawberries (Fragaria ananassa Duch.) 434
385 fruit (Fig. 1). were firmer than wild-type fruit (Jiménez-Bermúdez et al., 2002). 435
386 The removal of methyl groups from polyuronides is a common In contrast to PL, PG activity was unaffected by ULO storage, while 436
387 feature to all fruit species during maturation and ripening it was suppressed in calcium-treated fruit (Table 4). This may have 437
388 (Brummell et al., 2001). These demethylations are catalysed by contributed to better retention of uronic acids in the CDTAsf in these 438
389 PME, generally expressed to high levels before the onset of ripen- samples (Table 5), and thus to higher firmness levels. Although ULO- 439
390 ing but down-regulated by ethylene thereafter (Owino, Ambuko, storage did not directly affect PG activity, better preservation of the 440
391 & Mathooko, 2005). Accordingly, PME activity was promoted in branched structure of pectins resulting from decreased b-Gal and 441
392 ULO-stored apples, which produced the lowest amounts of ethyl- AFase activities (Table 4 and Fig. 4) might have limited the access 442
393 ene Fig. 1. In contrast, calcium dips did not enhance PME activity of this enzyme to its substrates, thus preventing a relevant role of 443
394 (Table 4), maybe due to less intense effects on ethylene production PG in the softening process of cold-stored ‘Golden Reinders’ apple 444
395 (Fig. 1). Higher PME activities were generally found for firmer fruit fruit. 445
396 (Table 2), suggesting that this enzyme activity was not directly re- Matrix glycans are susceptible to cleavage by b-Xyl and EGase 446
397 lated to fruit softening in ‘Golden Reinders’ apples, in agreement (Brummell et al., 2001; Goulao et al., 2008). Decreased b-Xyl activ- 447
398 with previous findings for ‘Golden Delicious’, its parental cultivar ity was found after ULO storage regardless of period, whereas cal- 448
399 (Klein, Hanzon, Irwin, Ben Shalom, & Lurie, 1995; Siddiqui et al., cium dips were effective uniquely after mid-term storage (Table 4). 449
400 2004). In fact, the demethylation of galacturonic acid residues Inhibiting effects might have arisen from an ethylene-dependent 450
401 can promote calcium-mediated pectin cross-links, thus contribut- expression pattern as suggested previously (Boquete, Trinchero, 451
402 ing to cell wall integrity and to increased tissue rigidity (Guillemin Fraschina, Vilella, & Sozzi, 2004; Di Santo, Pagano, & Sois, 2009). 452
403 et al., 2008; Jarvis, 2009). Accordingly, although in general no cal- The regression models showed an inverse relationship to fruit 453
404 cium-related differences were observed in PME activity, higher firmness, thus hinting a role for b-Xyl in apple softening (Fig. 3). 454
405 CDTAsf yields (Table 3) and better preservation of the middle la- The mechanism underlying this relationship remains unclear in 455
406 mella (Fig. 2) were found for calcium-treated fruit, consistent with the light of results, as no clear association to matrix glycan content 456
407 improved retention of uronic acids in the CDTA-soluble fraction was observed (Table 3). However, this enzyme activity has been re- 457
408 isolated from these samples (Table 5). ported to contribute to the removal of arabinosyl residues from 458
409 PME action, however, may also promote fruit softening, since rhamnogalacturonan polymers (Bustamante, Rosli, Añón, Civello, 459
410 previous demethylation of polyuronides is required for PG- and & Martínez, 2006; Hayama, Shimada, Fujii, Ito, & Kashimura, 460
411 PL-catalysed depolymerisation through cleavage of a-(1,4)-galac- 2006), apparently a key event for ‘Golden Reinders’ apple softening 461
412 turonan linkages or by b-elimination, respectively (Bennett & Labav- as discussed above. As to EGase activity, higher levels were found 462
413 itch, 2008). To date, the impact of PL activity on apple softening has for ULO-stored fruit, whereas no clear effects were detected in re- 463
414 received little attention. PLSR models developed herein revealed it sponse to calcium dips (Table 4). An inverse relationship to KOHsf 464
415 was strongly associated to CWM solubilisation and inversely related yields was observed (Fig. 3), suggesting a possible role in the mod- 465
416 to firmness (Fig. 3), suggesting a key role in ripening-related soften- ification of matrix glycans after storage of ‘Golden Reinders’ apples, 466
417 ing of ‘Golden Reinders’ fruit. ULO storage generally inhibited PL although with no apparent relationship to firmness levels. This en- 467
418 activity (Table 4), whereas in air-stored samples calcium dips zyme has been reported to act preferentially on the xyloglucan 468
19 April 2011
469 polymers bound to cellulose microfibrils (Brummell et al., 2001), Hagerman, A. E., & Austin, P. J. (1986). Continuous spectrophotometric assay for 540
plant pectin methyl-esterase. Journal of Agricultural and Food Chemistry, 34, 541
470 which are more abundant at earlier ripening stages, and thus its 542
440–444.
471 role in firmness loss during the post-harvest period of apples Harker, F. R., Kupferman, E. M., Marin, A. B., Gunson, F. A., & Triggs, C. M. (2008). 543
472 should be possibly deemphasised. Indeed, EGase has been rather Eating quality standards for apples based on consumer preferences. Postharvest 544
Biology and Technology, 50, 70–78. 545
473 associated to cell wall expansion and reported to decrease as fruit 546
Hayama, H., Shimada, T., Fujii, H., Ito, A., & Kashimura, Y. (2006). Ethylene regulation
474 attain full size (Goulao et al., 2007). of softening and softening-related genes in peach. Journal of Experimental 547
475 Results reported herein show that both ULO and calcium dips Botany, 57, 4071–4077. 548
Hwang, J., Pyun, Y. R., & Kokini, J. L. (1993). Sidechains of pectins: Some thoughts on 549
476 delayed fruit softening after storage of ‘Golden Reinders’ apples
their role in plant cell walls and foods. Food Hydrocolloids, 7, 39–53. 550
477 by deferring the ripening-related solubilisation of pectic polymers, Jaeger, S. R., Andani, Z., Wakeling, I. N., & MacFie, H. J. H. (1998). Consumer 551
478 but the biochemical mechanisms underlying preservation of fruit preferences for fresh and aged apples: A cross-cultural comparison. Food Quality 552
479 firmness were apparently different. Data suggest that the effects and Preference, 9, 355–366. 553
Jarvis, M. C. (2009). Plant cell walls: Supramolecular assemblies. Food Hydrocolloids, 554
480 of ULO storage arose primarily from generally delayed ripening 25, 257–262. 555
481 due to decreased ethylene production, while applied calcium had Jiménez-Bermúdez, S., Redondo-Nevado, J., Muñoz-Blanco, J., Caballero, J. L., López- 556
482 additional direct effects on the integrity of the middle lamella. Aranda, J. M., Valpuesta, V., et al. (2002). Manipulation of strawberry fruit 557
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483 Inhibited levels of b-Gal, AFase and PL activities were apparently 751–759. 559
484 relevant for firmness preservation after storage. Johnston, J. W., Hewett, E. W., & Hertog, M. (2002). Postharvest softening of apple 560
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485 Acknowledgements Klein, J. D., Hanzon, J., Irwin, P. L., Ben Shalom, N., & Lurie, S. (1995). Pectin esterase 563
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486 A. Ortiz is the recipient of a FPU grant from the Ministerio de Lara, I., & Vendrell, M. (1998). ACC oxidase activation by cold storage on ‘Passe- 566
487 Ciencia e Innovación (MICINN) of Spain. This work was supported Crassane’ pears: Effect of calcium treatment. Journal of the Science of Food and 567
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488 through the AGL2006-00345/ALI project, financed by the Ministe- 569
Lara, I., Echeverría, G., Graell, J., & López, M. L. (2007). Volatile emission after
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538 (2008). Firming of fruit tissues by vacuum-infusion of pectin methylesterase: degradation in ripening apple fruit. Postharvest Biology and Technology, 56, 623
539 Visualisation of enzyme action. Food Chemistry, 109, 368–378. 147–154. 624
625
CAPÍTULO XIII
Ortiz A, Echeverría G, Graell J, Lara I.

Cell wall-modifying enzyme activities after controlled atmosphere
storage of calcium-treated ‘Fuji’ apples.
Acta Horticulturae, 2010, 858, 213–216
181
182
Cell Wall-Modifying Enzyme Activities after Controlled Atmosphere
Storage of Calcium-Treated ‘Fuji’ Apples
Àrea de Post-Collita, XaRTA, UdL-IRTA
Rovira Roure 191, 25198 Lleida
Spain
Keywords: Malus × domestica, pectin, firmness, pectolytic enzymes, calcium

Abstract
In this work, ‘Fuji Kiku-8’ apples were treated with 2% (w/v) CaCl2 and stored
at 1°C and 92% RH under either air or two different controlled atmosphere (CA)
regimes for 7 months. Different cell wall-modifying enzyme activities were determined
7 d after removal from storage in order to assess relationships, if any, to changes in
fruit firmness and cell wall composition induced by CaCl2 treatment and/or storage
atmosphere. Applied calcium was effective in preserving firmness of air-stored fruit
but no significant differences in this attribute were found for samples kept under CA
regardless of calcium treatment. Results showed high correlation between firmness
and covalently-bound pectin content. To a lesser extent, non covalently-bound pectin
content was also related to firmness. Solubilisation of pectins, which leads to loss of
fruit firmness, may require a previous release of neutral sugar residues from the side-
chains of wall-bound pectins by the action of a number of glycosidases such as
-galactosidase ( -Gal) or -xylosidase ( -Xyl), which in turn was partially inhibited
by CA-conditions during cold storage.
INTRODUCTION
Refrigeration of apple (Malus domestica Borkh.) fruit has been widely used to
delay many ripening-related modifications, and thus to extend commercial life of fruit.
These changes include modifications in the cell wall structure, which are believed to
underlie changes in fruit firmness and texture, and are largely driven by different related
enzyme activities. Furthermore, a number of post-harvest procedures, including
treatments with CaCl2 solutions, have been tested to delay fruit softening (reviewed in
DeEll et al., 2001). The purpose of this work was to study changes in fruit firmness and
cell wall composition induced by calcium and/or storage atmosphere, with special
emphasis focused on some related pectolytic and non-pectolytic enzyme activities.
‘Fuji Kiku-8’ apple fruit were harvested at commercial maturity in IRTA-
Experimental Station of Lleida (NE Spain). Fruit were divided into six lots, three of
which were dipped in a 2% (w/v) CaCl2 solution for 5 min before storage for 7 months at
1ºC and 92% RH. Storage atmospheres were either air or two different controlled
atmosphere (CA) regimes: low oxygen (LO; 3 kPa O2 : 2 kPa CO2) and ultra low oxygen
(ULO; 1 kPa O2 : 2 kPa CO2). Analyses were carried out after 7 d at 20ºC and 92% RH
subsequent to cold storage. Firmness was measured on two opposite sides of 15 fruit
randomly selected per sample, with an Effegi hand-penetrometer (Facchini s.r.l.,
Alfonsine, RA, Italy; 11-mm diameter tip). Results are given as N. For calcium content
determination, freeze-dried pulp tissue obtained from 5 fruit per sample was submitted to
acid digestion and thereafter analysed by inductively coupled plasma emission
spectroscopy (ICP-OES) in a Horiba Jobin Yvon ACTIVA-M (HORIBA Jobin Yvon Inc.,
Madrid, Spain) spectrometer. Results were expressed as mg 100 gFM-1.
Samples of pulp tissue, frozen in liquid nitrogen, freeze-dried (Telstar, Sant Cugat
del Vallès, Spain) and powdered were used for extraction and assay of enzyme activities.
Polygalacturonase (PG), pectin methylesterase (PME), pectate lyase (PL) and endo-(1-4)-
-D-glucanase (EGase) extraction and assay were performed as described elsewhere
Proc. IIIrd IC Postharvest Unlimited 2008

Ed: W.B. Herppich 213
(Ortiz and Lara, 2008). -Xyl and -Gal activities were measured according to Cleemput
et al. (1997) and Vicente et al. (2005). Results were expressed as units mgprot-1.
Cell wall materials (CWM) were extracted in triplicate from fruit flesh according
to Redgwell et al. (1992), with some modifications (Lara et al., 2004). Lyophilised tissue
(3 g) was homogenised in phenol:acetic acid:water (2:1:1, w/v/v) (PAW). After
centrifugation (Hettich Lab Technology, Tuttlingen, Germany) of the homogenate, the
pellet was washed in water. The PAW and water wash supernatants were combined (PAW-
soluble fraction), dialysed, lyophilised and weighed. The pellet was washed in acetone,
filtered, lyophilised and weighed to determine yield of CWM (g 100 gFM-1). As previously
described (Selvendran and O’Neill, 1987), CWM from each sample was fractionated
sequentially with water, cyclohexane-trans-1,2-diamine tetra-acetate (CDTA), Na2CO3,
and KOH, in order to fractionate soluble, non covalently-bound, covalently-bound and
matrix glycans, respectively. Each fraction was filtered, dialysed, lyophilised and
weighed. Yields were expressed as g 100 gCMW-1. All reagents were provided by Sigma-
Aldrich Corp. (Tres Cantos, Spain) or Bio-Rad Laboratories Ltd. (Alcobendas, Spain).
Results were subjected to analysis of variance (GLM-ANOVA), followed by the
Fisher’s LSD test at P<0.05. Unscrambler vers. 6.11a software (CAMO ASA, 1997) was
used for Principal Component Analysis (PCA) of data.
Increases of fruit firmness due to CaCl2 treatments were found only in air-stored
apples, since both LO and ULO conditions by themselves were able to maintain
satisfactorily this attribute (Table 1). Moreover, CA-stored apples were significantly
firmer than those stored in air, regardless of calcium treatment.
In an attempt to characterise samples, a PCA model was developed with firmness,
enzyme activities, cell wall fractions and calcium content as explaining variables.
Samples were distributed mainly along PC1 according to storage atmosphere. Distribution
along PC2 was found, for each of the three storage atmospheres, as a function of calcium
content (Fig. 1). Thus, both storage and calcium treatment had an influence on
modifications of the cell wall of pulp tissue after a long-term storage, possibly resulting in
the observed differences in fruit firmness.
Although showing the lowest firmness values (Table 1), air-stored samples were
characterised by higher content of CWM than CA-stored fruit, suggesting composition
and structure of cell walls is a more important factor for textural characteristics of apples
than total amount of CWM. The PCA model (Fig. 1) showed a good correlation between
firmness and the Na2CO3-soluble fraction (Na2CO3sf), which suggests this fraction may
be a key factor for preserving firmness in apples. Accordingly, ULO-stored apples were
characterised by higher levels of Na2CO3sf concomitantly with better firmness
preservation after storage. Calcium-treated apples contained higher amounts of CDTAsf,
which was also related to improved fruit firmness, probably explained by the capacity of
calcium ions to bind to non-methylated uronic residues and form linkages between
adjacent polymers, therefore reinforcing cell walls (Lara et al., 2004). Actually, the yield
of CDTAsf was strongly related to calcium content, consistent with its role in preserving
middle lamella and thus cell-to-cell adhesion. Interestingly, PG activity appeared close to
calcium content (Fig. 1). Consequently, PG might not play a main role in apple softening.
In fact, apple has been generally reported to lack detectable endo-PG, which leads to a
more dramatic pectin depolymerization than exo-PG (reviewed in Goulao and Oliveira,
2008). Alternatively, -Xyl and -Gal activities might be important factors for firmness
loss: these two activities were associated to air-stored apples, which were significantly
softer than the rest. Thus, these two enzymes might be considered as key factors for fruit
softening, susceptible to be inhibited by CA-conditions. The importance of these enzyme
activities could lie on a possible requirement, for effective pectin solubilisation, for the
release of galactosyl, xylosyl or other neutral sugars residues from the side-chains of
pectic polymers (such as xylogalacturonan and some rhamnogalacturonans), or for some
rearrangement of the associations between polysaccharides leading to fruit softening. In
214
relation to this issue, Bartley (1976) found a loss of galactose residues from the cell wall
to manifestly accompany softening of ripening apples, probably due to -Gal action. In
tomato, transgenic suppression of a ripening-related -Gal, which could have led to a
delay in the porosity changes necessary for the access of degradative enzymes to their
substrates, was associated with better retention of fruit firmness (Smith et al., 2002). In
contrast to our results, -Xyl, the other enzyme activity apparently related to fruit
softening (Fig. 1) has not been previously detected in apples during ripening (Dick et al.,
1990). This fact could have arisen from low activities in this fruit, since large volumes of
crude enzyme extract were needed in this work to measure them. However, these low
activities might have been critical for firmness loss.
In summary, our results suggest that the release of neutral sugars residues present
in the side-chains of wall-bound pectins might be a limiting factor for apple softening.
This event, due to the action of some enzymes such as -Xyl and -Gal, appears to have
been to some extent prevented by CA-storage, which has been shown to partially inhibit
these enzyme activities. Calcium dips also improved retention of fruit firmness, probably
due to the strengthening of cell walls by increasing calcium bridges between adjacent
pectin polymers.
ACKNOWLEDGEMENTS
A. Ortiz is the recipient of a FPU grant from the Ministerio de Ciencia e
Literature Cited
Bartley, 1976. Changes in the glucans of ripening apples. Phytochem. 15:625-626.
Cleemput, G., Hessing, M., Van Oort, M., Deconynck, M. and Delcour, J.A. 1997.
Purification and characterization of a -D-Xylosidase and an Endo-Xylanase from
Wheat Flour. Plant Physiol. 113:377-386.
DeEll, J.R., Khanizadeh, S., Saad, F. and Ferree, D.C. 2001. Factors affecting apple fruit
firmness – A review. J. Amer. Pomol. Soc. 55:8-27.
Dick, A.J., Opoku-Gyamfua, A. and DeMarco, A.C. 1990. Glycosidases of apple fruit: a
multi-functional -galactosidase. Physiol. Plant. 80:250-256.
Goulao, L.F. and Oliveira, C.M. 2008. Cell wall modifications during fruit ripening: when
a fruit is not the fruit. Trends Food Sci. Tech. 19:4-25.
Lara, I., García, P. and Vendrell, M. 2004. Modifications in cell wall composition after
cold storage of calcium-treated strawberry (Fragaria ananassa Duch.) fruit.
Ortiz, A. and Lara, I. 2008. Cell wall-modifying enzyme activities after storage of
1 MCP-treated peach fruit. Acta Hort. 796:137-142.
Redgwell, R.J., Melton, L.D. and Brasch, D.J. 1992. Cell wall dissolution in ripening
material. p.25-153. In: D. Glick (ed.), Methods of Biochemical Analysis, vol. 32. John
Wiley Interscience, New York.
Smith, D.L., Abbott, J.A. and Gross, K.C. 2002. Down-regulation of tomato
-galactosidase 4 results in decreased fruit softening. Plant Physiol. 129:1755-1762.
Vicente, A.R., Costa, M.L., Martínez, G.A., Chaves, A.R. and Civello, P.M. 2005. Effect
of heat treatments on cell wall degradation and softening in strawberry fruit.
215
Tables
Table 1. Firmness (N) of ‘Fuji Kiku-8’ apples after 7 days at 20ºC following 7 months of
cold storage in different atmospheres.
Treatment AIR1 LO ULO

Untreated 55.3 Bb2 72.8 Aa 72.4 Aa
CaCl2 (2%, w/v) 63.1 Ab 72.0 Aa 72.3 Aa
1
AIR: 21 kPa O2 + 0.03 kPa CO2; LO: 3 kPa O2 + 2 kPa CO2; ULO: 1 kPa O2 + 2 kPa CO2
2
Means separation at p<0.05 (LSD test): Capital letters between calcium treatments, small letters between
storage atmospheres. Values represent means of 15 fruit.
Figurese
ULO
LO
AIR
Fig. 1. Biplot of PC1 vs. PC2 corresponding to a PCA model for cell wall-modifying
enzyme activities, cell-wall fractions, calcium content and flesh firmness in ‘Fuji
Kiku-8’ apples (HARV; harvest).
216
CAPÍTULO XIV
Ortiz A, Vendrell M, Lara I.

Softening and cell wall metabolism in late-season peach in response to
controlled atmosphere and 1-MCP treatment.
Journal of Horticultural Science & Biotechnology, 2011, 86, 175–181
187
188
Journal of Horticultural Science & Biotechnology (2011) 86 (2) 175–181
Softening and cell wall metabolism in late-season peach in response

to controlled atmosphere and 1-MCP treatment
By ABEL ORTIZ1, MIQUEL VENDRELL2 and ISABEL LARA1*

1
Departament de Química, Unitat de Postcollita-XaRTA, Universitat de Lleida, Alcalde Rovira
Roure 191, 25198 Lleida, Spain
2
Departament de Genètica Molecular de Plantes, Consorci CSIC-IRTA, Jordi Girona 18-26, 08034
Barcelona, Spain
(e-mail: lara@quimica.udl.cat) (Accepted 4 November 2010)
SUMMARY
Softening of peach (Prunus persica L. Batsch) fruit is generally rapid at ambient temperature, which limits
considerably the commercial life of the produce. Modifications in the cell wall are believed to underlie changes in fruit
firmness and texture. In this work, the effects of controlled atmosphere (CA) storage and 1-methylcyclopropene (1-
MCP) on cell wall composition and on the activities of some cell wall-modifying enzymes were assessed in ‘Tardibelle’
fruit, a late-season, melting-flesh peach cultivar. CA storage and 1-MCP treatment applied separately were effective
in delaying the softening of ‘Tardibelle’ peach during shelf-life at 20ºC following cold storage, apparently through
different mechanisms. CA storage partially inhibited the activities of some pectolytic enzymes and increased the
content of pectic polysaccharides, possibly related to delayed softening. 1-MCP treatment caused increased pectin
methylesterase activity, but other pectolytic enzymes were inhibited, resulting in an enrichment of chelate-soluble
pectin in the cell walls. In contrast, a combined treatment (i.e., CA plus 1-MCP) was unsatisfactory for retaining fruit
firmness, apparently due to the extensive degradation of pectin.
apid rates of softening in peach (Prunus persica L. Girardi et al., 2005), the use of ethylene-antagonising
R Batsch) fruit during the post-harvest period lead to
major losses throughout the marketing chain due to
chemicals could provide an alternative method to delay
fruit softening, in order to extend the storage potential of
over-ripeness, bruising, or increased susceptibility to peach. One such chemical is 1-methylcyclopropene (1-
infection. Refrigerated storage is therefore frequently MCP), which binds competitively to ethylene receptors,
used to preserve fruit firmness and to extend the shelf- thereby blocking ethylene-dependent responses
life potential of such produce. Since peach fruit are (Blankenship and Dole, 2003). Although 1-MCP
particularly prone to chilling injury, refrigerated storage treatments have proved useful in delaying peach
may lead, in some cases, to physiological disorders such softening, in many cases this response was limited to the
as woolliness, internal browning, or flesh bleeding. These period of treatment and a few days afterwards, or upon
can be prevented, in part, by storing fruit under a removal from subsequent cold storage (Mathooko et al.,
controlled atmosphere (CA), particularly in the presence 2001; Liguori et al., 2004; Dal Cin et al., 2006; Ziliotto et al.,
of high levels of CO2 (Lurie, 1992; Zhou et al., 2000). 2008).The purpose of this work was to assess the effects of
Structural and chemical changes in the middle lamella 1-MCP treatment and CA storage, alone or in
and in the primary cell wall, leading to cell separation and combination, on cell wall composition and on different
tissue softening, are thought to underlie the loss in cell wall-modifying enzyme activities related to loss of
firmness during ripening (Brummell and Harpster, 2001; firmness in ‘Tardibelle’ peach fruit. The late harvest date
Brummell, 2006). These chemical modifications include of this cultivar, when the availability of other commercial
the depolymerisation and solubilisation of cell wall peaches is considerably less, makes this cultivar a good
polysaccharides, as well as rearrangements of their subject for the implementation of post-harvest
associations (Rose et al., 1998; Goulao and Oliveira, 2008). technologies to extend its storage potential.
The contribution of each of these alterations to the
softening process varies significantly between species.
While solubilisation is considered to be a universal feature MATERIALS AND METHODS
of pectin modifications, depolymerisation of pectin and Plant material
other cell wall polymers also appears to occur in some Late-season peach (Prunus persica L. Batsch.
species, including peach (Girardi et al., 2005). These ‘Tardibelle’) fruit were picked in a commercial orchard
modifications in cell wall structure are driven mainly by at Torrelameu (Segrià, Spain) at commercial maturity
the co-operative actions of a number of related enzyme (18 September 2006), according to the usual production
activities. Given that some of these enzyme activities are standards in this area (i.e., fruit diameter ≥ 70 mm; 100%
ethylene-dependent (Brummell and Harpster, 2001; red surface). In total, 225 fruit were harvested,
corresponding to approx. 75 kg. A portion of these
*Author for correspondence. samples (25 peaches) were analysed immediately after
176 Cell wall disassembly in peach fruit
harvest. Half of the remaining sample (100 fruit) was (Na2CO3sf), and matrix glycans (hemicelluloses; KOHsf),
treated with 1 µl l–1 1-MCP (SmartFreshTM; Agrofresh respectively. Each fraction was dialysed extensively as
Inc., Valencia, Spain) at 1ºC for 24 h, whereas the above, then filtered through Miracloth, lyophilised, and
remaining 100 fruit were kept untreated at 1ºC. Both weighed. The yields of each fraction were expressed as %
untreated and 1-MCP-treated fruit were then stored for (w/w) CWM.
21 d at 0ºC and 92% relative humidity under air, or in a
CA (3 kPa O2 : 10 kPa CO2), then transferred to 20ºC to Extraction and assay of cell wall-modifying enzyme
simulate commercial shelf-life. Analyses were carried out activities
0 d and 7 d after removal from cold (0ºC) storage. For the extraction of polygalacturonase (exo-PG; EC
3.2.1.67 and endo-PG; EC 3.2.1.15), pectin
Analysis of ethylene production methylesterase (PME; EC 3.1.1.11), pectate lyase (PL;
Ethylene production was measured in three fruit EC 4.2.2.2) and endo-1,4-b-D-glucanase (EGase; EC
from each treatment. Each fruit was kept in a 3.2.1.4) activities, a 10% (w/v) homogenate of peach
respiration jar and aerated continuously with flesh was prepared by homogenising 100 mg of freeze-
humidified air at a rate of 5 l h–1. Samples of the effluent dried flesh tissue in an extraction buffer prepared
air (1.0 ml) were removed with a syringe and injected according to Lohani et al. (2004).
into a gas chromatograph (Model 6890N; Agilent PG activity was determined using apple pectin (degree
Technologies, Madrid, Spain) equipped with a flame of esterification 70 – 75%; Sigma-Aldrich, Steinheim,
ionisation detector and an alumina column (1.5 m 3 3 Germany) as described previously (Pathak and Sanwall,
mm). Analyses were conducted isothermally at 100ºC, 1998), using galacturonic acid (GalUA) as a standard.
with N2 as the carrier gas (at 45 ml min–1), in the One Unit of PG activity was defined as that causing the
presence of air and H2 at flow rates of 400 and 45 ml liberation of 1.0 µmol GalUA min–1.
min–1, respectively. The injector and detector were held PME activity was measured according to Hagerman
at 120ºC and 180ºC, respectively, and the results were and Austin (1986). The reaction mixture contained
expressed as µl C2H4 kg–1 FW h–1. enzyme extract, apple pectin, and bromothymol blue,
prepared as described previously (Alonso et al., 1997).
Determination of flesh firmness One Unit of PME activity was defined as that causing a
Fruit firmness (in N) was measured on opposite sides decrease of 1.0 A620 units min–1.
of 15 fruit per treatment using a hand-held Effegi PL activity was assayed using apple pectin as the
penetrometer (Model FT 327; Milan, Italy) equipped substrate, according to Moran et al. (1968), as modified
with an 8-mm diameter tip. by Lohani et al. (2004). One Unit of PL activity was
defined as that causing an increase of 1.0 A235 units min–1.
Extraction and fractionation of cell wall material To measure EGase activity, carboxymethylcellulose
Samples of flesh tissue (100 g) were taken from six was used as the substrate. The dinitrosalicylic acid
peaches per treatment (two fruit/replicate 3 three method (Miller, 1959) was used to determine the amount
replicates), frozen in liquid nitrogen, freeze-dried, and of reducing sugars released, with glucose used as a
powdered. Weight loss after lyophilisation was consistent standard. One Unit of EGase activity was defined as that
at approx. 85%. Cell wall material (CWM) was extracted causing the release of 1.0 µmol glucose min–1.
in triplicate from lyophilised tissue (3.0 g) according to For the extraction of b-galactosidase (b-Gal; EC
Redgwell et al. (1992), with some modifications. Samples 3.2.1.23) and a-L-arabinofuranosidase (AFase; EC
were homogenised in 20 ml 2:1:1 (w/v/v) phenol:acetic 3.2.1.55) activities, a 10% (w/v) homogenate of peach flesh
acid:water (PAW) for 20 min. After centrifugation of the was prepared by homogenising 100 mg of freeze-dried
homogenate at 4,000 3 g for 45 min at 4ºC, the pellet was flesh tissue in an extraction buffer prepared according to
resuspended in 10 ml Milli-Q water and centrifuged once Vicente et al. (2005). b-Gal and AFase activity assays were
again. The PAW and water-wash supernatants were undertaken on crude extracts, as described by Vicente
combined and dialysed intensively (mol. wt. cut-off 7,000 et al. (2005) and Wei et al. (2010), respectively. One Unit of
Da) for 2 d at 4ºC against 500 ml Milli-Q water. The b-Gal or AFase activity was defined as that resulting in the
dialysate was centrifuged at 4,000 3 g for 45 min at 4ºC liberation of 1.0 µmol p-nitrophenol min–1 from p-
to remove the precipitate formed during dialysis. The nitrophenyl-b-D-galactopyranoside or p-nitrophenyl-a-L-
supernatant (henceforth termed the PAW-soluble arabinofuranoside, respectively.
fraction; PAWsf) was recovered, lyophilised, and weighed. The total protein contents of the crude extracts were
The pellet obtained after PAW extraction and water- determined using the Bradford (1976) method, with
washing was later washed twice in 10 ml 100% (v/v) bovine serum albumin as a standard. All analyses were
acetone, recovered by vacuum-filtration through done in triplicate, and the results were expressed as
Whatman Grade 4 filter paper, lyophilised, and weighed specific activities (Units mg–1 protein).
to determine the yield of CWM. CWM was expressed as
% (w/w) FW. Statistical analysis
For further fractionation, 100 mg CWM from each A multifactorial design with storage atmosphere, 1-
replicate sample was extracted, sequentially, with water, MCP treatment, and shelf-life period as factors was used
0.05 M cyclohexane-trans-1,2-diamine tetra-acetate to analyse the results statistically. All data were tested by
(CDTA), 0.05 M Na2CO3, and 4.0 M KOH, as described analysis of variance (GLM-ANOVA Procedure) using
previously (Selvendran and O’Neill, 1987). This the 2002 SAS System 9.0 programme package (SAS
fractionated the water-soluble pectin (watersf), loosely- Institute, Cary, NC, USA). Means were separated using
bound pectin (CDTAsf), covalently-bound pectin Fisher’s LSD test at P ≤ 0.05.
A. ORTIZ, M. VENDRELL and I. LARA 177
TABLE I TABLE II
Firmness (N) of ‘Tardibelle’ peach fruit after cold storage for 21 d at 0ºC Ethylene production (µl kg–1 FW h–1) by ‘Tardibelle’ peach fruit after
cold storage for 21 d at 0ºC
Shelf-life‡
Air storage CA storage
Treatment Storage Day-0 Day-7 Days at 20ºC
after storage Untreated 1-MCP-treated Untreated 1-MCP-treated
At harvest – 66.2 –
Untreated Air 51.7 C† <5 0 0.83 a† 1.19 a 0.49 a 0.35 a
CA 61.5 Ba 6.3 Bb 1 15.16 a 7.01 b 6.92 b 10.18 ab
2 20.49 a 10.31 b 3.70 c 8.93 b
1-MCP treated Air 68.9 Aa 12.5 Ab 4 7.85 a 8.40 a 3.31 b 7.55 a
CA 56.0 BC <5 5 6.45 bc 19.33 a 3.91 c 8.00 b
†
Values represent the means of 15 replicates. Mean values followed by 7 6.12 bc 13.51 a 3.40 c 7.69 b
different capital letters within the same column are significantly †
Values represent the means of three replicates. Mean values followed
different at P ≤ 0.05 (LSD test). Mean values followed by a different by different lower-case letters within a row are significantly different at
lower-case letters within the same row are significantly different at P ≤ P ≤ 0.05 (LSD test).
0.05 (LSD test). Ethylene production at harvest was 2.0 µl kg–1 FW h–1.
‡
Days at 20ºC following cold storage.
RESULTS AND DISCUSSION treatment, in parallel to increased yields of PAWsf (Table

Fruit firmness (FF) decreased sharply during the shelf- III). However, the changes observed were dependent on
life period at 20ºC, regardless of 1-MCP treatment or the factors considered. CWM yields in air-stored fruit
storage atmosphere. In some cases, FF declined to values were not affected by 1-MCP treatment after either 0 d or
below the level of detection of the device used (Table I). 7 d at 20ºC.Yet this treatment was particularly effective in
For fruit stored in normal air, the application of 1-MCP decreasing the amount of solubilised material in these
resulted in firmer fruit after 0 d and 7 d at 20ºC, in fruit, as indicated by PAWsf yields. When 1-MCP
agreement with reports on other peach cultivars (Liguori treatment was combined with CA storage, CWM
et al., 2004; Girardi et al., 2005). In contrast, 1-MCP amounts were lower than those observed for control fruit,
treatment was not effective in delaying fruit softening although PAWsf yields also decreased. Similarly, untreated
when combined with CA storage, which contradicted samples stored in a CA showed lower PAWsf yields
previous observations for plum (P. salicina Lindl.; Menitti relative to the controls, even though higher amounts of
et al., 2006). This discrepancy is notable, especially in light CWM were observed upon removal from storage, but not
of the observation that CA storage alone also led to after 7 d at 20ºC. Since the highest FF values were found
improved preservation of FF compared to the air-stored for 1-MCP-treated fruit stored in air (Table I), despite
controls (Table I). Higher concentrations of 1-MCP, or similar CWM yields relative to the controls (Table III),
longer exposures, may be needed in order to delay these observations suggest that the chemical composition
softening in CA-stored fruit. Actually, whereas the of cell wall polysaccharides and the nature of the linkages
production of ethylene after storage was partially responsible for their associations, rather than the total
inhibited in CA-stored fruit, it was simply delayed by a amounts of CWM, were the most relevant factors
few days in 1-MCP-treated samples (Table II), in controlling the preservation of FF in ‘Tardibelle’ peach, in
agreement with previous reports (Liu et al., 2005). These agreement with previous work in other cultivars
data are also in accordance with earlier reports that a (Manganaris et al., 2006; Yang et al., 2009).
single dose of 1-MCP had little effect on the expression of Modifications in pectic polymers are thought to be a
those genes related to ethylene biosynthesis and to primary cause of softening and of other changes in the
ethylene responses in peach (Mathooko et al., 2001; Dal textural properties of fruit (Brummell, 2006; Yang et al.,
Cin et al., 2006). These observations led to the hypothesis 2009). Changes in the firmness of peach after storage
that new ethylene receptors could be synthesised within a have been suggested to be related primarily to the
short time in peach fruit, which would require continuous content of pectins bound covalently to the cell wall
or intermittent exposure to 1-MCP for effective (Fishman et al., 1993; Zhang et al., 2010). Significant
suppression of the expression of these genes. differences in the yields of the pectin-enriched fractions
were found in response to the treatments applied in this
Modifications in cell wall composition after storage work (Table IV). CA storage and 1-MCP treatment,
Significant decreases in CWM yields were observed alone and, to a lesser extent, in combination, resulted in
during the shelf-life period at 20ºC, regardless of higher Na2CO3sf values upon removal from cold storage.
TABLE III
Cell wall solubilisation (% FW)§ in ‘Tardibelle’ peach fruit after cold storage for 21 d at 0ºC
Fraction§ Shelf-life‡ Untreated 1-MCP-treated Untreated 1-MCP-treated
CWM 0 1.90 Ba† 1.92 Ba 2.02 Aa 1.59 Ca
7 1.61 Ab 1.53 Ab 1.15 Cb 1.34 Bb
PAWsf 0 0.66 Ab 0.20 Cb 0.16 Cb 0.43 Bb
7 1.19 Aa 0.50 Ca 0.87 Ba 0.91 Ba
†
Values represent the means of three replicates. Mean values followed by a different capital letter within the same row are significantly different
at P ≤ 0.05 (LSD test). Mean values followed by a different lower-case letter within the same column for a given fraction are significantly different at
P ≤ 0.05 (LSD test).
Yields of CWM and PAWsf at harvest were 2.0 and 0.71% (w/w), respectively.
‡
§
Fraction yields were recorded as g 100 g–1 fresh weight (FW).
CWM, insoluble cell wall material; PAWsf , fraction soluble in 2:1:1 (w/v/v) phenol:acetic acid:water.
TABLE IV
Yields (% CWM)§ of various cell wall fractions isolated from cell wall material of ‘Tardibelle’ peach fruit after cold storage for 21 d at 0ºC
Fraction§ At harvest Shelf-life‡ Untreated 1-MCP-treated Untreated 1-MCP-treated
Watersf 5.18 0 3.70 Bb† 2.32 Cb 3.30 Bb 4.58 Ab
7 4.89 Ca 6.41 Ba 7.35 Aa 5.37 Ca
CDTAsf 45.08 0 48.55 Aa 46.84 Aa 43.06 Ba 26.68 Ca
7 46.93 Aa 42.96 Bb 31.46 Cb 27.33 Da
Na2CO3sf 21.96 0 23.76 Ca 27.68 Aa 28.47 Aa 25.62 Ba
7 12.57 Cb 23.63 Ab 17.15 Bb 17.57 Bb
KOHsf 4.38 0 3.05 Ab 3.47 Ab 3.11 Ab 4.24 Ab
7 11.50 Aa 5.50 Ca 9.04 Ba 6.54 Ca
†
at P ≤ 0.05 (LSD test). Mean values followed by a different lower-case letter within the same column for a given fraction are significantly different at
P ≤ 0.05 (LSD test).
‡
§
Fraction yields were recorded as g 100 g–1 insoluble cell wall materials (CWM).
Watersf, fraction soluble in water; CDTAsf, fraction soluble in 0.05 M cyclohexane-trans-1,2-diamine tetra-acetate; Na2CO3sf, fraction soluble in 0.05 M
Na2CO3; KOHsf, fraction soluble in 4.0 M KOH.
Atomic force microscopy analysis of pectins in CA-stored Brummell et al., 2004), which agrees with previous
peach showed a lower frequency of small-width pectin reports on melon (Cucumis melo; Rose et al., 1998).
molecules compared to air-stored fruit (Yang et al., Therefore, the breakdown of hemicellulose may
2006a,b), demonstrating that the degradation of pectin in contribute, preferentially, to the onset of the softening
peach was inhibited under hypoxic conditions. 1-MCP process, while pectin degradation might be related to the
treatments have also been shown to reduce the extensive loss in FF at the more advanced stages of
depolymerisation of pectin in fruits of other species ripening, consistent with the apparent lack of any
(Jeong et al., 2002; Luo, 2007). Yields of Na2CO3sf also relationship between KOHsf yield and firmness (Table
remained higher in 1-MCP-treated and in CA-stored IV). Previous work on ripening in nectarine (P. persica L.
fruit, compared to the untreated controls, after being kept Batsch var. nectarina) fruit showed that a sharp decline
at 20ºC for 7 d (Table IV). 1-MCP treatment was in the yield of the KOHsf cell wall fraction occurred
particularly effective. These data suggest that this cell wall immediately prior to the onset of a melting-like decrease
fraction was preserved and protected in response to both in FF, while no large variations were observed thereafter
treatments, which may be related to the improved (Ortiz et al., 2010).
preservation of FF during shelf-life. However, although
the combined (CA + 1-MCP) treatment also led to higher Modifications in cell wall-modifying enzyme activities
Na2CO3sf yields in comparison to the untreated controls, after storage
no significant differences in FF were found (Table I). The structural changes observed may have been
Similarly, while no differences in the content of Na2CO3- promoted, in part, by the co-ordinated actions of several
soluble pectin were detected between CA-stored and 1- cell wall-modifying enzymes, the temporal pattern of
MCP-treated ‘Tardibelle’ peaches upon removal from which varies widely between species and cultivars
cold storage, the latter showed higher FF values. Thus, FF (Goulao and Oliveira, 2008). PG and PL depolymerise
must also be dependent on the contribution of other cell homogalacturonan chains through the cleavage of a-
wall fractions. Indeed, yields of the CDTA-soluble (1,4)-galacturonan linkages between demethylated
fraction, indicative of non-covalently-bound pectin, were residues, or by b-elimination, respectively. Both enzymes
higher in 1-MCP-treated peaches kept in air compared to act preferentially upon demethylated galacturonosyl
CA-stored fruit (Table IV), and the content of solubilised residues (Bennett and Labavitch, 2008). Therefore PME,
material was also lower in 1-MCP-treated samples, as the enzyme catalysing this demethylation, is considered
indicated by the yields of the water-soluble fraction. to play a major role in fruit softening. In this work, PME
Softening of melting-flesh peach is associated with a large activity in untreated fruit was inhibited by CA storage
increase in the solubilisation of polyuronides, followed by after 0 d and 7 d at 20ºC (Table V), which may have
progressive depolymerisation at the later stages of resulted in fewer sites available for the actions of PG and
ripening (Fishman et al., 1993; Brummell et al., 2004). PL. Because PG and PL activities were also lower than in
Therefore, the changes in CDTAsf may have arisen from a the controls, pectin depolymerisation might have been
redistribution of covalently-bound pectins. It has been prevented, leading to the enhanced preservation of FF in
suggested that only a small portion of the covalently- these fruit (Table I), as observed elsewhere (Manganaris
bound polyuronides becomes water-soluble during fruit et al., 2006). Moreover, the arabinogalactan side-chains of
ripening, while most remain associated with the cell wall pectins are thought to link the rhamnogalacturonan
via ionic bonds to other covalently-bound pectins backbone of pectins to hemicelluloses and to cellulose
(Dawson et al., 1992). (Hwang et al., 1993). Accordingly, the loss of galactosyl
The depolymerisation of matrix glycans is also and other neutral sugar residues from branched pectins
believed to contribute substantially to fruit softening. has been reported to contribute to the solubilisation of
However, in peach fruit, this event is mainly associated pectin (Wakabayashi, 2000; Brummell et al., 2004). b-Gal
with cell division and cell enlargement during fruit and AFase promote fruit softening by disrupting these
growth, and with the initial softening phase in young linkages, and thus increasing the solubility of these
mature fruit, prior to ripening (Wakabayashi, 2000; polymers. These enzymes can also contribute to increased
TABLE V
Specific activities (Units mg–1 protein) of cell wall-modifying enzymes in ‘Tardibelle’ peach fruit after cold storage for 21 d at 0ºC
Specific activity
Enzyme§ at harvest Shelf-life‡ Untreated 1-MCP-treated Untreated 1-MCP-treated
PME 68.69 0 75.94 Cb† 105.31 Ba 40.66 Db 121.82 Aa
7 104.09 Ba 106.11 Ba 66.95 Ca 123.34 Aa
PG 1.35 0 1.69 Aa 1.21 Db 1.35 Ca 1.56 Ba
7 1.72 Aa 1.39 Ca 1.40 Ca 1.53 Ba
PL 1.12 0 1.41 Aa 1.05 Ca 0.95 Cb 1.26 Bb
7 1.36 Aa 1.01 Ca 1.17 Ba 1.37 Aa
b-Gal 0.43 0 0.63 Ab 0.49 Ba 0.43 Bb 0.72 Ab
7 1.16 Aa 0.23 Cb 0.68 Ba 1.17 Aa
AFase 0.074 0 0.086 Aa 0.048 Cb 0.062 Bb 0.078 Aa
7 0.093 Aa 0.068 Ba 0.072 Ba 0.073 Ba
EGase 1.25 0 1.06 Bb 1.30 Ab 1.11 Ba 0.83 Ca
7 1.56 Aa 1.61 Aa 1.24 Ba 0.86 Ca
†
at P ≤ 0.05 (LSD test). Mean values followed by a different lower-case letter within the same column for a given enzyme activity are significantly
different at P ≤ 0.05 (LSD test).
‡
§
PME, pectin methylesterase; PG, polygalacturonase; PL, pectate lyase; b-Gal, b-galactosidase; AFase, a-L-arabinofuranosidase; EGase, endo-1,4-b-
D-glucanase.
porosity of cell walls by removing galactosyl and although it should also be noticed that the PG activity
arabinosyl residues, thereby allowing the access of other assay used in this work (Pathak and Sanwall, 1998) does
enzymes to their sites of action (Brummell, 2006). not permit any differentiation between the endo-PG and
Although no analyses of sugar composition or of the exo-PG activities which lead to the solubilisation or
molecular weight of pectins were undertaken in this depolymerisation of pectin, respectively.
study, recent work on ‘Snow Queen’ nectarine fruit (Ortiz In accordance with the hypothesis of increased cell-to-
et al., 2010), which displays a melting pattern of softening cell adhesion resulting in the improved preservation of
like ‘Tardibelle’, has demonstrated important losses of FF, yields of the CDTA-soluble fraction that is enriched
arabinosyl, galactosyl, and uronic acid residues from the in ionically-bound pectins, were higher in 1-MCP-treated
Na2CO3sf during the softening process. Partial inhibition peaches stored in air than in CA-stored samples (Table
of b-Gal and AFase activities was detected in the CA- IV). In contrast, when 1-MCP treatment was combined
stored samples (Table V), which may have contributed to with CA storage, PME activity increased further (Table
the improved preservation of the integrity of the cell V), but yields of the pectin-enriched fractions were
walls in these samples. For instance, the softening process significantly lower (Table V), probably as a result of the
in apple (Malus 3 domestica Borkh.) fruit has been enhancement in PG and PL activities relative to the
shown to be more closely related to b-Gal and AFase separate treatments (Table V). These changes were
activities than to either PG or PME activity (Wei et al., associated with lower CWM values (Table III), and to a
2010). Similarly, cell wall disassembly during on-tree significant drop in FF, similar to that in control fruit
ripening of ‘Snow Queen’ nectarines was preceded by the (Table I). Moreover, b-Gal and AFase activity levels in
elimination of galactan side-chains, which may have fruit samples submitted to the combined treatment were
facilitated the solubilisation of pectin (Ortiz et al., 2010). similar to those in control fruit. Thus, extensive softening
Anti-sense suppression of a ripening-related tomato in these samples might have arisen from the
(Solanum lycopersicum L.) b-Gal gene reduced fruit depolymerisation of demethylated pectins, facilitated by
softening (Smith et al., 2002), and the loss of galactose easier access to the substrate polymers, due to the higher
from cell walls was significantly lower in the non- porosity of the cell wall caused by the removal of
softening rin and nor mutants of tomato relative to wild- arabinogalactan side-chains.
type tomato (Gross, 1983; 1984). These combined treatment samples also showed the
1-MCP treatment was particularly effective in reducing lowest levels of EGase activity (Table V), suggesting a
the softening of ‘Tardibelle’ peach fruit upon removal minor role for this enzyme in the softening process.
from cold storage in air (Table I). Nevertheless, our data Although EGase is thought to contribute to the
suggest that the mechanisms underlying the preservation depolymerisation of matrix glycans (Lill et al., 1989), it is
of FF in these samples were different from those claimed to be involved in the initiation of processes
operating in CA-stored fruit. As in fruit stored in a CA, b- leading to peach softening rather than in the loss of FF
Gal and AFase activities were inhibited by 1-MCP- during the melting stage (Bonghi et al., 1998; Ortiz et al.,
treatment (Table V). However, upon removal from cold 2010). Therefore, the timing and contribution of the
storage, PME activity was significantly higher than in the breakdown of hemicellulose to the onset of the softening
controls (Table V), and was accompanied by a partial process remains a key issue to be investigated in the
inhibition of PG and PL activities. Thus, our data suggest future.
that increased demethylation of pectin, together with
reduced PG- and PL-catalysed depolymerisation of the
pectin backbone, might have increased the anionic charge CONCLUSIONS
on these polymers and thus their capacity to cross-link Our results suggest that CA storage delayed softening
through calcium bridges (Brummell and Harpster, 2001), in ‘Tardibelle’ peach due to partial inhibition of PME, PG,
PL, b-Gal, and AFase activities, which helped to preserve A. Ortiz was a recipient of an FPU grant from the
higher pectin contents. 1-MCP treatment increased PME Ministerio de Ciencia e Innovación (MICINN) of
activity, and apparently led to an enrichment in chelate- Spain. This work was supported by the Ministerio de
soluble pectin in the cell walls. The combined treatment Ciencia y Tecnología (MCyT), Spain (Project No.
(CA + 1-MCP) resulted in extensive pectin degradation AGL2003-01457), and by Research Contract No.
and thus did not delay fruit softening. This has obvious 2005SGR00630 funded by the Catalan Government.
commercial implications for the post-harvest Partial financial support through the ISAFRUIT
management of ‘Tardibelle’ peach. The reasons why cell Project, funded by the EU under the 6th Framework
wall-modifying enzyme activities were affected Programme of RTD (Contract No. FP6-FOOD–CT-
differently when CA or 1-MCP treatments were applied 2006-016279) is also gratefully acknowledged. The
individually, or in combination, are unclear and deserve authors are indebted to Pilar Sopeña for technical
further work. assistance.
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CAPÍTULO XV
Ortiz A, Sopeña P, Comabella E, Lara I.

Firmness loss and cell wall degradation after air- or CA-storage of ‘Rich
Lady’ peaches.
Acta Horticulturae, en prensa
197
198
Capítulo XV
Firmness loss and cell wall degradation after air- or CA-storage of ‘Rich
Lady’ peaches
A. Ortiz, P. Sopeña, E. Comabella, I. Lara
Unitat de Postcollita-XaRTA, Universitat de Lleida, Rovira Roure 191, 25198 Lleida,
Spain
Keywords: Prunus persica L. Batsch, controlled atmosphere, pectin, β-galactosidase, α-

L-arabinofuranosidase
Abstract
In this work, ‘Rich Lady’ peaches were picked at commercial maturity,
stored at 2ºC and 92% RH under air or CA (3 kPa O2 : 10 kPa CO2) for 3 or 15
days, and subsequently kept in air 1 day at 7ºC to simulate refrigerated transport.
After cold storage, samples were placed at 20ºC, and fruit firmness, cell wall
composition and some cell wall-degrading enzyme activities were analysed 0 and 3
days thereafter. In the light of the results obtained, no significant differences in the
flesh firmness were found between air- and CA-stored ‘Rich Lady’ peaches.
Firmness of these fruit was mainly dependent on the yield of covalently-bound
pectin in the cell wall, rather than total amount of cell wall materials. Among the
enzymes herein assessed, α-L-arabinofuranosidase and β-galactosidase are
suggested to play a key role in the softening process, probably through its capacity to
cleavage covalent linkages between pectin side-chains and other cell wall materials
and thus facilitating solubilisation of this cell wall polysaccharides.
INTRODUCTION
Handling and commercialisation of peach (Prunus persica L. Batsch) fruit are
limited by rapid softening and overall ripening, which results in short shelf life potential.
For this reason, refrigeration is one of the main tools used to extent commercial life of
produce. Nevertheless, cold storage of peaches under inappropriate conditions leads to a
range of chilling-induced disorders, which are manifested by textural disorders such as
woolliness or mealiness and by abnormal flesh colourations such as browning or bleeding
(Lurie and Crisosto, 2005). However, these disorders can be alleviated through storage
under controlled atmosphere (CA), especially under high CO2 levels (Crisosto et al.,
1999; Zhou et al., 2000).
It is generally assumed that firmness loss of fruits is the result of ripening-related
modifications in the cell wall, although current information in this regard is inconclusive.
The purpose of this work was to study changes in fruit firmness and cell wall metabolism
induced by CA-storage of peaches in order better understand this ripening-related event
and assist reaching improved postharvest handling procedures of produce.
Plant Material
Peach (Prunus persica L. Batsch) fruit of the melting cultivar ‘Rich Lady’ were
picked at a commercial orchard in Aitona (Segrià, NE Spain) at commercial maturity
according to the usual standards in the producing area (diameter ≥ 70 mm; 100% red
199
Capítulo XV
surface). After harvest, samples were stored at 2ºC and 92% RH under regular air or CA
(3 kPa O2: 10 kPa CO2) for 3 or 15 days, and subsequently kept in air 1 day at 7ºC in
order to simulate refrigerated transport (henceforth, 3+1 and 15+1 fruit, respectively).
After cold storage, samples were placed at 20ºC, and analyses were carried out 0 and 3
days thereafter.
Firmness determination
Once the skin tissue from two opposite sides of 20 fruit per sample was pared,
flesh firmness measurements were carried out using a hand-held Effegi penetrometer
equipped with an 8-mm diameter probe with a convex tip. Results are given as N.
Extraction, Fractionation and Analysis of Cell Wall Materials

Samples of flesh tissue were taken from six peaches per treatment (2 fruit/replicate
× 3 replicates), frozen in liquid nitrogen, freeze-dried, and powdered. Weight loss after
lyophilisation was consistently around 82%. Cell wall materials (CWM) were extracted
from lyophilised tissue (3 g) according to Redgwell et al. (1992). Samples were
homogenised in 20 mL phenol:acetic acid:water (2:1:1, w/v/v) (PAW) for 20 min. After
centrifugation at 4000 × g and 4ºC for 45 min, the pellet was resuspended in 10 mL water
and centrifuged again. The PAW and water wash supernatants were combined and
intensively dialysed (mol wt. cut-off 7000) for 2 days against Milli-Q water at 4ºC. The
dialysate was centrifuged at 4000 × g and 4 ºC for 45 min to sediment out the precipitate
formed during the dialysis. The supernatant (henceforth, PAW-soluble fraction; PAWsf)
was recovered, lyophilised and weighed. The pellet obtained after PAW extraction and
water wash was subsequently washed twice in acetone, recovered by vacuum-filtration
through Whatman grade 4 paper filters, lyophilised and weighed to determine yield of
CWM, expressed as % (w/w) FW.
For further fractionation, CWM (100 mg) from each replicate were extracted
sequentially with water, 0.05 M cyclohexane-trans-1,2-diamine tetra-acetate (CDTA),
0.05 M Na2CO3, and 4 M KOH as described previously (Selvendran and O’Neill, 1987),
in order to fractionate water-soluble pectin, loosely-bound pectin, covalently-bound
pectin and matrix glycans (hemicelluloses), respectively. Each fraction was dialysed (mol
wt. cut-off 7000) for 2 days against Milli-Q water at 4 ºC, filtered through Miracloth,
lyophilised and weighed. Yields are expressed as % (w/w) CWM.
Enzyme Extraction and Assay

For the extraction of polygalacturonase (exo-PG; EC 3.2.1.67 and endo-PG; EC
3.2.1.15), pectinmethylesterase (PME; EC 3.1.1.11), pectate lyase (PL; EC 4.2.2.2) and
endo-1,4-β-D-glucanase (EGase; EC 3.2.1.4), a 10% (w/v) flesh homogenate was
prepared by homogenising 100 mg of freeze-dried flesh tissue in an extraction buffer
prepared according to Lohani et al. (2004). PG activity was determined on apple pectin
(d.e. 70-75%) as described previously (Pathak and Sanwall, 1998), with galacturonic acid
(GalUA) as a standard. One unit (U) of PG activity was defined as the liberation of 1
µmol of GalUA min-1. PME activity was measured as described by Hagerman and Austin
(1986), and one unit (U) of this enzyme activity was defined as the decrease of one unit of
A620 min-1. PL activity was assayed according to Moran et al. (1968) as modified by
Lohani et al. (2004). One unit (U) of PL activity was defined as the increase of one unit of
A235 min-1. For the assessment of EGase activity, the DNS method (Miller, 1959), with
carboxymethylcellulose as the assay substrate, was used to determine the amount of
200
Capítulo XV
reducing sugars released, with glucose as a standard. One unit (U) of EGase activity was
defined as the release of 1 µmol of glucose min-1.
For the extraction of β-galactosidase (β-Gal; EC 3.2.1.23) and β-xylosidase (β-
Xyl; EC 3.2.1.37) activities, a 10% (w/v) flesh homogenate was prepared by
homogenising 100 mg of freeze-dried flesh tissue in an extraction buffer prepared
according to Vicente et al. (2005), and activity assays were undertaken in the crude
extract as described therein. One unit (U) of β-Gal or β-Xyl activity was defined as the
liberation of 1 µmol of p-nitrophenol min-1 from p-nitrophenyl-β-D-galactopyranoside or
1 nmol of p-nitrophenyl-β-D-xylopyranoside, respectively. The extraction and assay of α-
L-arabinofuranosidase (AFase; EC 3.2.1.55) was carried out according to Wei et al.
(2010). One unit (U) of AFase was defined as the liberation of 1 nmol of p-nitrophenol
min-1 from p-nitrophenyl-α-L-arabinofuranoside.
All assays were done in triplicate, and total protein content in the extracts was
determined with the Bradford method (1976), using BSA as a standard, and results were
expressed as specific activity (U mg protein-1).
Results were treated for multiple comparisons by analysis of variance (GLM-
ANOVA), followed by the least significant difference (LSD) Fisher’s test at p<0.05. To
provide a general visualisation of all the information contained in the data set obtained,
principal component analysis (PCA) was used. Partial least square regression (PLSR) was
used as a predictive method to relate a matrix of several dependent variables (Y) to a set
of explanatory variables (X) in a single estimation procedure. Samples were labelled XYZ,
where each digit takes values 1, 2 or 3 as described in Table 1.

Regardless of storage length or shelf life period, CA storage was not able to delay
softening of ‘Rich Lady’ peaches stored at 2ºC (Table 2). Although surprising, this results
are in agreement with previous works in which little differences in the flesh firmness of
peaches were found after CA-storage (Zhou et al., 2000; Girardi et al., 2005). On the late-
season cultivar ‘Tardibelle’, however, CA-storage improved retention of firmness after
cold-storage (Ortiz et al., 2010). Therefore, a main factor having a role on the
effectiveness of CA may be linked to the cultivar itself. Probably, late-season cultivars,
which are characterised by a slower ripening process, might be more reactive to CA
technology in terms of firmness preservation.
Because ripening-related softening occurs mainly as a result of cell wall disruption
(Goulao and Oliveira, 2008), CWM were extracted from fruit cortex and fractionated
sequentially. In general, the amount of CWM isolated from ‘Rich Lady’ peaches was
higher in CA-stored fruit (Table 3). So, these results were not in accordance with that of
firmness (Table 2) during the post-storage period. However, previous work (Brummell et
al., 2004; Peña and Carpita, 2004) has pointed out that modifications in composition and
linkages between polysaccharides, rather than the total amount of CWM, may be critical
traits influencing fruit softening. In an attempt to characterise samples, a PCA model was
developed with firmness, cell wall fractions and the enzyme activities herein assessed. In
this model, which was capable to explain up to 78% of total variability (Fig. 1), firmness
appeared strongly linked to the yields of Na2CO3sf , in turn indicative of the amount of
covalently-bound pectic polymers in the wall. The dependence of changes in fruit
firmness on the yield of this cell wall fraction was also supported by the data shown in
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Capítulo XV
Table 3, in which, similarly to fruit firmness, no differences in this value throughout shelf
life was observed between air- and CA-stored ‘Rich Lady’ peaches. Therefore, firmness
was mainly dependent on the content of covalently-bound pectin, rather than total amount
of CWM in the flesh tissue. Moreover, CA-storage of fruit helped preserving the amount
of non-covalently bound pectin, which was also well related to fruit firmness in the PCA
model, although this fact apparently did not induced changes in the ripening-related
softening process of cold-stored ‘Rich Lady’ peaches. The amount of covalently-bound
pectins dramatically decreased throughout the shelf life period, regardless of storage
atmosphere and period (Table 3).
It is generally accepted that most changes in the cell wall during ripening of fruits
are driven by a large number of enzyme activities. For this reason, a PLSR model was
developed, in which both firmness and yields of cell wall fractions were introduced as the
dependent variables, and a set of cell wall-modifying enzyme activities as the potentially
explanatory variables. A clear inverse relationship between both AFase and β-Gal
activities and firmness, which in turn remained clearly link to the yield of covalently-
bound pectins, was observed (Fig. 2). This suggest this two enzyme activities as having a
critical role in the solubilisation of pectins, probably through the removal of side-chain
polymers attached to the rhamnogalacturonans backbones, responsible to the linkage
between pectin polymers to other CWM such as matrix glycans. Probably explaining
changes in firmness values (Table 2) and the content of covalently-bound pectins (Table
3), both enzyme activities noticeably increased along the shelf life (Table 4), whereas
were little (AFase) or not affected (β-Gal) by storage atmosphere. Therefore, the lack of
effectiveness of CA-storage on preserving fruit firmness may have arisen from its failure
to inhibit or reduce the activity of this glycosidic enzymes.
ACKNOWLEDGEMENTS
Innovación (MICINN) of Spain. This work was supported by the European Comission
(ISAFRUIT project; contract no. FP6-FOOD-CT-2006-016279).
Literature cited
Bradford, M. 1976. A rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.
72:248-254.
Brummell, D.A., Dal Cin, V., Crisosto, C.H. and Labavitch, J.M. 2004. Cell wall
metabolism during maturation, ripening and senescence of peach fruit. J. Exp. Bot.
55:2029-2039.
Crisosto, C.H., Mitchell, F.G. and Ju, Z. 1999. Susceptibility to chilling injury of peach,
nectarine, and plum cultivars grown in California. Hortscience 34:1116-1118.
Girardi, C.L., Corrent, A.R., Lucchetta, L., Zanuzo, M.R., da Costa, T.S., Brackmann, A.,
Twyman, R.M., Nora, F.R., Nora, L., Silva, J.A. and Rombaldi, C.V. 2005. Effect of
ethylene, intermittent warming and controlled atmosphere on postharvest quality and
the occurrence of woolliness in peach (Prunus persica cv. Chiripá) during cold
storage. Postharvest Biol. Technol. 38:25-33.
Goulao, L.F. and Oliveira, C.M. 2008. Cell wall modifications during fruit ripening: when
a fruit is not the fruit. Trends Food Sci. Tech. 19:4-25.
Hagerman, A.E. and Austin, P.J. 1986. Continuous spectrophotometric assay for plant
pectin methyl-esterase. J. Agric. Food Chem. 34:440-444.
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Lohani, S., Trivedi, P.K. and Nath, P. 2004. Changes in activities of cell wall hydrolases
Postarvest Biol. Technol. 31:119-126.
Lurie, S., and Crisosto, C.H. 2005. Chilling injury in peach and nectarine. Postharvest
Biol. Technol. 31:195-208
Miller, G.L. 1959. Use of dinitrosalycilic acid reagent for determination of reducing
sugar. Anal. Chem. 31:426-428.
Moran, F., Nasuno, S. and Starr, M.P. 1998. Extracellular and intracellular
polygalacturonic acid trans-eliminase of Erwinia carotovora. Arch. Biochem.
Biophys. 123: 298-306.
Ortiz, A., Vendrell, M. and Lara, I. 2010. Modifications in cell wall composition after
storage of 1-MCP-treated peach fruit. Acta Hort. 858:221-224.
Pathak, N. and Sanwall, G.G. 1998. Multiple forms of polygalacturonase from banana
fruits. Phytochemistry 48:249-255.
Peña, M.J. and Carpita, N.C. 2004. Loss of highly branched arabinans and debranching of
rhamnogalacturonan I accompany loss of firm texture and cell separation during
prolonged storage of apple. Plant Physiol. 135:1305-1313.
Redgwell, R.J. Melton, L.D. and Brasch, D.J. 1992. Cell wall dissolution in ripening
material. p. 25-153. In: D. Glick (ed.), Methods of Biochemical Analysis, vol. 32.
John Wiley Interscience, New York.
Vicente, A.R., Costa, M.L., Martínez, G.A., Chaves, A.R. and Civello, P.M. 2005. Effect
of heat treatments on cell wall degradation and softening in strawberry fruit.
Wei, J., Ma, F., Shi, S., Qi, X., Zhu, X. and Yuan, J. 2010. Changes and postharvest
regulation of activity and gene expression of enzymes related to cell wall degradation
in ripening apple fruit. Postharvest Biol. Technol. 56:147-154.
Zhou, H.W., Lurie, S., Lers, A., Khatchitski, A., Sonego, L. and Ben Arie, R. 2000.
Delayed storage and controlled atmosphere storage of nectarines: two strategies to
prevent woolliness. Postharvest Biol. Technol. 18:133-141.
Tables
Table 1. Meaning of X-, Y- and Z-values for the sample generic labels.
1 2
a
X Air 3:10
Yb 3+1 15+1
c
Z 0 3
a
Storage atmosphere conditions (O2:CO2).
b
Storage period at 2ºC (days) + 1 day at 7ºC.
c
Shelf life period (days at 20ºC).
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Capítulo XV
Table 2. Firmness (N) of ‘Rich Lady’ peaches after storage at 2ºC.
Atmosphere Storage period + days at 20ºC

3+1a 15+1
0 3 0 3
Air 43.20 Aa 6.33 Ab 42.27 Aa <5
CA 44.48 Aa 6.45 Ab 43.98 Aa <5
Values represent means of twenty replicates. Means within the same row followed by
different letters are significantly different at p<0.05 (LSD test). Means within the same
column followed by different capital letters are significantly different at p<0.05 (LSD
test).
a
3+1: 3 days at 2ºC + 1 day at 7ºC; 15+1: 15 days at 2ºC + 1 day at 7ºC.
Table 3. Yield of insoluble CWM and PAWsf (% FW) and of CWM fractions (% CWM)
isolated from ‘Rich Lady’ peaches after storage at 2ºC.
Fraction Atmosphere Storage period + days at 20ºC

3+1a 15+1
0 3 0 3
CWM Air 1.256 Ba 1.273 Aa 1.315 Ba 1.270 Ba
CA 1.600 Aa 1.417 Aa 1.554 Aa 1.524 Aa
PAWsf Air 0.103 Ab 0.319 Ba 0.095 Bb 0.394 Aa
CA 0.092 Ab 0.514 Aa 0.224 Ab 0.439 Aa
Wsf Air 3.202 Bc 3.786 Bc 5.503 Ab 6.896 Aa
CA 5.824 Ab 5.918 Ab 6.101 Ab 7.471 Aa
CDTAsf Air 31.694 Ba 25.503 Bb 30.580 Aa 21.737 Bc
CA 44.775 Aa 30.767 Ab 32.379 Ab 24.064 Ac
Na2CO3sf Air 26.959 Ab 18.658 Ac 27.519 Aa 16.743 Ac
CA 27.626 Aa 18.853 Ab 25.693 Aa 16.899 Ab
KOHsf Air 4.954 Bb 5.264 Ab 9.617 Aa 9.525 Aa
CA 7.270 Aa 5.064 Ab 8.700 Aa 5.280 Bb
Values represent means of three replicates. Means within the same row followed by
different letters are significantly different at p<0.05 (LSD test). For each fraction, means
within the same column followed by different capital letters are significantly different at
p<0.05 (LSD test).
a
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Capítulo XV
Table 4. AFase and β-Gal specific activities (U mg protein-1) in the flesh tissue of ‘Rich
Lady’ peaches after storage at 2ºC.
Enzyme Atmosphere Storage period + days at 20ºC

3+1a 15+1
0 3 0 3
AFase Air 16.53 Ab 22.67 Aa 17.78 Ab 22.20 Aa
CA 14.77 Ab 18.57 Ba 14.49 Bb 19.59 Aa
β-Gal Air 0.70 Ac 0.77 Ab 0.80 Ab 0.94 Aa

CA 0.67 Ac 0.73 Ab 0.78 Ab 0.89 Aa
Values represent means of three replicates. Means within the same row followed by
different letters are significantly different at p<0.05 (LSD test). For each enzyme activity,
means within the same column followed by different capital letters are significantly
different at p<0.05 (LSD test).
a
Figures
Fig. 1. Biplot of PC1 vs. PC2 corresponding to a PCA model for firmness, cell wall
fractions and cell wall-modifying enzyme activities in cold-stored ‘Rich Lady’
peaches.
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Capítulo XV
Fig. 2. Loadings plots of PC1 versus PC2 corresponding to a PLSR model for firmness
and yields of CWM fractions and firmness (Y variables) versus cell wall
modifying enzyme activites (X variables) in cold-stored ‘Rich Lady’ peaches.
206
CAPÍTULO XVI
Ortiz A, Echeverría G, Graell J, Lara I.

Sensory evaluation of calcium-dipped ‘Fuji Kiku-8’ and ‘Golden
Reinders’ apples.
Acta Horticulturae, 2010, 877, 799–806
207
Capítulo XVII
208
Sensory Evaluation of Calcium-Dipped ‘Fuji Kiku-8’ and ‘Golden
Reinders’ Apples
Àrea de Post-Collita, XaRTA, UdL-IRTA
Rovira Roure 191, 25198
Lleida
Spain
Keywords: Malus domestica, calcium dips, cold storage, consumer acceptability,

trained panel, quality
Abstract
‘Fuji Kiku-8’ and ‘Golden Reinders’ apples, harvested at commercial
maturity, were dipped in calcium chloride (2% w/v) and stored at 1°C and 92% RH
for 4 or 7 months under air. The calcium concentration was higher in treated fruit,
showing that the treatment was effective in introducing calcium into the tissues. A
number of sensory attributes were assessed after cold storage plus 7 days at 20°C by
a panel of 9 trained judges. Simultaneously, a panel of 45 consumers was asked to
evaluate overall acceptance of samples according to a hedonic scale (1-9). Firmness,
soluble solids content and titratable acidity were instrumentally measured in order
to assess a possible correlation between parameters instrumentally and sensorily
evaluated. For both cultivars, calcium treatment resulted in higher crispness and
perceived hardness. Multivariate analysis of data revealed that perception of both
crispness and sweetness were the parameters most related to consumer acceptability.
Besides, perception of mealiness, which was reduced by calcium applications,
appeared to be detrimental for overall consumer acceptability. In consequence,
results suggest calcium treatments to be a good practice to enhance sensory quality
of cold-stored apples. On the other hand, weak correlations were found between
sensorily and instrumentally measured quality parameters, thus indicating the
usefulness of sensory tests to assess the effects of postharvest procedures on the
quality of produce.
INTRODUCTION
Currently, calcium treatment of apples (Malus domestica Borkh.) is often carried
out in order to maintain firmness, as well as to reduce the incidence of some physiological
disorders or to prevent losses due to decay-causing pathogens. These treatments also meet
with the increasing awareness of consumers on the benefits of incorporating calcium in
the diet. Fruit consumers demand produce exhibiting good organoleptic and health-
promoting characteristics, in addition to intrinsic traits related to safety. Consequently, the
demand for high sensory quality highlights the importance of combining the traditional,
instrumental methodologies used to monitor quality, focused largely on fruit firmness,
titratable acidity and soluble solids content, with the use of consumers’ tests.
The proposed quality assessment guiding principle is complex, since consumers
are themselves highly variable with regard to their response to a particular foodstuff,
normally as a result of genetic or social conditions (reviewed in Wyllie, 2008). However,
the use of sensory tests addressed to panels comprised of trained assessors can partially
avoid such a great variability between consumers. Hence, a combination of a trained
panel and consumer tests for postharvest quality evaluation represents a source of better
understanding of behaviour and expectation of final consumers, and allow producers to
truthfully judge the benefits and drawbacks of submitting certain produce to a particular
postharvest procedure. To date, little research has focused on the effects of the different
postharvest handling practices on fresh fruit. It is therefore convenient and required to
widen current knowledge on the modifications driven by manipulation and preservation
conditions on those factors contributing to sensory quality of fruit. Thus, the aim of this
Proc. 6th International Postharvest Symposium

Eds.: M. Erkan and U. Aksoy 799
work was to assess how postharvest calcium dips affect the sensory profile and the
consumers’ acceptability of cold-stored ‘Fuji Kiku-8’ and ‘Golden Reinders’ apples,
taking into account that the effects of postharvest treatments on sensory characteristics
can vary between species and also between cultivars. In order to establish possible
correlations with sensory attributes, fruit were also instrumentally assessed for firmness,
soluble solids content and titratable acidity.
Plant Material, Calcium Treatment and Storage Conditions

‘Fuji Kiku-8’ and ‘Golden Reinders’ apple fruit were harvested at commercial
maturity, from 5 and 6 year-old trees, respectively, at the IRTA-Experimental Station of
Lleida (NE Spain). For each cultivar and immediately after harvest, fruit were randomly
divided into two lots and one of them was dipped in a 2% (w/v) CaCl2 solution at ambient
temperature for 5 minutes. After treatment, treated and untreated apples were stored at
1ºC and 92% RH in air. Fruit samples were removed after 4 or 7 months of cold storage,
and placed at 20ºC to simulate commercial shelf-life. All analyses were carried out 7 days
thereafter.
Calcium Content Determination
For each cultivar, freeze-dried pulp tissue obtained from 5 fruit per batch (calcium
treatment storage period) was submitted to acid digestion and analysed by inductively
coupled plasma emission spectroscopy (ICP-OES). Analyses were carried out in triplicate
and results were expressed as mg 100 g FW-1.
Analysis of Standard Quality Parameters
For both ‘Fuji Kiku-8’ and ‘Golden Reinders’, fifteen apples per batch (calcium
treatment storage period) were used individually for the analysis of flesh firmness,
soluble solids content (SSC) and titratable acidity (TA). Flesh firmness was measured on
opposite sides of each fruit with a penetrometer (Effegi, Milan, Italy) fitted with an
11-mm diameter plunger tip; results were expressed in N. SSC and TA were measured in
juice pressed from the whole fruit. SSC was determined with a hand-held refractometer
(Atago, Tokyo, Japan), and results were expressed as % sucrose in an equivalent solution.
TA was determined by titrating 10 ml of juice with 0.1 N NaOH to pH 8.1 using 1% (v/v)
phenolphthalein; results were given as g malic acid/L.
Consumer Acceptability Assessment and Sensory Evaluation

Two analysis sessions for each cultivar (after all of both storage periods
considered) were conducted as described elsewhere (Echeverría et al., 2008a). A group
comprised of 45 consumers was asked to score apple slices according to a nine-point
hedonic scale (1, dislike extremely; 9, like extremely). Consumers were volunteers from
the staff working at the UdL-IRTA and students from the University of Lleida.
Simultaneously to the consumer acceptability evaluation, a nine-member trained panel
experienced in descriptive analysis of fruit was convened. Panellists were presented with
randomised three-digit coded samples asked to evaluate sweetness, sourness, crispness,
hardness, juiciness and overall flavour. Similarly to Echeverría et al. (2008b), attributes
were expressed on 15-cm unstructured line scales and quantified by measuring the
distance of the mark from the origin.
For each cultivar, a multifactorial design with storage period and calcium
treatment as factors was used to statistically analyse the results. All data were tested by
analysis of variance (GLM-ANOVA procedure) with the SAS program package (SAS
Institute, Cary, NC, USA, 1988). Means were separated with the LSD test at p 0.05.
Partial least square regression (PLSR) was used to investigate sensory attributes
800
predicting consumer acceptability. Unscrambler vers. 7.6 was used for developing the
PLSR model, which were validated by the full cross-procedure. For multivariate analysis,
samples were coded as XYZ, taking values of 1 or 2 as indicated in Table 1.

Dipping of apples resulted in significantly increased calcium content in the pulp of
fruit (Table 2). Average increases in calcium content were 25 and 53% in ‘Fuji Kiku-8’
and ‘Golden Reinders’ fruit, respectively, thus indicating that the chosen treatment
procedure was helpful in incorporating calcium into fruit tissues during postharvest cold
storage. Calcium treatments were also reflected in the standard quality parameters, as
observed in Table 3. As in a previous report (Glenn and Poovaiah, 1990), calcium-treated
fruit showed higher firmness and TA values after long-term (7 months) storage periods,
whereas after medium-term storage periods (4 months), these effects were only apparent
in ‘Fuji Kiku-8’ apples. Although an important criterion concerning eating quality of
apples, especially regarding firmness (Harker et al., 2008), description of standard quality
parameters alone is not enough to achieve a complete quality evaluation of produce, the
assessment of sensory attributes by trained panels being a useful, complementary
procedure to reach a better knowledge of the effects of a particular postharvest treatment,
on consumers’ acceptability.
A PLSR model was developed including both ‘Fuji Kiku-8’ and ‘Golden
Reinders’ samples in order to establish possible relationships between a number of
sensory attributes (X variables), as tested by a trained panel, and consumer acceptability
(Y variables), evaluated as explained above. The model was capable of explaining 76% of
total variability. As observed in the score plot corresponding to this model (Fig. 1A),
calcium-treated samples were located on the right of the PC1 axis (50% of explained
variability), while untreated samples appeared on the left, thus suggesting an influence of
calcium dips on perceived sensory attributes of fruit. Actually, calcium-treated apples
were observed to be characterised by higher acceptability scores, since this variable was
also located on the right of the loading plot (Fig. 1B). These results are partially in
agreement with previous report (Abbott et al., 2000) that calcium-treated ‘Golden
Delicious’ apples were preferred by consumers.
The developed PLSR model showed that consumer acceptability was well related
to the perception of crispness and sweetness. In contrast, perception of mealiness and
sourness were negatively related to acceptability (Fig. 1B). The positive relationship
found between crispness and consumer acceptability has been reported recently. From a
test comprising a large number of participants, Péneau et al. (2006) rated crispness as the
second most important factor for acceptability of apples, followed by the perception of
juiciness. Calcium dips enhanced perception of hardness and crispness on both ‘Fuji
Kiku-8’ and ‘Golden Reinders’ apples after cold storage, as well as perception of
juiciness in the case of fruit stored for 7 months (Table 4), thus supporting the idea of
calcium dips being useful for the enhancement of consumer acceptability of apple fruit.
Moreover, dipping fruit in CaCl2 solutions resulted in lower perception of mealiness after
long-term storage periods. Contrarily to crispness and juiciness, perception of mealiness
in apples has been suggested to be associated to low consumer acceptability (Péneau et
al., 2007), thus in agreement with observations herein (Fig. 1B). Mealy texture in apple
fruit is frequently induced at room temperature after cold-storage in air (De Smedt et al.,
2002). It is thought to result from an imbalance between the relative strength of the cell
wall and the strength of the middle lamella (De Smedt et al., 1998). Since calcium has
been reported to be effective in preventing the disassembly of the middle lamella in the
flesh tissue of fruit (Chardonnet et al., 2003), dipping fruit in CaCl2 solutions might be
able to lessen perception of mealiness when consuming apples, in accordance with our
results (Table 4).
In an attempt to establish possible relationships between instrumental quality
parameters (X variables) and the assessed sensory attributes (Y variables), a PLSR model
was developed. With the only exception of perceived hardness in relation to
801
penetrometrically measured firmness, weak correlations were found between sensory and
instrumental quality parameters (data not shown), thus supporting the usefulness of
sensory tests to achieve a better knowledge of effects of postharvest procedures on the
quality of produce.
In conclusion, calcium applications have been shown to be a good practice to
enhance sensory quality of cold-stored ‘Fuji Kiku-8’ and ‘Golden Reinders’ apples,
possibly as a consequence of higher perception of crispness and sweetness and the
reduced development of mealiness, maybe resulting from calcium-induced prevention of
disassembly of the middle lamella and cell wall structure during the postharvest life of
produce.
ACKNOWLEDGEMENTS
A. Ortiz is the recipient of an FPU grant from the Ministerio de Ciencia e
P. Sopeña and A. Latorre are acknowledged for technical assistance.
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802
Tables
Table 1. Codes of X-, Y- and Z- values for the generic sample labels.
1 2
Xa ‘Fuji Kiku-8’ ‘Golden Reinders’
Yb 4 7
Zc untreated 2
a
Cultivar
b
Storage period at 1ºC + 7 days at 20ºC
c
Calcium treatment (% CaCl2, w/v)
Table 2. Calcium contenta (mg 100g FW-1) in pulp tissue of ‘Fuji Kiku-8’ and ‘Golden
Reinders’ apples after 7 days at 20ºC following cold storage in air for 4 or 7 months.
4 months 7 months
untreated CaCl2-treated untreated CaCl2-treated
Fuji Kiku-8 2.9 C 3.9 B 4.1 B 4.8 A
Golden Reinders 2.8 C 3.4 B 2.9 BC 5.4 A
a
Values represent means of 3 replicates. Means in the same row followed by different letters
are significantly different at p 0.05 (LSD test).
Table 3. Standard quality parametersa of ‘Fuji Kiku-8’ and ‘Golden Reinders’ apples
stored in cold air for 4 or 7 months at 1ºC + 7 days at 20ºC.
4 months 7 months
Fuji Kiku-8
Firmness (N) 64.26 B 70.23 A 55.30 C 63.11 B
SSC (%) 16.81 A 16.86 A 16.05 B 16.63 AB
TA (mg malic acid/L) 1.50 B 2.13 A 0.91 D 1.16 C
Golden Reinders
Firmness (N) 58.00 A 56.32 A 49.24 C 53.29 B
SSC (%) 15.55 A 14.94 AB 15.14 AB 14.37 C
TA (mg malic acid/L) 2.47 A 2.34 A 1.42 C 1.89 B
a
Values represent means 15 individual fruits. Means in the same row followed by different
letters are significantly different at p 0.05 (LSD test).
803
Table 4. Sensory attributesa of ‘Fuji Kiku-8’ and ‘Golden Reinders’ apples stored in cold
air for 4 or 7 months at 1ºC + 7 days at 20ºC.
4 months 7 months
Fuji Kiku-8
Sweetness 8.14 A 8.19 A 8.10 A 8.75 A
Sourness 3.81 A 4.38 A 3.70 A 4.42 A
Crispness 5.71 B 7.21 A 5.10 B 7.80 A
Hardness 5.06 B 6.19 A 3.75 B 6.60 A
Juiciness 7.50 A 7.92 A 5.25 B 7.08 A
Mealiness 6.25 B 5.63 BC 8.92 A 3.75 C
Flavour 7.19 A 6.81 A 6.00 A 7.42 A
Golden Reinders
Sweetness 7.13 B 7.94 AB 8.56 A 7.50 AB
Sourness 4.14 A 4.69 A 2.06 B 2.44 B
Crispness 3.88 B 6.36 A 4.33 B 6.14 A
Hardness 5.07 B 6.79 A 3.64 C 4.83 B
Juiciness 5.00 AB 6.36 A 3.24 C 4.81 B
Mealiness 5.38 A 3.94 B 5.44 A 4.00 B
Flavour 6.25 AB 7.00 A 5.29 B 6.14 AB
a
Values represent means of 9 individual ratings on a 15-cm unstructured line scale. Means in the same row
followed by different letters are significantly different at p 0.05 (LSD test).
804
Figures
Fig. 1. Scores (A) and loadings (B) plot of PC1 vs. PC2 corresponding to a PLSR model
for consumer acceptability (Y variable) vs. sensory attributes (X variables) in ‘Fuji
Kiku-8’ and ‘Golden Reinders’ apple fruit stored in cold air for 4 or 7 months at
1ºC + 7 days at 20ºC. Samples are coded as indicated in Table 1.
805
806
CAPÍTULO XVII
Ortiz A, Lara I, López M. L., Graell J, Echeverría G.

The influence of storage period and temperature on consumer
acceptance and sensory properties of ‘Big Top®’ nectarines.
Acta Horticulturae, en prensa
217
Capítulo XVII
218
Capítulo XVII
The influence of storage period and temperature on consumer

acceptance and sensory properties of ‘Big Top®’ nectarines
A. Ortiz, I. Lara, M.L. López, J. Graell, G. Echeverría*
Institut de Recerca i Tecnologia Agroalimentàries (IRTA) and Universitat de Lleida
(UdL), Alcalde Rovira Roure, 191, 25198 Lleida (Spain)
Keywords: Consumer acceptance, Prunus persica, sensory attributes, standard quality

parameters, storage temperature
Abstract
'Big Top®' nectarines undergo rapid ripening and therefore have a limited
post-harvest life. Yet they have to be firm enough to withstand handling in the
packing house and during marketing (transport, storage and retail display). For this
reason, they are frequently harvested at early ripening stages and are consequently
not perceived as sufficiently satisfactory by the final consumer.
The objective of this study was to characterise the influence of maturity stage,
storage period and storage temperature on fruit acceptance and sensory attributes
as scored by a consumer panel (n=44). At harvest time, 'Big Top®' nectarines were
graded in three categories according to the IAD index (index of absorbance difference
= A670-A720) based on Vis spectroscopy. Fruit in the second maturity class defined
based on IAD were stored at 20, 10, 4 or -1 ºC for up to 49 days depending on storage
temperature. If at removal the fruit was of low quality (firmness ≤ 5 N and presence
of fungal decay) the experiment (for that temperature) was ended. In that case, fruit
were removed from the storage chamber and not analyzed or assessed sensorily.
Fruit quality parameters were instrumentally measured by quantifying soluble
solids concentration, titratable acidity and flesh firmness. After keeping at 20 ºC for
4 days, 'Big Top®' nectarines had the highest overall acceptance as scored by the
consumer panel. For longer storage periods, no significant differences among
storage temperatures were observed, except for fruit stored for 7 weeks, for which
higher consumer acceptance was found for fruit stored at -1 ºC in comparison to
those stored at 4 ºC. Results also suggested that higher acceptance scores were
associated mainly to more intense perception of the characteristic “peach” flavour.
INTRODUCTION
Total peach production in Spain in 2008 was more than one million tons; 36% of
which were nectarines. An important increase in nectarine production since 1992 to 2008
was observed because the percentage increased from 23 to 36%. ‘Big Top®’ nectarines
accounted for 25% of this production in Spain in 2008, 30% of which being produced in
Catalonia, thus being the predominant cultivar. This high production is the result of the
fact that ‘Big Top®’ nectarines are very appreciated by consumers due to its excellent
sweet taste. However, nectarines submitted to long cold storage are subjected to suffer
serious quality decay, detected at final consumer level, which symptoms are the lack of
flavor associated with unripe fruit (Gomez and Ledbeter, 1997), the development of
chilling injury (Fernádez-Trujillo et al., 1998), evidenced as mealiness and internal
browning (Lurie and Crisosto, 2005; Crisosto, 2006) among the most commons.
219
Capítulo XVII
Nectarine quality has always been measured in terms of the attributes of the products,
chiefly through evaluating the physical and chemical properties that better explain
maturation and ripening. Flesh firmness, ground color, soluble solids content (SSC), and
titratable acidity (TA) are the commonly used parameters for defining fruit quality
because they provide a common language among researchers, industry, and consumers
(Abbott, 1999). When quality is measured from the consumers’ perspective, these
parameters do not always match to what consumers take into account for deciding
whether the quality is good or poor. Thus, it is important to define quality on the basis of
consumer expectations (Predieri et al., 2006).
In this work, special attention was paid to the study of temperature and storage
period dependence in nectarine in order to know how these factors can affect standard
quality parameters, some sensory attributes and consequently consumer acceptance.
Plant Material
Yellow-fleshed nectarines (Prunus persica L. Batsch var. nectarine cv. ‘Big
Top®’) were harvested in a commercial orchard located in Massalcoreig (Segrià, NE
Spain) and selected for uniformity of size and absence of defects. A commercial
equipment (C2005d, Minolta, Valencia, Spain) was used to pre-sort nectarine non-
destructively by Vis spectroscopy of fruit according to the IAD index at harvest (index of
absorbance difference = A670-A720) (Ziosi et al., 2008). Following sorting the fruit was
classified into three different categories by decreasing values of the IAD (M1: IAD 0.17-
0.15; M2: IAD 0.14-0.12; M3: IAD 0.11-0.09) and stored at 20, 10, 4 or -1 ºC for up to 49
days depending on storage temperature. Standard quality parameters (flesh firmness, SSC
and TA) were measured for fruit from the three classes, while acceptability and sensory
attributes described by a consumer panel were scored only for fruit from the second
maturity class. All of them were assessed at harvest and periodically throughout storage.
Analysis of Standard Quality Parameters

Fifteen nectarines at harvest and per combination of factors (storage temperature ×
storage period) were used individually for the analysis of flesh firmness, SSC and TA.
Flesh firmness was measured on opposite sides of each fruit with a penetrometer (Effegi,
Milan, Italy) fitted with an 8-mm diameter plunger tip; results were expressed in N. SSC
and TA were measured in juice pressed from the whole fruit. SSC was determined with a
hand-held refractometer (Atago, Tokyo, Japan), and results were expressed as ºBrix. TA
was determined by titrating 10 mL of juice with 0.1 N NaOH to pH 8.1 using 1% (v/v)
phenolphthalein; results were given as g malic acid/L.
Consumer Acceptance and Sensory attributes Assessment

Sensory evaluations were conducted as described elsewhere (Echeverría et al.,
2008). A group comprised of 44 consumers was asked to score nectarine slices according
to a nine-point hedonic scale (1, dislike extremely; 9, like extremely). Consumers were
volunteers from the staff working at the UdL-IRTA and students from the University of
Lleida. In addition, consumers were asked to score the intensity of sweetness, sourness,
firmness, juiciness and flavour according to a five-point hedonic scale (1, very low
intensity; 5, very high intensity).
220
Capítulo XVII
A multifactorial design with storage period and temperature as factors was used to
statistically analyse the results. All data were tested by analysis of variance (GLM-
ANOVA procedure) with the SAS/STAT 9.1 procedures (SAS Institute Inc., 2004).
Means were separated with the LSD test at p ≤ 0.05.
Standard Quality Parameters at Harvest

At harvest, ‘Big Top®’ nectarines graded according to IAD showed significant
differences in flesh firmness and SSC. However, no differences in TA were recorded, as
shown in Table 1.
Consumer Acceptance and Sensory Attributes at Harvest

Differences between classes of fruit at harvest were detected by consumers.
Consumer’s acceptability improved with maturity stage at harvest. Fruit from the third
class obtained the highest acceptability score (Table 2). Otherwise, it is important to point
out that all the classes obtained consumer scores of 6 or higher, therefore meaning that all
fruit was well accepted. Consumers also detected differences in sweetness, hardness,
juiciness and flavour. Fruit from the second and third maturity classes obtained higher
scores for these attributes, except for hardness attribute, which showed higher value in
fruit from the first class. This hardness decline as more advanced maturity stage was
concurrent with a significant increase in juiciness. No differences in sourness were found
by consumer.
Standard Quality Parameters during Storage

Fig. 1 illustrates the softening rates of ‘Big Top®’ nectarines during storage at
different temperatures. The results clearly show that nectarines softened faster at higher
temperatures. Fruit stored at -1°C did not soften at all throughout the storage period,
although they decayed by fungal infection. In general, the firmness loss rates among
different maturity classes did not exhibit significant differences, probably due to the high
variability in physiological maturity. M3 fruits, which were most mature, softened faster
compared to less mature fruits (M1 and M2), most clearly seen at the highest temperature
(20°C). The other quality parameters (SSC and TA) were not different for the different
maturity classes during storage. SSC did not change much during storage at the four
different temperatures and TA slightly decreased (data not shown). These no clear
differences observed for TA or SSC among maturity stages, as defined by the IAD index,
might suggest firmness is a more reliable index of storage potential for ‘Big Top’
nectarine.
Consumer Acceptance and Sensory Attributes during Storage

Fig. 2 shows consumer acceptance scores for M2 fruit. On the one hand, it can be
observed that consumer acceptance scores were higher for fruits stored at higher
temperatures and on the other hand that overall degree of liking remained acceptable
regardless of temperature.
Regarding sensory attributes (Table 3), it can be seen that the higher the storage
temperature, the higher the sweetness, the flavour and the juiciness are. In contrast, the
higher the temperature, the lower the hardness scores are. On the subject of the effect of
221
Capítulo XVII
storage period on sensory attributes, it can be said that, in general, the longer the period,
the higher the juiciness and the flavour scores and the lower the firmness scores are,
except for fruits stored at -1 ºC. Storage period didn’t have any effect on sensory
attributes of fruit stored at -1 ºC. Moreover, no clear influence of temperature and period
on sourness was found.
Comparing Fig. 2 and Table 3, with the aim of elucidating the influence of sensory
attributes on consumer acceptance, it can be observed that higher consumer acceptance
scores were associated mainly to higher perception of sweetness, of juiciness and of
peach flavour by consumers.
ACKNOWLEDGEMENTS
Innovación (MICINN) of Spain. This work was supported by the European Comission
(ISAFRUIT project; contract no. FP6-FOOD-CT-2006-016279). The authors are indebted
to Mrs. A. Latorre and Mrs. P. Sopeña for technical assistance.
Literature Cited
Abbott, J. 1999. Quality measurement of fruits and vegetables. Postharvest Biol. Technol.
15: 207-225.
Crisosto, C. 2006. Peach quality and postharvest technology. Acta Hort. 713: 479-487.
Echeverría, G., Graell, J., Lara, I. and López, M.L. 2008. Physicochemical measurements
in ‘Mondial Gala®’ apples stored at different atmospheres: Influence on consumer
acceptability. Postharvest Biol. Technol. 50: 135-144.
Fernández-Trujillo, J.P., Cano, A. and Artés, F. 1998. Physiological changes in peaches
related to chilling injury and ripening. Postharvest Biol. Technol. 13: 109-119.
Gomez, E. and Ledbetter, C. 1997. Development of volatile compounds during fruit
maturation: characterization of apricot and plum x apricot hybrids. J. Sci. Food Agric.
74: 541-546.
Lurie, S. and Crisosto, C. 2005. Chilling injury in peach and nectarine. Postharvest Biol.
Technol. 37: 195-208.
Predieri, S., Ragazzini, P. and Rondelli, R. 2006. Sensory evaluation and peach fruit
quality. Acta Hort. 713: 429-434.
SAS Institute, Inc. 2004. SAS/STAT© 9.1 User’s Guide. Cary, NC: SAS Institute Inc.
Ziosi, V., Noferini, M., Fiori, G., Tadiello, A., Trainotti, L., Casadoro, G. and Costa, G.
2008. A new index based on vis spectroscopy to characterize the progression of
ripening in peach fruit. Postharvest Biol Technol 49: 319-329.
Tables
Table 1. Changes in firmness, SSC and TA of ‘Big Top®’ nectarines at harvest. M1, M2
and M3 represent fruits classes according IAD (see Plant Material section).
M1 M2 M3
Firmness (N) 5.97 B 6.47 AB 6.91 A
SSC (ºBrix) 2.32 B 2.91 A 3.15 A
TA (g/L malic acid) 3.06 A 3.03 A 2.50 A
222
Capítulo XVII
Table 2. Changes in consumer liking degree and sensory attributes of ‘Big Top®’
nectarines at harvest. M1, M2 and M3 represent fruits with different IAD values (see
Plant Material section).
M1 M2 M3
Overall degree of liking 5.97 B 6.47 AB 6.91 A
Sweetness 2.32 B 2.91 A 3.15 A
Sourness 3.06 A 3.03 A 2.50 A
Hardness 4.00 A 3.41 B 3.12 B
Juiciness 2.44 B 3.35 A 3.35 A
Nectarine flavour 2.52 B 3.00 AB 3.38 A
Table 3. Changes in sensory attributes scores for fruit from M2 during storage of ‘Big
Top®’ nectarines at four different constant temperatures.
Sensory Storage Storage days

attributes temperatures 4 7 14 21 28 35
Sweetness - 1 ºC 2.6 bc 3.0 b 2.7 b 2.5 b 2.6 a 2.5 a
4 ºC 3.0 ab 2.7 bc 2.6 b 3.4 a 3.0 a 2.9 a
10 ºC 2.5 c 2.5 c 3.1 a 3.4 a
20 ºC 3.3 a 3.7 a
Sourness - 1 ºC 3.4 a 2.4 b 3.2 a 3.2 a 3.3 a 2.6 a
4 ºC 2.5 b 2.9 b 2.7 a 2.3 b 2.7 b 2.7 a
10 ºC 2.9 b 3.4 a 2.9 a 2.7 a
20 ºC 2.9 b 2.6 b
Hardness - 1 ºC 3.6 a 3.6 a 3.8 a 4.4 a 4.0 a 4.3 a
4 ºC 3.4 a 3.4 a 3.5 a 2.8 b 2.6 b 2.4 b
10 ºC 3.8 a 3.8 a 2.3 b 1.6 c
20 ºC 2.6 b 1.4 b
Juiciness - 1 ºC 2.7 b 2.4 c 2.8 b 2.1 c 2.4 b 2.1 b
4 ºC 3.0 b 2.9 b 2.8 b 3.2 b 3.2 a 3.4 a
10 ºC 2.6 b 2.6 b 3.6 a 4.2 a
20 ºC 3.8 a 4.4 a
Flavour - 1 ºC 2.8 b 2.7 b 2.9 a 2.7 b 3.0 a 2.5 a
4 ºC 2.6 b 3.0 b 2.8 a 2.9 ab 2.7 a 2.6 a
10 ºC 2.5 b 2.7 b 3.3 a 3.4 a
20 ºC 3.4 a 3.9 a
223
Capítulo XVII
Figures
Firmness(N) of fruits stored at -1 ºC Firmness (N) of fruitsstored at 4 ºC

70 70
60 60
50 50
40 40
M1 M1
30 30
M2 M2
20 M3 20 M3
10 10
0 0
0 7 14 21 28 35 42 49 0 4 8 12 16 20 24 28 32 36 40 44 48
Firmness (N) of fruits stored at 10 ºC Firmness (N) of fruits stored at 20 ºC

70 70
60 60
50 50
40 40
M1 M1
30 30
M2 M2
20 M3 20 M3
10 10
0 0
0 3 6 9 12 15 18 21 24 27 0 1 2 3 4 5 6 7 8 9 10 11
Fig. 1. Flesh firmness changes during storage of ‘Big Top®’ nectarines at four different
constant temperatures.
9 -1 ºC
4 ºC
8 10 ºC
a 20 ºC
Sensory acceptance
7 a a
(hedonic scale: 1-9)
b ab b bb aa
ab b
b b a a
6 a a
1
4 7 14 21 28 35
Storage days
Fig. 2. Consumer acceptance scores for fruit from M2 during storage of ‘Big Top®’
nectarines at four different constant temperatures.
224
DISCUSIÓN GENERAL
225
226
Discusión general
1. PRODUCCIÓN DE COMPUESTOS VOLÁTILES AROMÁTICOS DURANTE

LA MADURACIÓN EN CAMPO.
1.1. Manzana (Malus × domestica Borkh, cv. ‘Golden Reinders’ y ‘Fuji Kiku-8’).
En esta Tesis se han estudiado dos variedades de manzana importantes

económicamente en el área frutícola del Segrià, caracterizadas por un potencial de
comportamiento post-cosecha muy distinto. En ambos casos, se observaron importantes
modificaciones en la composición de la fracción volátil emitida a lo largo de la maduración.
Para la variedad ‘Golden Reinders’ se realizaron muestreos periódicamente a lo largo de siete
semanas (97-146 ddpf), detectándose hasta un total de 28 ésteres y 8 alcoholes (cap. I, Tabla
2) según la fecha. De entre todos los compuestos detectados, siete ésteres (acetato de etilo, 2-
metilbutanoato de etilo, acetato de hexilo, propanoato de hexilo, butanoato de hexilo, 2-
metilbutanoato de hexilo y hexanoato de hexilo) fueron los que se emitieron a mayores
concentraciones. Según Plotto et al. (1999, 2000), la preeminencia cuantitativa de los ésteres
de hexilo confiere a la fracción volátil el ‘aroma a manzana’ característico. Sin embargo, para
manzanas ‘Golden Delicious’, variedad de la que procede ‘Golden Reinders’, se ha observado
que los compuestos mayoritarios son el acetato de butilo, el butanoato de butilo y el
hexanoato de butilo (Song y Bangerth, 1996). Esta diferencia entre ambos cultivares indica la
importancia del factor varietal en el perfil aromático en manzanas, a pesar de sus similitudes
fenotípicas.
La influencia del estado de madurez sobre la producción de compuestos volátiles fue

diferente en cada caso, y pudieron definirse tres estados de madurez fisiológica para los frutos
bajo estudio según la emisión de determinados compuestos. Los frutos menos maduros (97-
111 ddpf) se caracterizaron por una mayor producción de acetato de propilo, acetato de 2-
metilpropilo, butanoato de etilo y 2-metilbutanoato de etilo (cap. I, Fig. 1). En un estado de
madurez más avanzado (118-132 ddpf), el perfil aromático estuvo caracterizado por mayores
emisiones de acetato de pentilo, propanoato de 2-metilbutilo, 2-metilpropanoato de 2-
metilbutilo y 2-metilbutanoato de 2-metilbutilo, mientras que la fracción volátil de los frutos
maduros comercialmente (139-146 ddpf) se caracterizó por una mayor producción del resto
de los compuestos detectados, especialmente de acetato de 2-metilbutilo. Esta diferenciación
indica, así, que la emisión de compuestos volátiles es un índice importante que informa del
estado fisiológico de madurez de los frutos y que podría utilizarse como un índice de madurez
227
Discusión general
no destructivo, como se ha sugerido anteriormente (Dirinck y Schamp, 1989). Por ejemplo, se

ha propuesto el acetato de 2-metilbutilo como posible indicador del estado de madurez en
frutos de la variedad ‘Bisbee Delicious’ (Mattheis et al., 1991), que también podría ser útil
para otras variedades como ‘Pink Lady®’ o ‘Mondial Gala’, en las que la emisión de este
compuesto fue significativamente superior en madurez comercial (Villatoro et al., 2008; Lara
et al., 2008).
En relación a la variedad ‘Fuji Kiku-8’, el estudio fue más prolongado, realizándose

un seguimiento de más de 10 semanas (130-202 ddpf) durante su maduración en campo. En
función de la fecha de muestreo, se detectaron un total de 31 ésteres y 7 alcoholes (cap. II,
Tabla 2). Los estados de madurez más tempranos se caracterizaron por mayores emisiones de
determinados compuestos (octanoato de etilo, butanoato de 2-metilpropilo, 2-metilpropanoato
de 2-metilbutilo y 2-metilbutanoato de 2-metilbutilo), algunos de los cuales no fueron
detectados en el estadio de madurez comercial de los frutos. El resto de los compuestos
identificados, en general, aumentaron progresivamente con el estado de madurez de las
muestras, siendo algunos de ellos detectados únicamente en los estadios más avanzados. De
entre ellos, los acetatos de butilo, de 2-metilbutilo y de hexilo, y dos ésteres de butilo
(hexanoato y 2-metilbutanoato) fueron los que mostraron un mayor incremento de emisión en
madurez comercial, por lo que podrían utilizarse asimismo como indicadores no destructivos
del estado de madurez. Con la excepción del acetato de butilo, la mayor emisión de estos
compuestos en madurez comercial también fue observada en ‘Golden Reinders’ (cap. I, Tabla
2). El acetato de 2-metilbutilo, por ejemplo, ha sido identificado como un compuesto volátil
relevante en manzanas ‘Fuji’ (Fellman et al., 2000; Echeverría et al., 2004a, b),
recomendándose por ello como un buen indicador para estimar la fecha óptima de
recolección. Como se ha indicado anteriormente, este mismo compuesto podría utilizarse
también como indicador de madurez comercial en manzanas ‘Golden Reinders’.
A pesar de la importancia de los niveles de emisión, la contribución de un compuesto

al aroma de un fruto depende también de su umbral de percepción olfativa (Baldwin et al.,
2000), de tal modo que sólo los compuestos que presentan valores positivos para el logaritmo
de las unidades de olor (concentración de un compuesto dividida por su umbral de percepción
olfativa) se consideran como ‘compuestos impacto’ que contribuyen al aroma, mientras que el
resto contribuyen únicamente proporcionando lo que se denomina ‘notas de fondo’. Así, el 2-
metilbutanoato de etilo, el acetato de 2-metilbutilo, el 2-metilbutanoato de butilo y tres ésteres
228
Discusión general
de hexilo (acetato, propanoato y 2-metilbutanoato) (cap. I, Tabla 2), serían los compuestos
que más contribuyen al perfil aromático de ‘Golden Reinders’ en el momento de cosecha
comercial, confiriendo en conjunto al fruto notas verdes y afrutadas (Mehinagic et al., 2006).
Algunos de estos compuestos ya se habían identificado anteriormente como contribuidores
importantes al aroma de diferentes variedades del grupo ‘Golden’ (López et al., 2000; Rizzolo
et al., 2006a). Por otro lado, 12 ésteres (los acetatos de 2-metilpropilo, de butilo, de 2-
metilbutilo, de pentilo y de hexilo, los butanoatos de metilo y de etilo, los 2-metilbutanoatos
de butilo y de hexilo, y los propanoatos de butilo, de 2-metilbutilo y de hexilo) serían los
compuestos que más contribuirían al aroma de manzanas ‘Fuji Kiku-8’ alrededor del
momento de cosecha (cap. II, Tabla 3).
En el caso de la variedad ‘Fuji Kiku-8’, el estudio de maduración en campo se llevó a

cabo en una campaña (2008) diferente a la correspondiente a los estudios de
frigoconservación (2006/2007). El efecto campaña, y en concreto las temperaturas y
pluviometrías registradas durante la maduración de los frutos, es también un factor importante
que determina en parte la composición de fracción volátil emitida (Yamada et al., 1994;
Mattheis et al., 1995; Motosugi et al., 1995; Echeverría et al., 2004a), tanto cuantitativa como
cualitativamente. Así, los compuestos que, en estados próximos a la madurez comercial,
contribuyeron al aroma en manzanas ‘Fuji Kiku-8’ durante la campaña 2006/2007 difirieron
respecto al año 2008. En dicha campaña, los compuestos que mostraron mayor contribución al
aroma de los frutos fueron los acetatos de butilo, de 2-metilbutilo y de hexilo, los propanoatos
de tert-butilo, de butilo, de 2-metilbutilo, de pentilo y de hexilo, los butanoatos de metilo y de
etilo, y los 2-metilbutanoatos de etilo, de butilo y de hexilo (cap. IV, Tabla 2).
Está demostrado que la mayor parte de los compuestos volátiles emitidos por
manzanas se sintetizan en la piel de los frutos (Rudell et al., 2002). Sin embargo, la actividad
AAT en este tejido durante la maduración en campo de ambas variedades de manzana no
mostró una relación aparente con la emisión de ésteres volátiles. Esta falta de correlación
directa se ha observado anteriormente en otras variedades como ‘Fuji Nagafu-6’ (Echeverría
et al., 2004b), ‘Mondial Gala’ (Lara et al., 2008) o ‘Pink Lady®’ (Villatoro et al., 2008). Estos
datos sugieren que la actividad AAT, aunque obviamente necesaria, no es el factor principal
que modula la biosíntesis de ésteres, lo que indica claramente que la disponibilidad de
substratos, dependiente de otros enzimas situados más atrás en la ruta de producción, es un
aspecto clave que controla la composición final de la fracción volátil emitida por los frutos.
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Discusión general
Para ‘Golden Reinders’, los resultados muestran una fuerte relación entre las
actividades PDC y ADH, especialmente en la piel de los frutos, y la producción final de
ésteres (cap. I, Fig. 2B). Esto sugiere que el incremento en la producción de ésteres durante la
maduración de los frutos sería consecuencia de un mayor suministro de alcoholes y acil-CoAs
para la actividad AAT. De hecho, en este experimento se observó que, en general, mayores
concentraciones de alcoholes se tradujeron en mayores producciones de la correspondiente
familia de ésteres durante la maduración (cap. I, Fig. 1B). A su vez, la producción de
alcoholes como el 2-metil-1-butanol, el 1-pentanol o el 2-etil-1-hexanol mostró en líneas
generales una buena relación con la actividad ADH en la piel del fruto (cap. I, Tabla 2). La
producción de ésteres de tipo acetato se mostró claramente dependiente, asimismo, del
contenido de acetaldehído en los frutos (cap. I, Fig. 5), lo que igualmente subraya la
importancia del suministro de los correspondientes acil-CoAs para la acción catalítica de
AAT.
La relevancia del suministro de precursores, por encima de la de la actividad AAT en

sí, se vio apoyada también por los resultados del experimento correspondiente a la variedad
‘Fuji Kiku-8’. La importancia de este factor tiene también interesantes aplicaciones prácticas
que se pusieron de manifiesto en los resultados de este estudio. Así, el seguimiento de la
emisión de compuestos volátiles durante la maduración se complementó en este caso con el
estudio de los efectos de aplicaciones precosecha de calcio sobre este atributo. Los resultados
demuestran que la mayoría de los compuestos que contribuyen al aroma en esta variedad
alrededor de la fecha de cosecha comercial se emitieron a mayor concentración en los frutos
tratados (cap. II, Tabla 3). Con la excepción del acetato de pentilo, la emisión del resto de
acetatos aumentó significativamente en respuesta al tratamiento (cap. II, Tabla 2B), en
paralelo al aumento en la concentración de su precursor acetaldehído (cap. II, Fig. 1A). A su
vez, otros precursores necesarios para la biosíntesis de ésteres como los alcoholes 1-butanol,
2-metil-1-butanol y 1-hexanol también aumentaron su emisión en frutos tratados con cloruro
cálcico (cap. II, Tabla 4), lo que de nuevo pone de manifiesto la relevancia de este factor para
la biosíntesis de ésteres, y por tanto para la composición de la fracción volátil emitida por los
frutos. Exceptuando el etanol, la concentración de los alcoholes detectados estuvo asociada
claramente con las actividades PDC y ADH, lo cual subraya la importancia de las mismas
para la composición de los ésteres emitidos. Esta relación explica también el efecto favorable
del tratamiento con cloruro cálcico sobre la emisión de ésteres en fechas próximas a la
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Discusión general
madurez comercial, puesto que las actividades PDC y ADH fueron más altas en frutos
tratados (cap. II, Tabla 5). PDC y ADH son enzimas asociados al metabolismo anaeróbico en
los frutos y, por tanto, el incremento de su actividad en respuesta al tratamiento podría ser
consecuencia de una menor concentración de O2 en los tejidos. De hecho, se ha demostrado
que el tratamiento de manzanas con cloruro cálcico aumenta el gradiente de concentración de
O2 a través de los tejidos de los frutos, debido principalmente a una mayor dificultad a la
difusión del mismo al medio (Rajapakse et al., 1992), provocando por tanto un aumento en la
concentración de CO2 (Hewett y Thompson, 1992; Saftner et al., 1998), que induciría un
incremento en la actividad de estos enzimas, tal como se ha observado en este trabajo. Estas
observaciones tienen una clara relevancia para el sector frutícola, ya que sugieren un método
sencillo y económico para mejorar la calidad gustativa del fruto. Dado que la mayor parte de
la producción de manzana está destinada a su frigoconservación durante períodos
relativamente largos, los frutos suelen cosecharse antes de alcanzar la plena madurez con
objeto de mejorar su resistencia a la manipulación y su potencial de conservación. No
obstante, puesto que la emisión de compuestos volátiles depende del estado fisiológico del
fruto, dicha práctica resulta con frecuencia en una calidad gustativa deficiente que no satisface
al consumidor. Los resultados de este trabajo indican, por el contrario, que las aplicaciones de
cloruro de calcio en pre-cosecha permitirían un retraso en la pérdida de firmeza y acidez,
favorable para el almacenamiento del producto (cap. II, Tabla 1), evitando la merma en su
calidad aromática, o incluso mejorándola (cap. II, Tabla 3).
1.2. Melocotón (Prunus persica L. Batsch, cv. ‘Rich Lady’).
Se observaron igualmente importantes modificaciones en la composición de la

fracción volátil emitida a lo largo de la maduración de melocotones ‘Rich Lady’. Se realizó
un total de seis muestreos, a intervalos de tres o cuatro días, a lo largo de tres semanas
alrededor de la cosecha comercial, detectándose hasta 34 compuestos volátiles, 28 de los
cuales ésteres. A diferencia de las manzanas, entre los ésteres se identificaron en este caso
algunos de tipo lactona (γ-hexalactona, γ-octalactona, δ-decalactona y γ-dodecalactona),
previamente descritos como importantes contribuidores al aroma típico de los melocotones y
las nectarinas (Engel et al., 1988a,b; Aubert et al., 2003; Rizzolo et al., 2006b). Los
compuestos mayoritarios cuantitativamente fueron cuatro ésteres de hexilo (acetato, 2-
metilbutanoato, butanoato y propanoato), dos ésteres de butilo (hexanoato y 2-metilbutanoato)
y el acetato de 2-metilbutilo. Sin embargo, al igual que en manzana, la emisión de los mismos
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Discusión general
a lo largo del período experimental no siguió un patrón uniforme. En concordancia con

observaciones anteriores (Visai y Vanoli, 1997), los compuestos de tipo lactona se emitieron a
mayores concentraciones en el estadio de madurez comercial, lo que explicaría su asociación
con el aroma típico de estos frutos. Sin embargo, la emisión de compuestos volátiles de otros
tipos no siguió en todos los casos este mismo patrón. La mayor emisión de compuestos
volátiles totales, así como de ésteres volátiles totales, no coincidió con la fecha de madurez
comercial. Esto no está en contradicción con una mejor calidad aromática de los frutos en la
fecha de madurez comercial, pues como se ha mencionado para el caso de manzanas, mayor
emisión de volátiles no implica necesariamente una mayor contribución de éstos al aroma
global del fruto, ya que también se debe tener en cuenta el umbral de percepción olfativa de
cada compuesto.
También de manera similar a lo observado en muestras de manzana, las

modificaciones en la actividad AAT no estuvieron relacionadas directamente con la
producción de ésteres (cap. III, Fig. 2). Las actividades enzimáticas responsables del
suministro de precursores para la reacción de esterificación catalizada por AAT también
demostraron ser un factor de capital importancia para la composición de la fracción volátil
emitida por los frutos. Tal y como se ha comentado anteriormente, los compuestos
predominantes cuantitativamente en la fracción volátil emitida por frutos de melocotón ‘Rich
Lady’ fueron distintos ésteres de hexilo y de butilo, cuya emisión mostró una fuerte
correlación con la actividad LOX en la pulpa (cap. III, Fig. 3). La actividad ADH en la pulpa
también mostró un papel significativo en la composición de la fracción volátil, ya que
presentó alta correlación con la producción de alcoholes (cap. III, Fig. 4), a su vez precursores
inmediatos para la biosíntesis de ésteres volátiles. Estos resultados confirman la relevancia de
la actividad de enzimas situados previamente a AAT en la ruta biosintética para las
características de la fracción volátil emitida por los frutos.
Resultados de un experimento adicional en melocotón ‘Tardibelle’ (Tabla D1), una

variedad tardía que muestra un patrón similar de pérdida de firmeza, confirman algunos
aspectos observados en ‘Rich Lady’. En este caso, se recolectaron los frutos en tres fechas
distintas (18, 21 y 27 de septiembre de 2006). Se detectó un total de 35 compuestos volátiles,
entre los cuales 26 ésteres que incluían dos lactonas. La mayor emisión total de ésteres no se
correspondió con el estadio de madurez más avanzado, lo que muestra la importancia de la
fecha de cosecha para asegurar una buena calidad aromática. Las familias mayoritarias
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Discusión general
cuantitativamente en la fracción volátil emitida por estos melocotones fueron los ésteres de
acetato y de etilo, y en ambos casos la emisión máxima correspondió a la segunda cosecha
(Tabla D1). La emisión máxima de ésteres se correspondió con la máxima disponibilidad de
alcoholes, mientras que la concentración de acetaldehído aumentó entre la segunda y la
tercera cosecha, lo que sugiere que el suministro del substrato alcohol puede ser más limitante
que el del acil CoA para la reacción de esterificación.
Tabla D1. Emisión de compuestos volátiles (µg kg-1) en melocotón ‘Tardibelle’ (año 2006)
en tres fechas de cosecha (datos no publicados).
18 Septiembre 21 Septiembre 27 Septiembre

Ésteres totales 33,62 b 82,28 a 48,48 b
Alcoholes totales 8,73 c 46,51 a 20,48 b
Acetaldehído 0,56 b 1,45 a 1,77 a
Ésteres de acetato 24,84 c 65,57 a 42,58 b
Ésteres de metilo 2,70 b 2,83 b 6,07 a
Ésteres de etilo 13,58 c 59,36 a 30,15 b
Ésteres de 2-metilbutilo 3,10 a 2,95 a 1,33 b
Ésteres de butilo 2,88 a 2,88 a 1,57 b
Ésteres de hexilo 4,22 b 6,06 a 5,71 a
Los valores representan la media de cuatro (ésteres y alcoholes) o 15 (acetaldehído) repeticiones. Letras
diferentes en una misma fila denotan diferencias significativas (P < 0’05; test LSD).
De manera similar a lo ya comentado para otros frutos, las variaciones en la actividad

AAT (Tabla D2) no estuvieron de acuerdo con los datos sobre emisión de ésteres volátiles, lo
que nuevamente refuerza la idea de la importancia del suministro de precursores.
Tabla D2. Actividad alcohol o-aciltransferasa (U mg-1 proteína) en melocotón ‘Tardibelle’

(año 2006) en tres fechas de cosecha (datos no publicados).
Tejido 18 Septiembre 21 Septiembre 27 Septiembre

AAT Piel 0,185 b 0,156 b 0,267 a
Pulpa 0,365 a 0,198 b 0,178 b
Los valores representan la media de tres repeticiones. Letras diferentes en una misma fila denotan diferencias
significativas (P < 0’05; test LSD).
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Discusión general
2. PRODUCCIÓN DE COMPUESTOS VOLÁTILES AROMÁTICOS EN

RESPUESTA A LA MANIPULACIÓN POST-COSECHA.
La producción de compuestos volátiles en manzana está altamente influenciada por la

tecnología de almacenamiento de los frutos. La mayor parte de la producción de manzana de
cada campaña se conserva en atmósferas controladas, que mantienen la calidad estándar del
producto y minimizan la aparición de ciertos desórdenes fisiológicos (Brackmann et al., 1994;
Graell et al, 1997; Graell y Ortiz, 2003; Kupferman, 2003). En este sentido, los resultados
obtenidos en la presente Tesis confirmaron la mejora de estos aspectos gracias al uso de este
tipo de tecnología, tanto para ‘Fuji Kiku-8’ (cap. IV, Tabla 4) como para ‘Golden Reinders’
(cap. V, Tabla 6). Sin embargo, múltiples estudios han demostrado que las atmósferas
controladas (especialmente aquéllas con bajo nivel de oxígeno) provocan generalmente una
reducción en la producción total de compuestos volátiles, así como alteraciones en la
composición de la fracción volátil emitida (Mattheis et al., 1995, 2005; Fellman et al., 2000;
Plotto et al., 2000; Dixon y Hewett, 2001; Rudell et al., 2002; Argenta et al., 2004; Echeverría
et al., 2004a; Lara et al., 2006, 2007; Villatoro et al., 2009; Raffo et al., 2009). En el presente
trabajo, se observó asimismo que la utilización de atmósferas controladas para la
conservación de manzanas ‘Fuji Kiku-8’ y ‘Golden Reinders’ disminuyó la concentración de
volátiles en frutos mantenidos durante 7 días a 20 ºC tras la frigoconservación. Sin embargo,
se completó el estudio evaluando también el efecto de los tratamientos postcosecha con
cloruro cálcico sobre la fracción volátil emitida, un aspecto sobre el que apenas existían
trabajos previos.
Tal y como se ha comentado en el apartado 1.1. de esta discusión, los compuestos que
contribuyeron al aroma de manzana ‘Fuji Kiku-8’ en la campaña 2006/2007 fueron los
acetatos de butilo, de 2-metilbutilo y de hexilo, los propanoatos de tert-butilo, de butilo, de 2-
metilbutilo, de pentilo y de hexilo, los butanoatos de metilo y de etilo, y los 2-metilbutanoatos
de etilo, de butilo y de hexilo (cap. IV, Tabla 2). Se analizaron las modificaciones en la
emisión de estos compuestos, además del butanoato de butilo debido a sus altas
concentraciones, y del acetato de etilo como indicador de posibles procesos fermentativos en
los frutos conservados en hipoxia, con objeto de estudiar el efecto de las atmósferas
controladas con muy bajo nivel de oxígeno (ULO) y de los tratamientos post-cosecha con
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Discusión general
cloruro cálcico sobre el aroma en manzanas conservadas a 1 ºC. La utilización de la

tecnología ULO (en este caso 1 kPa O2 : 2 kPa CO2) para la frigoconservación provocó una
disminución en la producción de los citados compuestos aromáticos volátiles (cap. IV, Tabla
5), en concordancia con anteriores trabajos en manzanas ‘Fuji’ (Echeverría et al., 2004a;
Altisent et al., 2008).
Tras un período de almacenamiento de 4 meses, los frutos tratados con calcio

mostraron un aumento en la emisión de algunos de los compuestos identificados como de
impacto (acetatos de etilo y de 2-metilbutilo, propanoatos de butilo y de pentilo, 2-
metilbutanoato de etilo) independientemente de la atmósfera utilizada, lo que sugiere que este
tratamiento ayuda a mejorar la calidad aromática en esta variedad después de períodos medios
de frigoconservación, incluso si ésta se lleva a cabo en condiciones ULO. Estos resultados
concuerdan parcialmente con los resultados obtenidos por Saftner et al. (1999), que
observaron aumentos en la emisión de algunos de los compuestos de la fracción volátil de
manzanas ‘Golden Delicious’ tratadas con calcio previamente a su almacenamiento.
Similarmente a lo observado durante la maduración en campo de manzanas ‘Fuji Kiku-8’
(cap. II), la actividad AAT no mostró una relación directa con la producción de ésteres, pero
sí se observó una fuerte correlación entre la emisión de ésteres y alcoholes y las actividades
ADH y PDC en la pulpa (cap. IV, Fig. 1). Por lo tanto, también después de la
frigoconservación, los efectos favorables del tratamiento con cloruro cálcico sobre la calidad
aromática de los frutos vendrían mediados por el incremento de las actividades PDC y ADH,
debido posiblemente a una mayor dificultad en la difusión de gases desde el interior de los
frutos. Efectivamente se observó un incremento en las mismas tras 4 meses de
frigoconservación en los frutos tratados (cap. IV, Tabla 6), con el consiguiente aumento en la
producción de la mayoría de los alcoholes detectados (cap. IV, Tabla 7) y de acetaldehído
(cap. IV, Tabla 4), lo que supone una mayor disponibilidad de substratos para la actividad
AAT.
Sin embargo, tras un período de almacenamiento de 7 meses, el efecto del tratamiento

post-cosecha con cloruro cálcico sobre la composición de la fracción volátil de manzanas
‘Fuji Kiku-8’ sí estuvo modulado por el tipo de atmósfera de frigoconservación. La emisión
de los acetatos de etilo y de 2-metilbutilo, de los propanoatos de tert-butilo y de 2-metilbutilo,
del butanoato de butilo, y de los 2-metilbutanoatos de butilo y de hexilo se incrementó a
consecuencia del tratamiento con cloruro cálcico después un almacenamiento en frío normal
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Discusión general
durante 7 meses (cap. IV, Tabla 4), pero no después de la conservación en condiciones ULO,
en cuyo caso incluso se observó una disminución en la emisión de algunos compuestos en los
frutos tratados. De hecho, no se hallaron efectos significativos del tratamiento con calcio
sobre las actividades PDC y ADH (cap. IV, Tabla 5), aunque sí se detectó un incremento de la
actividad AAT en frutos tratados y conservados en frío normal, lo que podría explicar el
incremento en la producción de algunos ésteres en estas muestras. Así, después de períodos
largos de frigoconservación también la capacidad intrínseca de los tejidos para sintetizar
ésteres podría verse afectada, representando un factor importante, además de la disponibilidad
de substratos, para la composición de la fracción volátil de los frutos. Este incremento en la
actividad AAT podría haber resultado de una mayor producción de etileno (cap. IV, Tabla 4),
ya que se considera que la actividad AAT en manzana es etileno-dependiente (Defilippi et al.,
2005).
Cabía la posibilidad de que los resultados del tratamiento post-cosecha con cloruro
cálcico fueran distintos para manzanas ‘Golden Reinders’, que tiene un potencial y
comportamiento post-cosecha distinto a ‘Fuji Kiku-8’. Por eso, se repitió el mismo
experimento que en ‘Fuji Kiku-8’ introduciendo adicionalmente una modalidad de
frigoconservación en atmósfera controlada estándar (ACS; 3 kPa O2 : 2 kPa CO2), también a 1
ºC. El número de compuestos volátiles que contribuyeron activamente al aroma de la variedad
‘Golden Reinders’ tras la frigoconservación en frío normal fue superior respecto a la fecha de
cosecha comercial. Junto con los compuestos ya citados anteriormente (apartado 1.1 de la
presente discusión), siete ésteres lineales (acetatos de butilo y pentilo, propanoato de butilo,
butanoatos de etilo, butilo y hexilo, y hexanoato de etilo) y dos ramificados (acetato de 2-
metilpropilo y 2-metilbutanoato de butilo) también contribuyeron al aroma en esta variedad
(cap. V, Tabla 3), a causa de un incremento en la emisión de dichos compuestos tras la
frigoconservación. El aumento en la concentración de volátiles tras la frigoconservación con
respecto al momento de cosecha comercial ha sido observado también en variedades como
‘Golden Delicious’ (López et al., 2000), ‘Fuji’ (Echeverría et al., 2004a) o ‘Pink Lady®’
(Villatoro et al., 2009).
Como en la variedad ‘Fuji Kiku-8’, el efecto del tratamiento post-cosecha con cloruro
cálcico en manzanas ‘Golden Reinders’ fue dependiente de la atmósfera y el período de
frigoconservación. Tras un período de almacenamiento a 1 ºC en frío normal durante 4 meses
y 7 días a 20 ºC, la producción de la mayoría de ésteres lineales fue superior en frutos tratados
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Discusión general
(cap. V, Tabla 3). A diferencia de lo observado para la variedad ‘Fuji Kiku-8’, ello comportó
también un aumento en el número de compuestos impacto, pues la producción de butanoato
de metilo y hexanoato de butilo superó sus umbrales de percepción olfativa, lo que significa
que el impacto del tratamiento fue aún más marcado para la variedad ‘Golden Reinders’. El
efecto beneficioso del tratamiento con cloruro cálcico sobre la producción de compuestos
volátiles fue extensivo también a las manzanas almacenadas en ACS y ULO, aunque el
número de compuestos afectados fue menor cuanto más baja la concentración de oxígeno
durante la frigoconservación (cap. V, Tabla 3). Así, el tratamiento también ayudó a disminuir
los efectos negativos del almacenamiento en atmósfera controlada sobre la calidad aromática
en esta variedad.
Como se ha comentado ya repetidamente a lo largo de esta discusión, tampoco en

manzanas ‘Golden Reinders’ almacenadas durante 4 meses se halló una correspondencia clara
entre la actividad AAT y la producción de ésteres volátiles, y en la piel del fruto incluso se
observó la inhibición de los niveles de actividad en las muestras tratadas (cap. V, Tabla 5).
Sin embargo, en general el tratamiento causó un aumento en las actividades PDC y ADH,
similarmente a lo observado en ‘Fuji Kiku-8’, posiblemente en relación con las
concentraciones superiores de acetaldehído (cap. V, Tabla 6) y alcoholes (cap. V, Tabla 4)
observadas para los frutos tratados, nuevamente apuntando a una mayor disponibilidad de
substratos como factor clave para la producción de los ésteres que conforman el perfil
aromático.
En esta variedad (‘Golden Reinders’), no obstante, el tratamiento con cloruro cálcico

resultó en la disminución de la producción de ésteres tras 7 meses en frío normal (cap. V,
Tabla 3). Aún así, esta disminución no conllevó una reducción en el número de compuestos
impacto, y posiblemente resultó de una menor disponibilidad de alcoholes (cap. V, Tabla 4)
como consecuencia de la inhibición de la actividad ADH en la pulpa (cap. V, Tabla 6). Sin
embargo, la actividad AAT en la piel, tejido donde se sintetiza la mayor parte de los
compuestos volátiles, disminuyó en los frutos tratados (cap. V, Tabla 6), lo que sugiere que la
capacidad intrínseca para llevar a cabo la esterificación pudo haber representado también un
factor importante en la disminución de producción de ésteres. A su vez, la inhibición parcial
de la actividad AAT podría haber sido consecuencia de la menor producción de etileno en
estas muestras (cap. V, Tabla 6). Sin embargo, la reducción en la producción de volátiles a
causa del tratamiento no se observó en los frutos almacenados en atmósfera controlada
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Discusión general
durante 7 meses. En este caso, la producción de los propanoatos de hexilo y de 2-

metilbutanoato, dos de los compuestos impacto para esta variedad, se incrementó (cap. V,
Tabla 4) a causa del tratamiento, aunque para el resto de los ésteres emitidos no se observó en
general ningún efecto significativo.
Por tanto, los baños post-cosecha en una solución de cloruro cálcico ofrecen un buen
potencial para la mejora de la calidad gustativa en ambas variedades de manzana estudiadas,
al menos para períodos medios de frigoconservación, y de hecho la aceptabilidad de consumo
de los frutos aumentó en respuesta al tratamiento (datos no mostrados). Los resultados
sugieren que, para períodos de frigoconservación no muy prolongados, las aplicaciones post-
cosecha de cloruro cálcico representarían una alternativa muy recomendable, además de
mucho más sencilla y económicamente más viable, al uso de atmósferas controladas. Para
períodos de frigoconservación largos (7 meses), sin embargo, los efectos del tratamiento sobre
la calidad aromática del producto no serían tan claros. Para frutos almacenados en frío
normal, la mejora de la calidad aromática en respuesta al tratamiento se mantuvo en la
variedad ‘Fuji Kiku-8’, mientras que se vio comprometida en la variedad ‘Golden Reinders’.
No obstante, cabe destacar que, aunque menor que en los frutos no tratados, la emisión de
ésteres volátiles en las muestras tratadas con calcio fue significativamente mayor que en las
conservadas en atmósfera controlada (cap. V, Tabla 3), por lo que el tratamiento mejoraría la
calidad aromática incluso en estas condiciones.
2.2. Melocotón (Prunus persica L. Batsch, cv. ‘Rich Lady’ y ‘Tardibelle’).
El melocotón y la nectarina son frutos que se caracterizan por su rápido proceso de

maduración, además de ser especialmente propensos al desarrollo de múltiples desórdenes
fisiológicos (harinosidad, pardeamiento de la pulpa, enrojecimiento interno, etc.) durante su
frigoconservación (Lurie y Crisosto, 2005). Con la finalidad de minimizar la incidencia de
estas fisiopatías en estos frutos, diversos autores (Lurie, 1992; Retamales et al., 1992;
Crisosto et al., 1995; Zhou et al., 2000) han recomendado el uso de atmósferas controladas
para su conservación, especialmente con altas concentraciones de CO2. Sin embargo, igual
que en manzanas, su calidad aromática puede verse comprometida tras la conservación en
atmósfera controlada. Así, dos de los experimentos incluidos en la presente Tesis tuvieron por
objeto estudiar las modificaciones en el perfil aromático y la calidad estándar de melocotones
conservados en atmósfera controlada (3 kPa O2: 10 kPa CO2). Se estudiaron dos variedades en
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Discusión general
la campaña 2006, una de recolección temprana (‘Rich Lady’) y otra de recolección tardía
(‘Tardibelle’).
Para la variedad ‘Rich Lady’, se estudió el efecto de dichas modificaciones sobre la

aceptabilidad de consumo de los frutos. Los resultados obtenidos indican que la conservación
en atmósfera controlada no tuvo efectos significativos sobre la aceptabilidad sensorial del
fruto para períodos de frigoconservación cortos (3 días a 2 ºC + 1 día a 7 ºC) (cap. VI, Fig. 1).
Sin embargo, para períodos de frigoconservación más largos (15 días a 2 ºC + 1 día a 7 ºC),
los frutos conservados en atmósfera controlada recibieron puntuaciones mayores en cuanto a
aceptabilidad sensorial por parte de un panel de consumidores, lo que sugiere que esta
tecnología es potencialmente beneficiosa para prolongar la calidad gustativa y la vida
comercial del producto. La aceptabilidad sensorial se correlacionó positivamente con la
percepción por parte de los consumidores del ‘sabor característico’ a melocotón y de la
jugosidad, y también con la percepción de dulzor, que a su vez estaba inversamente
correlacionada con la acidez percibida (cap. VI, Fig. 2). Se observó asimismo que la
percepción del sabor característico a melocotón estaba relacionada con la emisión de algunas
lactonas (γ-octalactona, δ-decalactona y γ-dodecalactona) (cap. VI, Fig. 3B), de acuerdo con
estudios anteriores en que las lactonas fueron identificadas como ‘compuestos impacto’ en el
aroma del melocotón (Aubert et al., 2003; Lavilla et al., 2002; Rizzolo et al., 2006). Por tanto,
mayores emisiones de este tipo de compuestos serían indicativas de mejor calidad gustativa,
como consecuencia de su contribución al atributo sensorial ‘aroma a melocotón’ (Horvat et
al., 1990; Rizzolo et al., 2006). La percepción del sabor característico del melocotón también
mostró una relación positiva con el contenido en sólidos solubles en el fruto (cap. VI, Fig.
3B), posiblemente en relación con la asociación entre la aceptabilidad sensorial y la
percepción de dulzor (cap. VI, Fig. 2). Por otro lado, los resultados obtenidos indican que una
gran parte de los compuestos volátiles emitidos por el melocotón ‘Rich Lady’, y entre ellos la
γ-hexalactona, no tuvieron una influencia directa en la percepción del sabor y,
consecuentemente, tampoco en la aceptabilidad sensorial del fruto. Precisamente, aunque los
frutos almacenados en atmósfera controlada recibieron mayor puntuación en cuanto a
aceptabilidad, la producción de la mayoría de los volátiles detectados fue mayor en los
melocotones almacenados en frío normal, lo que concuerda también con lo observado en esta
Tesis para frutos de manzana. Esto sugiere que un equilibrio adecuado entre los distintos
compuestos volátiles emitidos por el fruto es más relevante para la aceptabilidad sensorial que
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Discusión general
una producción elevada de los mismos, sin olvidar que en dicha aceptabilidad influyen otros
compuestos distintos a los volátiles (azúcares, ácidos, etc.).
Dada la corta vida útil del melocotón, es lógico que el período de almacenamiento
fuera también un factor importante para la emisión de compuestos volátiles. Ésta no se vio
afectada significativamente por la conservación en atmósfera controlada durante períodos
cortos (cap. VI, Fig. 4A), pero sí para almacenamientos más prolongados, lo que concuerda
con lo observado para la aceptabilidad sensorial (cap. VI, Fig. 1). Los melocotones
frigoconservados durante 15 días mostraron mayores emisiones de la mayoría de los
compuestos volátiles detectados (cap. VI, Fig. 4). Estos mismos frutos se caracterizaron por
mayores emisiones de δ-decalactona y γ-dodecalactona cuando se conservaban en atmósfera
controlada, lo que vuelve a indicar el papel fundamental de estos compuestos en la
aceptabilidad de consumo de melocotón, ya que estas muestras recibieron mejores
puntuaciones por parte de los panelistas participantes en el estudio, de acuerdo con resultados
anteriores (Horvat et al., 1990). No obstante, los niveles de γ-octalactona, uno de los
compuestos correlacionados positivamente con la aceptabilidad sensorial, fueron superiores
para frutos almacenados durante tres días, al igual que los de linalool y acetaldehído, como se
ha observado anteriormente (Robertson et al., 1990; Bonghi et al., 1999).
La emisión de las tres lactonas que contribuyeron a la percepción del ‘sabor a

melocotón’ (γ-octalactona, δ-decalactona y γ-dodecalactona) fue muy dependiente de la
actividad AAT (cap. VI, Fig. 5), especialmente en la pulpa, al igual que la de otros
compuestos (acetatos de hexilo y de 2-metilbutilo, butanoato de hexilo, hexanoatos de pentilo
y de butilo, y octanoato de butilo). Se observó igualmente la importancia de una fecha de
recolección adecuada para la calidad aromática del fruto: aunque se consideraron tres fechas
de cosecha en el presente estudio, fue la segunda la que se caracterizó por una mayor emisión
de ésteres (cap. VI, Fig. 6) y por mayores niveles de actividad AAT en la pulpa (cap. VI,
Tabla 4). Esto sugiere que la recolección en un estadio sobremaduro puede comprometer
igualmente la calidad aromática de los melocotones tras su frigoconservación. La actividad
AAT aumentó durante el período a 20 ºC posterior a la frigoconservación, lo que indica la
regeneración de la capacidad de producir ésteres. Cabe destacar que esta regeneración de la
capacidad productiva sólo se observó para los frutos que habían sido almacenados en
atmósfera controlada. Aún así, las variaciones en la actividad AAT, al igual que en manzana,
no explican por sí solas los cambios en la producción de ésteres, lo que vuelve a poner de
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Discusión general
manifiesto la relevancia del suministro de substratos. Efectivamente, un 57% de la

variabilidad observada en la emisión de ésteres resultó ser dependiente de dicho suministro
(cap. VI, Fig. 6), del cual es parcialmente responsable ADH.
En la siguiente campaña (2007), además del período de almacenamiento se evaluaron

también la influencia de la temperatura de conservación sobre la producción de compuestos
volátiles aromáticos y sobre la aceptabilidad de consumo de melocotones ‘Rich Lady’. La
temperatura de frigoconservación afectó claramente a la aceptabilidad de los frutos, de modo
que únicamente los conservados a 12 ºC obtuvieron una puntuación satisfactoria por parte de
los consumidores (cap. VII, Fig. 1). La aceptabilidad, sin embargo, fue declinando a medida
que se prolongaba el período de conservación, y a partir de tres semanas de almacenamiento
ya no hubo diferencias significativas según la temperatura.
La aceptabilidad de consumo de estos melocotones estuvo en estrecha relación con la

percepción del ‘sabor característico’ a melocotón y del dulzor (cap VII, Fig. 2). A su vez, la
percepción del ‘sabor característico’ a melocotón mostró una correlación inversa con la
firmeza y la acidez medidas instrumentalmente (cap VII, Fig. 3). Sin embargo, al igual que en
la campaña anterior (2006), la emisión de compuestos volátiles tuvo influencia sobre el grado
de satisfacción de los consumidores. Así, los frutos almacenados a 12 ºC, que fueron los
mejor valorados por los consumidores, se caracterizaron por una mayor producción de ésteres
volátiles (cap. VII, Fig. 3), lo que confirma la importancia de la calidad aromática en la
calidad gustativa global del fruto. Estas muestras tuvieron también mayor contenido en
sólidos solubles y mayor emisión de δ-decalactona y γ-hexalactona, entre otros, confirmando
la relevancia de estas lactonas para la percepción del ‘sabor característico’ a melocotón
observada en la campaña anterior (2006) para la misma variedad, así como en trabajos de
otros autores (Engel et al., 1998a, b; Aubert et al., 2003). Como se ha observado
repetidamente en los experimentos que integran esta Tesis, las modificaciones en la
producción de ésteres tampoco se correspondieron con las observadas en la actividad AAT
(cap. VII, Tabla 2), pero sí con la disponibilidad de alcoholes (cap. VII, Tabla 3), resaltando
nuevamente la importancia de este factor para la producción de compuestos aromáticos
volátiles.
En cuanto a la variedad ‘Tardibelle’, se estudiaron los efectos de un tratamiento con 1-

MCP (1 µL L-1 durante 24 horas a 1 ºC), tanto individualmente como en combinación con la
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Discusión general
conservación en atmósfera controlada, sobre la producción de compuestos volátiles

aromáticos emitida por los frutos. Los frutos se analizaron después de 0 y 7 días a 20 ºC tras
21 días a 1 ºC. No se pudo realizar análisis sensorial en este estudio, ni por ello comprobar la
relevancia de los posibles cambios para la aceptabilidad de consumo, ya que el 1-MCP no está
autorizado por la normativa española para su uso en frutos de hueso. Se identificaron 28
compuestos (15 ésteres lineales, 7 ésteres ramificados, 1 lactona, 4 alcoholes y aldehído) (cap.
VIII, Tabla 3) en la fracción volátil emitida por las muestras. El perfil de volátiles resultó
alterado en respuesta a los factores considerados, si bien dichas alteraciones fueron diferentes
según el compuesto y según el tratamiento aplicado. Tanto el almacenamiento en atmósfera
controlada como la aplicación de 1-MCP disminuyeron la producción total de ésteres lineales
al final del período de vida útil a 20 ºC (cap. VIII, Tabla 4). En cambio, no se apreciaron
efectos significativos sobre la producción total de ésteres ramificados, con la excepción de
cierta disminución en el día de salida de cámara después de la conservación en atmósfera
controlada.
El tratamiento con 1-MCP afectó especialmente a los ésteres de etilo y a los acetatos,
con un fuerte incremento en el momento de la salida de cámara (cap. VIII, Tabla 5), en
paralelo a una mayor producción de etanol (cap. VIII, Tabla 6). La producción de estos
ésteres, no obstante, disminuyó posteriormente, al igual que la de los ésteres de hexilo. Esto
indica que el aroma del fruto debió verse afectado, ya que por ejemplo la producción de
acetato de hexilo, que contribuye aportando notas de olor frutales y dulces, resultó
drásticamente disminuida en respuesta al tratamiento. De igual manera, la emisión de ésteres
de metilo, que también contribuyen notas dulces y afrutadas, se redujo considerablemente en
estas muestras durante su vida comercial a 20 ºC. Esto es importante desde el punto de vista
del sector ya que, a pesar de los efectos beneficiosos del tratamiento sobre la firmeza y sobre
otros parámetros de calidad estándar considerados habitualmente (cap. VIII, Tabla 2), los
resultados de este trabajo sugieren que la calidad aromática del fruto se vio seriamente
comprometida, y por tanto también su calidad gustativa global, dada la fuerte asociación entre
la aceptabilidad sensorial y la percepción del sabor característico observado para la variedad
‘Rich Lady’ en ambas campañas (2006 y 2007), que posiblemente se dé también en otras
variedades de melocotón. De ello se desprende la conveniencia de evaluar en conjunto los
efectos de un determinado tratamiento post-cosecha, con particular énfasis sobre el aroma
como atributo clave en la calidad sensorial del producto, algo esencial para fidelizar al
consumidor.
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Los factores causales de la disminución en la producción de ésteres volátiles en las

muestras tratadas con 1-MCP apuntan, al igual que en los demás casos comentados en esta
Discusión, a la reducción en el suministro de alcoholes y de acil CoAs necesarios para la
reacción de síntesis. Algunas actividades enzimáticas relacionadas con dicho suministro,
como LOX y HPL, resultaron inhibidas parcialmente en respuesta al tratamiento (cap. VIII,
Tabla 7), en concordancia con la prevalencia de los ésteres de cadena lineal en la fracción
volátil emitida por el fruto, que se consideran derivados de ácidos grasos. Esto sugiere
también una regulación etileno-dependiente de la expresión y/o actividad de estos enzimas.
Los resultados sugieren también un papel importante para factores como la limitación en la
disponibilidad de portadores activados de energía como consecuencia de la disminución en las
tasas respiratorias, o las preferencias de substrato de las isoformas de AAT presentes en los
tejidos.
3. METABOLISMO DE LAS PAREDES CELULARES Y PÉRDIDA DE

FIRMEZA DURANTE LA MADURACIÓN EN CAMPO.
Uno de los cambios más generalizados durante la maduración de los frutos es la

pérdida de firmeza de la pulpa. Este ablandamiento de los tejidos, aunque resulta hasta cierto
punto deseable para su palatabilidad, puede generar importantes pérdidas económicas durante
su comercialización, dado que los frutos más blandos son más sensibles a los daños
mecánicos durante su manipulación y a la aparición de enfermedades ocasionadas por hongos.
Para el estudio del metabolismo de las paredes celulares en manzanas, un factor directamente
implicado en el proceso de ablandamiento de los frutos (Johnston et al., 2002), se escogieron
dos variedades (‘Golden Reinders’ y ‘Fuji Kiku-8’) con un comportamiento en post-cosecha
marcadamente diferente.
Durante la maduración en campo (90-147 ddpf) de la variedad ‘Golden Reinders’, la

firmeza de los frutos decreció en 25 N (cap. IX, Fig. 1), mientras que en el caso de ‘Fuji Kiku-
8’ (130-202 ddpf), la firmeza disminuyó en 40 N (cap. X, Fig. 1A). Esta pérdida de firmeza
resultó, al menos en parte, de modificaciones en la composición de las paredes celulares de la
pulpa, y de hecho en ambas variedades el contenido en materiales de pared celular insolubles
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Discusión general
fue menor en los frutos menos firmes (cap. IX, Fig. 2; cap. X, Fig. 2B). Los frutos con menor
firmeza se caracterizaron igualmente por contener niveles más altos de material de pared
soluble en fenol: ácido acético: agua (FAA).
Los cambios en las paredes celulares durante la maduración de los frutos incluyen
tanto la solubilización y despolimerización de sus componentes como la modificación en el
tipo de enlaces que se establecen entre sus moléculas (Redgwell et al., 1997; Brummell y
Harpster, 2001; Goulao et al., 2008). Para ambas variedades, a medida que los frutos
maduraban, se observó un descenso significativo en el contenido de la fracción soluble en
CDTA, enriquecida en pectinas unidas a la pared por enlaces no covalentes (cap. IX, Fig. 2;
cap. X, Tabla 2). En consecuencia, la solubilización de este tipo de polímeros representaría un
factor con una influencia importante en la pérdida de firmeza en manzanas, en concordancia
con un trabajo anterior (Yoshioka et al., 1992). En cuanto a las pectinas unidas a la pared por
enlaces covalentes, representadas por la fracción soluble en Na2CO3, su contenido disminuyó
en manzanas ‘Fuji Kiku-8’ a lo largo del período considerado (cap. X, Tabla 2), por lo que
también jugarían un papel importante en el mantenimiento de la firmeza durante la
maduración en campo. En la variedad ‘Golden Reinders’, sin embargo, las modificaciones en
el contenido de esta fracción aparentemente no estuvieron en relación directa con el
ablandamiento de los frutos (cap. IX, Fig. 2). Se extrajo también la fracción soluble en KOH,
que contiene principalmente hemicelulosas. Al igual que la fracción soluble en Na2CO3, las
modificaciones observadas en la fracción enriquecida en hemicelulosas aparente no guardaron
relación directa con la pérdida de firmeza en manzanas ‘Golden Reinders’ (cap. IX, Fig. 2),
mientras que para la variedad ‘Fuji Kiku-8’ sí se halló una disminución en su contenido a lo
largo del período estudiado (cap. X, Tabla 3). Las diferencias observadas entre las dos
variedades en los patrones de solubilización, tanto de las pectinas unidas a la pared por
enlaces covalentes como de las hemicelulosas, podrían por tanto representar una posible
explicación para el diferente potencial de vida post-cosecha que las caracteriza.
Los cambios producidos en las paredes celulares de los frutos se deben en buena parte
a la acción conjunta de un gran número de enzimas. Entre ellos, PG y PME han sido los más
ampliamente estudiados y a los que tradicionalmente se les ha asignado un rol clave en la
solubilización y despolimerización de los polímeros que conforman las paredes celulares
(Goulao et al., 2008). Sin embargo, los experimentos en que se ha modificado genéticamente
la expresión de PG o PME han resultado generalmente poco conclusivos respecto a un posible
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Discusión general
papel clave de los mismos en la pérdida de firmeza (Goulao y Oliveira, 2008), lo que ha
hecho descartar la idea de un único enzima como causante principal del proceso, en línea con
los resultados de experimentos similares realizados para otros genes relacionados con las
modificaciones en la pared celular. Se considera actualmente, por el contrario, que los
cambios en las paredes celulares que subyacen en la pérdida de firmeza de un fruto durante su
maduración resultan de la acción conjunta y coordinada de diferentes proteínas, tanto
enzimáticas como no enzimáticas. Por tanto, en la presente Tesis se ha estudiado también la
actividad de otros enzimas como posibles factores relacionados con el proceso de
ablandamiento de los frutos.
A lo largo de la maduración en campo, la actividad PME no evolucionó de manera

paralela a la pérdida de firmeza ni en manzanas ‘Golden Reinders’ (Tabla 3) ni en ‘Fuji Kiku-
8’ (cap. X, Fig. 3A), si bien en ambas variedades se observó un pico de actividad en un estado
de madurez temprano, en concordancia con lo observado para la variedad ‘Mondial Gala’
(Goulao et al., 2007). La actividad PME, entonces, no guardaría una relación directa con la
pérdida de firmeza en manzanas, como apuntan trabajos anteriores (Klein et al., 1995;
Siddiqui et al., 2004). No obstante, la actividad PME condiciona la de otros enzimas, ya que
la desmetilación de los residuos de ácido galacturónico presentes en las pectinas las convierte
en sustratos para las actividades PG y PL (Brummell, 2006; Bennett y Labavitch, 2008). La
actividad PME, además, modifica el pH del apoplasto, lo que a su vez modula los niveles de
actividad de otros enzimas. Así, aunque no de forma directa sobre la firmeza, los efectos
derivados de la actividad PME pueden implicar ciertas modificaciones en los atributos que
influyen sobre la textura de las manzanas, tal como se ha observado en otras especies (Tieman
y Handa, 1994; Fraeye et al., 2009).
Durante la maduración en campo de manzanas ‘Golden Reinders’ se observaron

patrones distintos para las actividades PG y PL (cap. IX, Tabla 1). Mientras que la actividad
PL disminuyó progresivamente a lo largo del período considerado, la actividad PG mostró la
tendencia contraria. La evolución de estas dos actividades en manzanas ‘Fuji Kiku-8’ difirió
respecto a ‘Golden Reinders’: además de aumentar en estados de madurez próximos al
comercial, se hallaron también niveles altos de actividad PG en estados de madurez muy
tempranos, mientras que la actividad PL aumentó ligeramente hasta un mes antes de la
cosecha comercial y se mantuvo constante posteriormente (cap. X, Fig. 3B). La pérdida de
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Discusión general
firmeza en esta variedad no mostró una relación clara con estos cambios (cap. X, Fig. 1A), lo
que confirma la existencia de otros factores implicados en el ablandamiento de los frutos.
Tabla D3. Actividades pectinmetilesterasa (PME), β-galactosidasa (β-Gal) y β-xilosidasa (β-

Xyl) (U mg-1 proteína) en manzana ‘Golden Reinders’ (año 2007) durante su
maduración en campo (datos no publicados).
Fecha Pectinmetilesterasa β-galactosidasa β-xilosidasa

10 Julio 76,689 e 0,108 e 0,086 a
17 Julio 77,874 e 0,108 e 0,065 a
24 Julio 135,506 b 0,298 c 0,060 ab
31 Julio 262,542 a 0,339 c 0,020 c
07 Agosto 139,294 b 0,317 c 0,016 c
14 Agosto 119,681 c 0,481 b 0,025 c
21 Agosto 114,496 cd 0,617 a 0,028 c
29 Agosto 101,785 d 0,484 b 0,021 c
04 Septiembre 72,846 e 0,236 d 0,031 bc
Los valores representan la media de tres repeticiones. Letras diferentes en una misma columna denotan
diferencias significativas (P < 0’05; test LSD).
Se considera que, en manzana, los polímeros de pared celular no experimentan una

despolimerización muy intensa (Brummell, 2006; Goulao y Oliveira, 2008), lo que restaría
relevancia a las actividades PG y PL en el metabolismo de las paredes celulares. Además de
estos enzimas, en la presente Tesis se han estudiado las modificaciones sufridas por β-Gal y
AFasa durante la maduración en campo de manzanas y su posible implicación en la pérdida
de firmeza de los frutos. En manzanas ‘Golden Reinders’, la actividad β-Gal aumentó
significativamente hasta el momento de madurez comercial, a partir del cual disminuyó
(Tabla D3). En ‘Fuji Kiku-8’ se observó un aumento en las actividades β-Gal y AFasa en
estados de madurez más avanzados (cap. X, Fig. 4). Estos aumentos estarían posiblemente
relacionados con la progresiva disminución en el contenido de pectinas unidas
covalentemente a las paredes (cap. X, Tabla 2), y por tanto evidenciarían una implicación
directa en la pérdida de firmeza de los frutos.
Como se ha indicado anteriormente, en la variedad ‘Golden Reinders’ la pérdida de

firmeza no estuvo relacionada de manera directa con las alteraciones en la fracción rica en
hemicelulosas y, de hecho, la actividad EGasa (cap. IX, Tabla 1) no experimentó
modificaciones significativas durante el período considerado. En la variedad ‘Fuji Kiku-8’,
por el contrario, sí se observó un aumento en estas actividades conforme los frutos se
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Discusión general
aproximaban a la madurez comercial (cap. X, Fig. 5), lo que explicaría en parte el menor
rendimiento de la fracción enriquecida en hemicelulosas al final del período experimental
(cap. X, Tabla 2) e indicaría una posible implicación con la pérdida de firmeza de esta
variedad durante la maduración en campo.
Una de las consecuencias de la actividad PME es la generación de cargas negativas en

los homogalacturonanos, lo que confiere a estos polímeros la capacidad de formar puentes de
calcio y un mecanismo para el mantenimiento de la firmeza de los tejidos del fruto (Jarvis,
2011). Por ello, se evaluaron también los efectos de la aplicación de calcio en pre-cosecha
sobre la firmeza y el metabolismo de las paredes celulares en la variedad ‘Fuji Kiku-8’. En el
momento de cosecha comercial, los valores de firmeza fueron mayores en los frutos tratados
(cap. X, Fig. 1A), confirmando la implicación del calcio en la definición de este atributo. La
mejora en los valores de firmeza podría haber resultado del mayor contenido total de
materiales insolubles de pared celular en los frutos tratados, junto con un menor rendimiento
de la fracción soluble en FAA (cap. X, Fig. 1B) como consecuencia del retraso en la
disminución en las fracciones solubles en CDTA y KOH (cap. X, Tabla 2). Así, los
tratamientos pre-cosecha con soluciones de calcio, además de resultar beneficiosos para la
calidad aromática de los frutos (cap. II), también lo son para el mantenimiento de la firmeza y
por lo tanto resultan altamente recomendables para mejorar la calidad comercial de los
mismos.
Además de sus efectos sobre las pectinas unidas de manera no covalente y el

consiguiente beneficio para el mantenimiento de la firmeza, los tratamientos con calcio en
pre-cosecha inhibieron significativamente las actividades de algunos de los enzimas
implicados en el metabolismo de las paredes celulares. Así, las actividades PME, PL, AFasa,
β-Gal y β-Xyl, implicadas de manera importante en el proceso, se vieron afectadas durante la
segunda mitad del período experimental, lo que también explicaría el mayor contenido en
material de pared celular en los frutos. La causa de esta inhibición no está del todo clara,
aunque podría guardar relación con la disminución de la producción de etileno en los frutos
tratados (Lieberman y Wang, 1982; Lara y Vendrell, 1998).
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Discusión general
3.2. Nectarina (Prunus persica L. Batsch var. nectarina, cv. ‘Snow Queen’ y ‘Big Top’)
y melocotón (Prunus persica L. Batsch, cv. ‘Rich Lady’).
A diferencia de la manzana, cuya pérdida de firmeza es moderada y relativamente

lenta, los melocotones y las nectarinas se caracterizan por una rápida y abrupta pérdida de
firmeza durante la maduración, hecho que condiciona significativamente la fecha de
recolección, el potencial de conservación post-cosecha y las posibilidades de manipulación.
De entre las múltiples variedades de nectarina existentes, ‘Snow Queen’ es una de las que
presentan tasas de ablandamiento más marcadas en fechas próximas a la madurez comercial,
por lo que resulta de gran interés estudiar los factores que intervienen en este proceso, tanto
desde el punto de vista del conocimiento básico como aplicado. Además de ‘Snow Queen’, se
estudiaron también el melocotón ‘Rich Lady’ y la nectarina ‘Big Top’, que son importantes
económicamente en la zona frutícola del Segrià. En los tres casos se observó una acusada
pérdida de firmeza en estadios próximos a la madurez comercial, que tuvo lugar en un corto
período de tiempo (cap. XI, Fig. 1A; Fig. D1; Fig. D2).
Al igual que en manzana (cap. IX y X), se observaron importantes modificaciones en

las paredes celulares a lo largo del proceso. El contenido en material insoluble de pared
celular en la variedad ‘Snow Queen’ disminuyó significantemente durante el período
experimental (cap. XI, Fig. 1B). Sin embargo, aparentemente esta disminución no estuvo
relacionada con la solubilización de la fracción soluble en CDTA, que se mantuvo constante
(cap. XI, Tabla 1). Tampoco en la variedad ‘Rich Lady’ estuvieron de acuerdo los
rendimientos de esta fracción con la pérdida de firmeza (Fig. 1). Esta observación difiere de lo
observado en manzanas, frutos en los cuales se apreció un descenso significativo en esta
fracción que explicaría en gran medida la pérdida de firmeza (cap. XI y X). Estas diferencias
en las modificaciones observadas para la fracción enriquecida en pectinas unidas a la pared
por enlaces no covalentes, por tanto, podrían ser uno de los factores subyacentes en el
diferente comportamiento de ambas especies en cuanto a pérdida de firmeza se refiere. No
obstante, en nectarina ‘Big Top’ sí se apreció un paralelismo entre las modificaciones en el
rendimiento de la fracción soluble en CDTA y los niveles de firmeza (Fig. 2), lo que indica la
existencia de diferencias importantes en el metabolismo de las paredes celulares entre
distintas variedades de una misma especie.
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Discusión general
Figura D1. Firmeza y rendimientos de las fracciones enriquecidas en pectinas en

melocotones ‘Rich Lady’ (datos no publicados) durante su maduración en
campo (año 2007). Los valores representan la media de 15 (firmeza) ó 3
repeticiones. (LSDfirmness = 5.2; LSDCDTA = 3.4; LSDNa2CO3 = 2.5).
Figura D2. Firmeza y rendimientos de las fracciones enriquecidas en pectinas en nectarinas

‘Big Top’ (datos no publicados) durante su maduración en campo (año 2008).
Los valores representan la media de 15 (firmeza) ó 3 repeticiones. (LSDfirmness =
4.9; LSDWsf = 4.1; LSDCDTA = 2.3; LSDNa2CO3 = 1.7; LSDKOH = 2.4).
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Discusión general
Las modificaciones observadas en los rendimientos de las fracciones solubles en

Na2CO3 y KOH siguieron un patrón similar a la pérdida de firmeza en la variedad ‘Snow
Queen’ (cap. XI, Tabla 1). Esto podría explicar la estabilidad del contenido en pectinas unidas
por fuerzas no covalentes en estos frutos, ya que generalmente se asume que sólo una parte de
las pectinas procedentes de la fracción soluble en Na2CO3 se solubiliza durante la maduración,
permaneciendo la mayor parte de la misma ligada a la pared por fuerzas no covalentes
(Dawson et al., 1992; Brummell and Harpster, 2001). Estas pectinas unidas iónicamente, a su
vez, tienen la capacidad de hidratarse formando geles, lo que aporta a estos frutos la textura
fundente que los caracteriza (Brummell and Harpster, 2001; Brummell et al., 2004). En la
variedad ‘Snow Queen’, esto quedó demostrado al observarse un enriquecimiento en el
contenido de azúcares neutros en la fracción soluble en CDTA a costa de una pérdida en la
fracción soluble en Na2CO3 (cap. XI, Tabla 2).
En concordancia con trabajos previos (Wakabayashi, 2000; Brummell and Harpster,

2001), la actividad PME declinó a medida que avanzaba el estado de madurez de los frutos
(cap. XI, Fig. 3), de modo que, al igual que en manzanas (cap. IX y X), esta actividad no
parece representar un factor clave para la pérdida de firmeza. Sin embargo, la desmetilación
de las pectinas favorecería la formación de geles típica de los frutos de textura fundente. En
este experimento, además, se apreció claramente la dependencia respecto a PME de las
actividades PG y PL, ilustrada por el paralelismo observado en sus modificaciones a lo largo
del período considerado (cap. XI, Fig. 3; cap. XI, Tabla 4). Las actividades AFasa y β-Gal
también fueron declinando a medida que el fruto se aproximaba a la senescencia (cap. XI,
Tabla 4). En vista de estos resultados, el rápido ablandamiento de estas nectarinas (cap. XI,
Fig.1A) podría haber resultado de un incremento puntual en las actividades de algunos
enzimas involucrados en la degradación de la pared celular, que desencadenaría una serie de
mecanismos, principalmente la eliminación de las ramificaciones ricas en azúcares neutros
presentes en la fracción péctica soluble en Na2CO3, que de forma irreversible causaría la
formación de geles en la fracción soluble en CDTA, aportando la textura fundente
característica de este tipo de frutos.
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Discusión general
4. METABOLISMO DE LAS PAREDES CELULARES Y PÉRDIDA DE

FIRMEZA EN RESPUESTA A LA MANIPULACIÓN POST-COSECHA.
En la presente Tesis, además de estudiarse las modificaciones en la producción de

compuestos volátiles aromáticos emitidos por manzanas ‘Golden Reinders’ y ‘Fuji Kiku-8’ en
respuesta al almacenamiento en atmósfera controlada y a los tratamientos post-cosecha con
calcio, se estudiaron también los efectos de estos factores sobre el metabolismo de las paredes
celulares y sobre la firmeza de los frutos, dada la importancia de la textura para la
aceptabilidad de consumo de los frutos.
Tanto el almacenamiento en atmósfera controlada como los baños post-cosecha en

cloruro cálcico resultaron efectivos para frenar la pérdida de firmeza en ambas variedades
estudiadas (‘Golden Reinders’: cap. XII, Tabla 3; ‘Fuji Kiku-8’: cap. IV, Tabla 4). Sin
embargo, la combinación de ambos tratamientos no conllevó una mejora significativa
respecto a su aplicación individual. Los datos, por tanto, indican que tanto el almacenamiento
en atmósferas bajas en O2 como el aporte de calcio exógeno tuvieron influencia sobre el
metabolismo de las paredes celulares de los frutos. En manzanas ‘Golden Reinders’ el
contenido en material insoluble de pared celular fue superior en general respecto a los
controles, tanto en frutos conservados en atmósfera controlada como en los tratados con
calcio, durante dos semanas a 20 ºC tras la conservación a 1 ºC (cap. XII, Tabla 4), lo que
estaría en relación con los mayores valores de firmeza observados para estas muestras (cap.
XII, Tabla 3). En la mayoría de los casos no se hallaron efectos aditivos de ambos
procedimientos sobre el contenido de materiales insolubles, similarmente a lo observado para
la firmeza. En la variedad ‘Fuji Kiku-8’, por el contrario, el almacenamiento en atmósfera
controlada no mejoró los rendimientos de materiales insolubles de pared celular (Tabla D4),
lo que constituye una importante diferencia respecto a ‘Golden Reinders’. El tratamiento con
calcio, en cambio, sí aumentó los rendimientos de esta fracción, con la única excepción de los
frutos almacenados durante 7 meses en atmósfera controlada.
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Discusión general
Tabla D4. Rendimiento de material insoluble de pared celular (MPC; % peso fresco) y de las
fracciones (% MPC) obtenidas a partir del mismo en pulpa de manzanas ‘Fuji
Kiku-8’ tras 7 días a 20 ºC después de la frigoconservación (datos no publicados).
Fracción Atmósfera Tratamiento Período de frigoconservación

4 meses 7 meses
MPC FN Control 1.604 c 1.805 ab
CaCl2 1.856 b 1.929 a
ULO Control 1.675 c 1.763 b
CaCl2 1.960 a 1.706 b
Aguafs FN Control 3.491 a 3.474 a
CaCl2 2.280 b 3.224 ab
ULO Control 1.889 b 2.580 b
CaCl2 1.747 b 3.674 a
CDTAfs FN Control 28.066 c 26.248 c
CaCl2 34.248 a 32.336 b
ULO Control 32.269 ab 38.994 a
CaCl2 31.943 b 40.617 a
Na2CO3fs FN Control 17.594 c 16.526 b
CaCl2 19.314 b 16.682 b
ULO Control 19.125 b 18.331 a
CaCl2 20.846 a 18.299 a
KOHfs FN Control 6.981 b 5.586 a
CaCl2 7.488 b 6.542 a
ULO Control 8.756 a 6.178 a
CaCl2 8.638 a 6.550 a
Los valores representan la media de tres repeticiones (MPC: material insoluble de pared celular; fs: fracción
soluble). Letras diferentes en una misma columna para una determinada fracción denotan diferencias
significativas (P < 0’05; test LSD).
Estos resultados, especialmente en lo que se refiere a la variedad ‘Fuji Kiku-8’,

indican que los valores de firmeza en frutos de manzana durante la post-cosecha no dependen
únicamente de la cantidad de material de pared celular, sino también de la composición y del
tipo de enlaces establecidos entre los distintos polímeros, como ya se había observado durante
la maduración en campo (apartado 3.1) y también en otros trabajos (Gross y Sams, 1984;
Murayama et al., 2002; Brummell et al., 2004; Peña y Carpita, 2004; Vicente et al., 2007;
Goulao y Oliveira, 2008). Así, en la variedad ‘Fuji Kiku-8’ se observó una fuerte asociación
entre el contenido en pectinas ligadas covalentemente a la pared y la firmeza (cap. XIII, Fig.
1), mientras que no se apreció asociación alguna con el contenido global de material de pared
celular. El contenido en pectinas ligadas por enlaces no covalentes también mostró cierta
relación con el mantenimiento de la firmeza (cap. XIII, Fig. 1). Al mismo tiempo, esta
fracción estuvo fuertemente asociada al contenido en calcio, indicando que es ésta la fracción
252
Discusión general
en que se establecen preferentemente los puentes de calcio entre polímeros que contribuyen a
mantener la firmeza en los frutos. Esta fuerte asociación entre las fracciones ricas en pectina y
el mantenimiento de la firmeza también se observó para la variedad ‘Golden Reinders’ (cap.
XII, Fig. 2), confirmando por tanto que durante la post-cosecha de manzanas el contenido en
estas fracciones es un factor clave que influye en este atributo. La fracción enriquecida en
hemicelulosas, en cambio, no presentó ninguna asociación significativa con la firmeza en
ninguna de las dos variedades (‘Golden Reinders’: cap. XII, Fig. 2: ‘Fuji Kiku-8’: cap. XIII,
Fig. 1), del mismo modo que lo observado durante la maduración en campo (‘Golden
Reinders’: cap. IX; ‘Fuji Kiku-8’: cap. X).
Al analizar en detalle los efectos de los factores estudiados sobre el metabolismo de

las paredes celulares, se pudo observar que el tratamiento con calcio mejoró
significativamente el contenido en pectinas unidas a la pared por enlaces no covalentes
(‘Golden Reinders: cap. XII, Tabla 4; ‘Fuji Kiku-8’: Tabla D4), lo que, como se ha indicado
anteriormente, aumentaría la posibilidad de formar puentes de calcio, ralentizándose de este
modo la disolución de la lámina media. El efecto del tratamiento con calcio sobre las pectinas
ligadas a la pared por enlaces covalentes fue menor. En manzanas ‘Golden Reinders’ se
observó una mejora en el rendimiento de esta fracción tras 31 semanas de frigoconservación
en ULO (cap. XII, Tabla 4), hallándose un efecto sinérgico entre ambos factores que también
se observó para la firmeza de los frutos después de dos semanas a 20 ºC (cap. XII, Tabla 3).
En cambio, en la variedad ‘Fuji Kiku-8’ este efecto se observó tras un período de
frigoconservación medio (4 meses), e independientemente de la atmósfera utilizada durante el
almacenamiento (Tabla D4).
Las aplicaciones de calcio exógeno, además de incrementar la posibilidad del

establecimiento de puentes de calcio entre residuos de ácido galacturónico desmetilados,
inhibieron también parcialmente la actividad de algunos enzimas pectolíticos. Así, la
actividad PL en manzanas ‘Fuji Kiku-8’ después de 7 meses de frigoconservación resultó
inhibida por el tratamiento (cap. XIII, Fig. 1), mientras que para la variedad ‘Golden
Reinders’, además de PME, se observaron también disminuciones significativas en las
actividades PG y PL (cap. XII, Tabla 5). La inhibición de las actividades PG y PL conllevó
mayor retención de ácidos urónicos en la fracción soluble en CDTA, lo que posiblemente
resultó en mayor número de interacciones entre los polímeros con la consiguiente mejora en
la integridad de las paredes celulares y por tanto en la firmeza. El tratamiento con calcio
253
Discusión general
también inhibió la actividad β-Gal en manzanas ‘Golden Reinders’, si bien únicamente

durante la primera semana a 20 ºC tras una frigoconservación prolongada (cap. XII, Tabla 5).
En general, la conservación en ULO causó asimismo la inhibición de las actividades

PL, β-Gal y AFasa (cap. XII, Tabla 5), aunque no de la actividad PG. No obstante, el
mantenimiento de la actividad PG respecto a los controles fue probablemente poco relevante
para la estructura de la pared celular, ya que la disminución en los niveles de las actividades
β-Gal y AFasa, que se considera que contribuyen a regular la porosidad de las paredes
celulares (Brummell et al., 2001), podría haber limitado el acceso de PG a su substrato, lo que
concordaría también con el mayor contenido en ácidos urónicos en la fracción soluble en
Na2CO3 observado para estas muestras (cap. XII, Tabla 6). Estos resultados coinciden con
publicaciones anteriores (Wei et al., 2010) en que se apuntó la hipótesis de que la pérdida de
firmeza de manzanas ‘Golden Delicious’ y ‘Fuji’ podría estar más influenciada por las
actividades β-Gal y AFasa que por la actividad PG. Esto explicaría también la fuerte
asociación entre los niveles de firmeza y el contenido en pectinas unidas a la pared por
enlaces covalentes (‘Golden Reinders’: cap. XII, Fig. 2; ‘Fuji Kiku-8’: cap. XIII, Fig. 1), ya
que las uniones covalentes de las pectinas a la pared celular se establecen preferentemente en
las cadenas ramificadas de los ramnogalacturonanos (Hwang et al., 1993), que son ricas en
residuos de galactosa y arabinosa (Caffall y Mohnen, 2009).
Tanto el almacenamiento en ULO como el tratamiento con calcio fueron en general

efectivos para ralentizar el desensamblaje de las paredes celulares en ambas variedades de
manzana, lo que resulta en la mejor preservación de la firmeza de los frutos tras su
frigoconservación. Esta mejora podría explicarse en parte por la inhibición de algunas
actividades enzimáticas (PG, PL, AFasa y β-Gal), que podrían por tanto considerarse claves
en el metabolismo de las paredes celulares durante la maduración del fruto. Sin embargo, los
mecanismos mediante los que ambos procedimientos post-cosecha condujeron a la inhibición
de estas actividades enzimáticas no quedan claros a partir de los datos presentados, aunque
podrían estar relacionados en parte con la reducción en la producción de etileno de los frutos
tratados respecto a los controles (cap. XII, Tabla 2), ya que se ha observado que la regulación
de las actividades PG, AFasa y β-Gal en manzana ‘Golden Delicious’ es etileno-dependiente
(Wei et al., 2010).
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Discusión general
Un resultado interesante de estos estudios es que el tratamiento con calcio de

manzanas ‘Golden Reinders’ y ‘Fuji Kiku-8’ no únicamente mejoró la firmeza de los frutos, e
incluso en algunos casos su calidad aromática, sino que los análisis de calidad organoléptica
realizados mediante un panel de jueces entrenados mostraron que otros atributos con
influencia sobre la textura también se vieron afectados. Para ambas variedades, las manzanas
tratadas se caracterizaron, en general, por una mejora en la crocanticidad, jugosidad y dureza
percibidas tras 4 ó 7 meses en frío normal, así como por una menor harinosidad (cap. XVI,
Tabla 4). Por todo ello, la aceptabilidad de consumo fue superior en los frutos tratados con
calcio, lo que concuerda con estudios anteriores en ‘Golden Delicious’ (Abbott et al., 2000),
en que los consumidores prefirieron los frutos tratados con calcio. Así, este tipo de
tratamiento, de fácil aplicación y bajo coste, resulta altamente recomendable para la mejora
global de la calidad organoléptica de las manzanas ‘Golden Reinders’ y ‘Fuji Kiku-8’, incluso
tras una frigoconservación de larga duración (7 meses).
4.2. Melocotón (Prunus persica L. Batsch, cv. ‘Rich Lady’ y ‘Tardibelle’).
La abrupta pérdida de firmeza que tiene lugar durante la vida post-cosecha en

variedades de melocotón de tipo ‘melting’, como ‘Rich Lady’ y ‘Tardibelle’, es el factor que
más limita su potencial de conservación. En el área del Segrià, la conservación en frío normal
es la modalidad de almacenamiento más utilizada para este tipo de frutos. Con la
conservación en frío normal se consigue un cierto retraso en la pérdida de firmeza del fruto,
aunque aún así el período de vida post-cosecha sigue siendo limitado. Por ello, es de gran
interés identificar alternativas al almacenamiento en frío normal que permitan prolongar los
períodos de almacenamiento preservando los parámetros de calidad comercial y sensorial del
producto, incluyendo la firmeza. Por ello, en esta Tesis se estudiaron los efectos del
almacenamiento en atmósfera controlada sobre el metabolismo asociado a la pérdida de
firmeza en melocotones ‘Rich Lady’ y ‘Tardibelle’. Para esta última variedad, además, se
estudió el posible impacto de la aplicación de 1-MCP.
Tras 3 ó 15 días a 2 ºC en atmósfera controlada, los melocotones ‘Rich Lady’ no

mostraron niveles superiores de firmeza respecto a los frutos conservados en frío normal (cap.
XV, Tabla 2). Esto se ha observado anteriormente en otros trabajos (Zhou et al., 2000; Girardi
et al., 2005), en que las atmósferas controladas tampoco fueron efectivas para la preservación
de la firmeza en melocotones y nectarinas. Por el contrario, el almacenamiento en atmósfera
255
Discusión general
controlada sí mantuvo niveles de firmeza significativamente más altos en melocotón

‘Tardibelle’, una variedad de recolección tardía (cap. XIV, Tabla 1). Esto indica que el
genotipo sería un factor influyente que determina la eficacia de las atmósferas controladas
sobre éste u otros atributos de calidad. El uso de 1-MCP fue también una buena alternativa
para la mejora de la firmeza en la variedad ‘Tardibelle’ (cap. XIV, Tabla 1), de acuerdo con lo
observado en otras variedades (Liguori et al., 2004; Girardi et al., 2005). Sin embargo, la
combinación de ambos tratamientos no mejoró significativamente la firmeza de los frutos, que
se mantuvo en valores similares a los frutos control almacenados en frío normal. Esta
observación está en discrepancia con un trabajo previo en ciruela (Prunus salicina Lindl.), en
el que la combinación de ambos factores sí supuso una mejora en el mantenimiento de la
firmeza (Menniti et al., 2006). La causa por la cual la combinación de atmósfera controlada y
1-MCP no fue efectiva para la preservación de la firmeza no queda clara, si bien dosis
mayores de 1-MCP o períodos de exposición más largos podrían ser necesarios para obtener
efectos significativos en frutos almacenados en atmósfera controlada. Si bien tanto la
conservación en atmósfera controlada como el tratamiento con 1-MCP, aplicados por
separado, mejoraron la firmeza de melocotones ‘Tardibelle’, las modificaciones en el
metabolismo de las paredes celulares inducidas en cada caso, y por tanto los mecanismos
bioquímicos subyacentes en dicha mejora, fueron distintas para cada uno de los dos
procedimientos.
Las diferencias de firmeza entre las muestras no guardaron relación aparente con los
rendimientos de material insoluble de pared celular obtenidos, ni en ‘Tardibelle’ (cap. XIV,
Tabla 3) ni en ‘Rich Lady’ (cap. XV, Tabla 3). Esto indica que la composición de las paredes
celulares, y no el contenido total de materiales insolubles, sería el factor con mayor influencia
sobre la firmeza de los frutos, al igual que en manzanas (apartado 4.1). Para la variedad ‘Rich
Lady’, se observó una fuerte asociación entre la firmeza y la fracción rica en pectinas ligadas
covalentemente a la pared (cap. XV, Fig. 1), lo que está en acuerdo con otros trabajos
(Fishman et al., 1993; Zhang et al., 2010). Las actividades AFasa y β-Gal (cap. XV, Tabla 4),
en general, no sufrieron modificaciones en muestras conservadas en atmósfera controlada, lo
que podría explicar la similitud de los rendimientos de la fracción soluble en Na2CO3 y de los
niveles de firmeza respecto a los frutos conservados en frío normal (cap. XV, Tabla 3) en
melocotones ‘Rich Lady’.
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Discusión general
Al contrario que en ‘Rich Lady’, la utilización de atmósferas controladas para la

frigoconservación de melocotones ‘Tardibelle’ se tradujo en un mejor mantenimiento de la
fracción soluble en Na2CO3 (cap. XIV, Tabla 4), posiblemente a causa de la inhibición de las
actividades AFasa y β-Gal (cap. XIV, Tabla 5), además de PL y PG, lo que explicaría por
tanto la eficacia de este procedimiento para preservar la firmeza en esta variedad (cap. XIV,
Tabla 1). Al término del período de frigoconservación, no se observaron diferencias
significativas entre los rendimientos de fracción soluble en Na2CO3 entre las muestras
almacenadas en atmósfera controlada y las tratadas con 1-MCP y conservadas en frío normal
(cap. XIV, Tabla 4). No obstante, los valores de firmeza fueron más elevados en los frutos
tratados con 1-MCP que en los controles conservados en atmósfera controlada (cap. XIV,
Tabla 1), lo que indica que el contenido en otras fracciones podría tener también una
influencia importante sobre este atributo en la variedad ‘Tardibelle’. De hecho, los frutos
tratados con 1-MCP presentaron rendimientos mayores de la fracción soluble en CDTA, rica
en pectinas ligadas a la pared por enlaces no covalentes (cap. XIV, Tabla 3). Por otro lado, la
actividad PME al término de la frigoconservación fue superior en los frutos tratados con 1-
MCP (cap. XIV, Tabla 5), para los que además se observaron actividades PG y PL
significativamente más bajas. Así, una desmetilación más intensa (PME) de los polímeros
pécticos, acompañada de menor despolimerización (PG y PL) de los mismos, incrementaría la
posibilidad de formación de puentes de calcio y conduciría a mayores contenidos de pectinas
ligadas a la pared por enlaces no covalentes, lo que explicaría la mejor preservación de la
firmeza de los frutos tratados con 1-MCP respecto al resto de tratamientos considerados.
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262
CONCLUSIONES
263
264
Conclusiones
PRODUCCIÓN DE COMPUESTOS VOLÁTILES AROMÁTICOS DURANTE LA

MADURACIÓN EN CAMPO DE MANZANA Y MELOCOTÓN:
1. En la fecha de cosecha comercial, tres ésteres de hexilo (acetato, propanoato y

2-metilbutanoato), junto con el 2-metilbutanoato de etilo, el acetato de 2-
metilbutilo y el 2-metilbutanoato de butilo fueron los compuestos con más
contribución al aroma en la variedad de manzana ‘Golden Reinders'.
2. En el estado de madurez correspondiente a la cosecha comercial, seis acetatos

(de 2-metilpropilo, de butilo, de 2-metilbutilo, de pentilo y de hexilo), dos
butanoatos (de metilo y de etilo), 2-metilbutanoatos (de butilo y de hexilo), y
tres propanoatos (de butilo, de 2-metilbutilo y de hexilo) fueron los
compuestos con más contribución al aroma en la variedad de manzana ‘Fuji
Kiku-8’.
3. En melocotón ‘Rich Lady’ se observó una fuerte relación entre la aceptación

sensorial, la percepción del aroma característico y la emisión de algunas
lactonas. No obstante, los compuestos mayoritarios cuantitativamente en la
fracción volátil emitida fueron cuatro ésteres de hexilo (acetato, 2-
metilbutanoato, butanoato y propanoato).
4. En general, los cambios en la actividad AAT (en piel y en pulpa) no

mostraron relación directa con la emisión de ésteres volátiles, lo que sugiere
que, aunque necesaria, dicha actividad no es suficiente para la biosíntesis de
estos compuestos en frutos de manzana y melocotón. La disponibilidad de los
precursores necesarios y/o la especificidad de sustrato de las isoformas
presentes en los tejidos pueden tener mayor relevancia para el proceso.
5. Los tratamientos pre-cosecha con cloruro cálcico en manzana pueden ayudar

a mejorar la calidad aromática de los frutos en el momento de cosecha
comercial, aparentemente como consecuencia de mayores actividades PDC y
ADH que resultarían en mayor disponibilidad de alcoholes y acil CoAs. Ello
permitiría cosechar los frutos en estadios más adecuados para su potencial de
conservación sin perjuicio para su calidad aromática.
265
Conclusiones
PRODUCCIÓN DE COMPUESTOS VOLÁTILES AROMÁTICOS DURANTE LA

POST-COSECHA DE MANZANA Y MELOCOTÓN:
6. El tratamiento post-cosecha con cloruro cálcico incrementó la emisión de la

mayoría de los ésteres volátiles que contribuyen al aroma en manzanas ‘Fuji
Kiku-8’ y ‘Golden Reinders’ después de 4 meses de frigoconservación a 1 ºC,
y mejoró su aceptabilidad de consumo. Estos efectos resultaron del aumento
en las actividades PDC y ADH, que causó una mejora en el suministro de
precursores para la actividad AAT, y fueron menos marcados en frutos
conservados en atmósfera controlada.
7. Para períodos largos (7 meses) de frigoconservación, en manzana ‘Fuji Kiku-

8’ sólo se observaron efectos positivos del tratamiento con cloruro de calcio
sobre la emisión de ésteres volátiles en los frutos almacenados en frío normal,
en este caso como resultado de un incremento en la actividad AAT en la
pulpa. En la variedad ‘Golden Reinders’ el calcio exógeno no tuvo efectos
claros en los frutos conservados en atmósfera controlada, pero causó una
disminución en la emisión de ésteres volátiles en los conservados en frío
normal, paralelamente a una menor actividad ADH. Aún así, no se redujo el
número de compuestos impacto, y la producción de ésteres volátiles fue
superior en los frutos tratados que en los no tratados conservados en hipoxia.
8. La percepción del sabor característico estuvo fuertemente relacionada con la

aceptabilidad de melocotones ‘Rich Lady’ tras la frigoconservación. El
almacenamiento en atmósfera controlada durante 15 días mejoró la
percepción de este atributo sensorial respecto a la conservación en frío
normal, debido al mantenimiento de mayores contenidos en sólidos solubles y
a la mayor emisión de algunos compuestos volátiles, principalmente lactonas.
9. La emisión de ésteres volátiles tras la frigoconservación de melocotón ‘Rich

Lady’ estuvo asociada a la disponibilidad de precursores de tipo alcohol para
la reacción de esterificación. Sin embargo, la emisión de las lactonas que
contribuyeron a la percepción del sabor característico a melocotón (γγ-
266
Conclusiones
octalactona, δ-decalactona y γ-dodecalactona) fue notablemente dependiente

de la actividad AAT en la pulpa de los frutos.
10. Tanto el almacenamiento en atmósfera controlada como la aplicación de 1-

MCP disminuyeron la producción total de ésteres de cadena lineal en
melocotón ‘Tardibelle’ tras 21 días de frigoconservación. El tratamiento con
1-MCP inhibió parcialmente las actividades LOX y HPL, lo que pudo
comprometer el suministro de precursores derivados de ácidos grasos, y
sugiere que dichas actividades son etileno-dependientes. Por el contrario, ni el
tratamiento con 1-MCP ni la conservación en atmósfera controlada causaron
cambios importantes en la producción de ésteres ramificados.
PÉRDIDA DE FIRMEZA Y METABOLISMO DE LAS PAREDES CELULARES

DURANTE LA MADURACIÓN EN CAMPO DE MANZANA Y MELOCOTÓN:
11. Para ambas variedades de manzana consideradas, la pérdida de firmeza

durante la maduración en el árbol estuvo asociada a un descenso en el
contenido de pectinas ligadas a la pared por enlaces no covalentes, paralelo a
la evolución de las actividades PG y PL. En la variedad ‘Fuji Kiku-8’, los
frutos menos firmes estuvieron caracterizados también por menores
contenidos de pectinas unidas por enlaces covalentes, lo que podría explicarse
por el progresivo aumento de las actividades AFasa y β -Gal, responsables de
la despolimerización de las ramificaciones laterales de las pectinas, que
representan el sitio para la unión covalente al resto de polímeros de la pared.
12. Los tratamientos pre-cosecha con soluciones de cloruro de calcio en manzanas

‘Fuji Kiku-8’ inhibieron las actividades PME, PL, AFasa, β-Gal y β-Xyl, y
mejoraron los niveles de firmeza en el momento de cosecha comercial. La
disminución de estas actividades enzimáticas, y la mayor disponibilidad de
calcio para el establecimiento de uniones no covalentes entre polímeros
adyacentes, podrían haber sido clave para el mantenimiento del contenido de
este tipo de pectinas y de hemicelulosas. Los frutos tratados tuvieron
asimismo mayores contenidos de ácidos urónicos y azúcares neutros.
267
Conclusiones
13. Para nectarina ‘Snow Queen’ y melocotón ‘Rich Lady’, la pérdida de firmeza
durante la maduración en el árbol estuvo asociada a la disminución en el
contenido de pectinas ligadas a la pared por enlaces covalentes, posiblemente
a causa de los altos niveles de actividad AFasa y β -Gal en estadios previos al
inicio de la fase aguda de ablandamiento propia de las variedades de tipo
‘melting’.
PÉRDIDA DE FIRMEZA Y METABOLISMO DE LAS PAREDES

CELULARES DURANTE LA POST-COSECHA DE MANZANA Y
MELOCOTÓN:
14. Tanto el almacenamiento en atmósfera controlada como las aplicaciones post-

cosecha de cloruro de calcio frenaron la pérdida de firmeza en manzanas
‘Golden Reinders’ y ‘Fuji Kiku-8’ tras la conservación a 1 ºC durante 4 ó 7
meses. Sin embargo, la combinación de ambos factores no produjo, en
general, efectos aditivos.
15. Para manzana ‘Golden Reinders’, los frutos más firmes tras la conservación a
1 ºC se caracterizaron por mayores contenidos de las fracciones ricas en
pectinas. Los baños post-cosecha en soluciones de calcio inhibieron la
actividad PG, y previnieron la solubilización de las pectinas ligadas a la pared
por enlaces no covalentes. El almacenamiento en atmósfera controlada, en
cambio, inhibió las actividades PL, β -Gal y AFasa. En ambos casos, estas
modificaciones habrían ayudado a mantener la integridad de las paredes
celulares y por tanto a mejorar la firmeza de los frutos.
16. Para manzana ‘Fuji Kiku-8’, los frutos más firmes tras 7 meses de
conservación a 1 ºC mostraron mayores niveles pectinas ligadas a la pared
por enlaces covalentes. Las muestras conservadas en atmósfera controlada
tuvieron actividades β -Gal y β -Xyl significativamente menores. El contenido
de calcio en la pulpa estuvo fuertemente asociado con el contenido de pectinas
ligadas por enlaces no covalentes, que a su vez mostró también alguna
268
Conclusiones
relación con la firmeza, lo que explicaría los efectos beneficiosos de las

aplicaciones post-cosecha de calcio sobre este atributo.
17. El tratamiento post-cosecha con calcio mejoró la crocanticidad, jugosidad y

dureza percibidas por un panel entrenado de consumidores, al tiempo que
disminuyó la sensación de harinosidad. Todo ello, junto con los efectos
positivos del tratamiento sobre la emisión de ésteres volátiles, contribuyó a
una mayor aceptabilidad de consumo.
18. Para melocotón ‘Rich Lady’, la frigoconservación en atmósfera controlada

durante 3 ó 15 días no mejoró la firmeza respecto a la conservación en frío
normal. Por el contrario, esta tecnología sí resultó efectiva para preservar la
firmeza en la variedad tardía ‘Tardibelle’ tras 3 semanas de conservación. El
tratamiento con 1-MCP también mejoró notablemente la firmeza de estos
frutos tras la frigoconservación. Sin embargo, la combinación de ambos
tratamientos resultó en niveles de firmeza similares a los de los controles.
19. La conservación de melocotones ‘Tardibelle’ en atmósfera controlada inhibió

las actividades AFasa, β-Gal, PL y PG, que pudo resultar en menor
solubilización y despolimerización de las pectinas ligadas a la pared por
enlaces covalentes, lo que explicaría el mantenimiento de la firmeza.
20. La aplicación de 1-MCP en melocotón ‘Tardibelle’ inhibió la mayoría de las

actividades pectolíticas estudiadas y retuvo mayores contenidos de pectinas
unidas por enlaces covalentes. Asimismo, la actividad PME fue más alta en los
frutos tratados, que mantuvieron también niveles más altos de pectinas
ligadas por fuerzas no covalentes, posiblemente como consecuencia de la
formación de puentes de calcio entre estos polímeros. Todo ello explicaría el
mejor mantenimiento de la firmeza respecto al uso de atmósferas controladas.
269

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Preharvest Calcium Sprays Improve Volatile Emission at

KEYWORDS: Alcohol o-acyltransferase; alcohol dehydrogenase; apple; aroma; preharvest calcium

© 2010 American Chemical Society Published on Web 12/02/2010 pubs.acs.org/JAFC

compound RIb RIc OTHd H1 H2 H3 H4 H5 H6 H7 H8 H9 H10

compound RIb RIc OTHd H1 H2 H3 H4 H5 H6 H7 H8 H9 H10

tissue activity H1 H2 H3 H4 H5 H6 H7 H8 H9 H10

Figure 2. Loadings plot of PC1 versus PC2 corresponding to a PLSR

skin tissue of calcium-treated samples up to H6 maturity stage

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Calcium Dips Enhance Volatile Emission of Cold-Stored

KEYWORDS: Alcohol o-acyltransferase; alcohol dehydrogenase; calcium applications; controlled

© 2009 American Chemical Society Published on Web 05/18/2009 pubs.acs.org/JAFC

methyl acetate 854 - 8300 (a) 21.21 a -2.59 48.25 a -2.24

at harvest atmosphere untreated CaCl2 untreated CaCl2

atmosphere untreated CaCl2 untreated CaCl2

atmosphere untreated CaCl2 untreated CaCl2

LOX air 5.519 Aa 4.381 Ba 3.875 Aa 2.925 Bb

to produce acetaldehyde, which can be processed subsequently by

CaCl2-treated fruit as a consequence of the beneficial role of this ACKNOWLEDGMENT

Contents lists available at ScienceDirect

Postharvest Biology and Technology

The emission of flavour-contributing volatile esters by ‘Golden Reinders’ apples is

1. Introduction quality (Goldberg, 1984) and in preservation of firmness and cell

Eight kg of intact apples (2 kg/replicate × 4 replicates) were

Volatile compound RIa OTHb Amountc log OAVd

Methyl acetate 854 8300(1) 15.2

Total emission 785.3

Volatile compound Treatment Storage period

Air SCA ULO Air SCA ULO

Propyl acetate Untreated 10.3 Ba 4.1 Bb 4.3 Bb 31.1 Aa 3.0 Ac 4.7 Ab

Methyl butanoate Untreated 4.3 Ba 3.3 Bb 3.1 Bb n.d. <0.5 n.d.

Ethyl butanoate Untreated *1.1 Ba 0.6 Bb 0.5 Bb *3.1 Aa 0.5 Bb 0.5 Bb

Propyl propanoate Untreated 1.6 Ba 0.5 Ab <0.5 4.4 Aa 0.6 Ab n.d.

Butyl acetate Untreated *425.8 Ba *84.7 Bb *25.5 Ac *1473.1 Aa *75.9 Ab *25.4 Ac

Butyl propanoate Untreated *38.7 Ba 10.1 Ab 3.1 Ac *87.9 Aa 8.7 Ab 1.8 Ac

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Ethyl butanoate Untreated 1.1 Ba 0.6 Bb 0.5 Bb 3.1 Aa 0.5 Bb 0.5 Bb

Butyl acetate Untreated 425.8 Ba 84.7 Bb 25.5 Ac 1473.1 Aa 75.9 Ab 25.4 Ac

Butyl propanoate Untreated 38.7 Ba 10.1 Ab 3.1 Ac 87.9 Aa 8.7 Ab 1.8 Ac

Pentyl acetate Untreated 16.0 Ba 12.1 Bb 4.8 Ac 76.0 Aa 12.1 Ab 3.8 Ac

Butyl butanoate Untreated 198.6 Ba 26.9 Ab 10.4 Ab 417.1 Aa 32.1 Ab 11.5 Ab

Ethyl hexanoate Untreated 1.5 Ba <0.5 n.d. 2.5 Aa 0.8 Ab n.d.

Hexyl acetate Untreated 499.8 Ba 179.9 Ab 52.2 Ac 1255.3 Aa 155.8 Ab 80.8 Ac

Hexyl propanoate Untreated 80.7 Ba 28.9 Bb 0.9 Bc 166.4 Aa 44.4 Bb *12.7 Ac

Hexyl butanoate Untreated 411.9 Ba 101.6 Bb 58.2 Ab 838.1 Ba 170.2 Ab 61.8 Ac

2-Methylpropyl acetate Untreated 15.7 Ba 6.4 Bb 3.6 Ac 65.0 Aa 7.7 Ab *4.8 Ac

Ethyl 2-methylbutanoate Untreated 10.9 Aa 7.7 Bb 7.3 Bb 4.0 a <0.5 *2.1 b

2-Methylbutyl acetate Untreated 153.5 Ba 152.9 Ba 57.5 Ab 178.4 Aa 116.2 Ab 83.1 Ac

Butyl 2-methylbutanoate Untreated 76.2 Ba 36.7 Ab 10.4 Ac 145.0 Aa 29.9 Ab 9.3 Ac

Hexyl 2-methylbutanoate Untreated 225.9 Ba 188.1 Ab 70.1 Ac 206.5 Aa 174.4 Bb 46.9 Ba