Modificaciones En Factores Relacionados Con El Aroma Y La Textura De La Manzana, Melocotón Y Nectarina Durante La Maduración Y La Post- Cosecha
Modificaciones en factores relacionados con el aroma y la textura de la manzana, melocotón y nectarina durante la maduración y la post- cosecha
Abel Ortiz Catalán
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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 Quı ´ mica, Unitat de Postcollita-XaRTA, Universitat de Lleida, Alcalde Rovira Roure 191, 25198 Lleida, Spain, and ‡Departament de Tecnologı ´ a 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 CaCl 2 (1.6%, w/v). Calcium treatment of apples is a widely used practice, which Treatment period was 23 June to 4 August 2008, corresponding to 81 and 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 (CaCl 2) 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: [email protected]. 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 Harvest a 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 -1 using 1% (v/v) phenolphthalein; results were given as g of malic acid L . 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) I 2 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 Quı ´mica, S.A. (Castellar del Vall es, 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 e b - Determination of Calcium Content. Lyophilized tissue (1 g) was parameter are significantly different at P 0.05 (LSD test ). EUROFRU 1 10 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, -1 ACTIVA’ spectrometer, and results were expressed as mg 100 g FW . 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 -1 into an 8-L Pyrex container, and an air-stream (900 mL min ) 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 -1 -1 (400 mL min ) and H 2 (32 mL min ). 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 - H9, and H10 fruit ( Table 1 ) were suitable for storage according system (Agilent Technologies 6890N 5973N) was used for compound -1 confirmation, in which the same capillary column was used as in the GC to local recommendations (TA<4 g L , 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 - Table 2. Emission ( μg kg 1) of Straight- (A) and Branched-Chain (B) Esters by ‘Fuji Kiku-8’ Apples during On-Tree Maturation a A compound RI b RI c OTH d 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 (LSD = 7.1 ) calcium 34.8 a 50.3 a 104.9 a 110.3 a 119.9 a 119.4 a 132.3 a 130.2 a 70.7 a 71.6 a 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 (LSD = 1.0 ) calcium 1.1 a 1.7 a 1.4 a 1.0 a 2.4 a 1.5 a 5.0 a 5.1 a 4.7 a 5.4 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 (LSD = 3.2 ) calcium 1.4 b 2.1 b 1.7 b 2.4 b 2.7 b 12.6 a 13.7 a 20.7 a 29.0 a 47.3 a 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 (LSD = 4.4 ) calcium 16.3 a 16.8 a 17.0 a 24.2 a 23.6 a 25.7 a 26.3 a 30.5 a 51.4 a 67.5 a 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 (LSD = 5.2 ) calcium 27.4 a 28.0 a 29.4 a 21.9 a 19.5 a 9.5 a 6.6 a 14.4 a 30.7 a 40.5 a 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 compound RI b RI c OTH d 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 (LSD = 4.5 ) calcium 14.3 a 20.7 a 20.1 a 17.0 a 19.3 a 14.2 a 12.3 a 12.4 a 10.2 a 10.4 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 (LSD = 29.8 ) calcium 0.6 a 3.3 a 7.7 a 8.9 a 37.2 a 44.8 a 90.1 a 214.6 a 395.1 a 531.4 a 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 (LSD = 3.8 ) calcium 4.1 a 4.8 a 4.2 a 5.2 a 10.4 a 9.0 a 13.1 a 17.6 a 18.1 a 25.4 a 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 (LSD = 6.6 ) calcium 11.8 b 11.1 b 8.8 b 7.8 b 3.1 b 1.6 a 0.8 a 1.2 a 4.2 a 9.1 a 338 J. Agric. Food Chem., Vol. 59, No. 1, 2011 Ortiz et al. Table 2. Continued B compound RI b RI c OTH d 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 (LSD = 6.0 ) calcium 29.1 a 29.6 a 27.3 a 27.8 a 36.2 a 39.6 a 39.0 a 41.0 a 46.5 a 74.6 a 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. Log 10 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-8 0 Apples around the a fraction emitted by fruit during the experimental time, although Commercial Harvest 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 by treatment, while significant differences between treated and methyl butanoate control <0 <0 0.14 a untreated fruit were observed in other instances. In some cases, calcium <0 <0 0.15 a 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 esters (2-methylpropyl acetate, 2-methylbutyl acetate, 2-methyl- calcium 0.27 a 0.42 a 0.58 a butyl propanoate, and hexyl 2-methylbutanoate) was increased butyl propanoate control <0 <0 0.33 a 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 Twelve out of the 31 volatile esters identified during the experi- hexyl 2-methylbutanoate control 0.74 b 0.79 b 1.01 b mental period were found to have log odor units (OU)>0 by the calcium 0.83 a 0.89 a 1.09 a time of commercial harvest ( Table 3 ) and thus deemed as likely to a 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 e compounds, with the exception of 2-methylpropyl and pentyl different letters are significantly different at P 0.05 (LSD test ). 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, Article J. Agric. Food Chem., Vol. 59, No. 1, 2011 339 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 CaCl 2 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 O 2 gradients across apple tissues in response to the treatment, due to higher difficulty for O 2 diffusion ( 22 ) and to augmented internal CO 2 levels ( 23 -25 ), causing hypoxia-like induction of PDC and ADH. PDC uses a 2-oxoacid to render CO 2 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 Maturation a compound RI b RI c 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 (LSD = 10.6 ) calcium 70.2 a 67.8 a 57.7 a 50.3 a 43.1 a 30.2 a 22.1 a 14.7 a 11.6 a 10.4 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 ). 340 J. Agric. Food Chem., Vol. 59, No. 1, 2011 Ortiz et al. Table 5. Flavor-Related Enzyme Activities (U mg protein -1) in ‘Fuji Kiku-8’ Apples during On-Tree Maturation a 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 AAT b 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 AAT b 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 tissue ( Table 5 ). 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Factors affecting apple aroma/flavour 620 -622. volatile concentration: a review. N. Z. J. Crop Hort. Sci. 2000 , 28 , (10) Ortiz, A.; Echeverrı ´ a, G.; Graell, J.; Lara, I. Calcium dips enhance 155 -173. aroma volatile emission of cold-stored ‘Fuji Kiku-8’ apples. J. Agric. (26) Gilliver, P. J.; Nursten, H. E. The source of the acyl moiety in the Food Chem. 2009 , 57 , 4931 -4938. biosynthesis of volatile banana esters. J. Sci. Food Agric. 1976 , 27 , (11) Ortiz, A.; Echeverrı ´ a, G.; Graell, J.; Lara, I. The emission of flavour- 152 -158. contributing volatile esters by ‘Golden Reinders’ apples is improved (27) Vancanneyt, G.; Sanz, C.; Farmaki, T.; Paneque, M.; Ortego, F.; after mid-term storage by postharvest calcium treatment. Posthar- Castan ˜ era, P.; Sa ´ nchez-Serrano, J. J. Hydroperoxide lyase depletion vest Biol. Technol. 2010 , 57 , 114 -123. in transgenic potato plants leads to an increase in aphid perfor- (12) Ke, D.; Yahia, E. M.; Mateos, M.; Kader, A. A. Ethanolic mance. Proc. Natl. Acad. Sci. U.S.A. 2001 , 98 , 8139 -8144. fermentation of ‘Barlett’ pears as influenced by ripening stage and (28) Ferreira, L.; Perestrelo, R.; Caldeira, M.; C amara,^ J. S. Character- atmospheric composition. J. Am. Soc. Hort. Sci. 1994 , 119 , 976 -982. ization of volatile substances in apples from Rosaceae family by (13) Lara, I.; Miro ´ , R. M.; Fuentes, T.; Sayez, G.; Graell, J.; Lo ´ pez, M. L. headspace solid-phase microextraction followed by GC -qMS. Biosynthesis of volatile aroma compounds in pear fruit stored under J. Sep. Sci. 2009 , 32 , 1875 -1888. long-term controlled atmosphere conditions. Postharvest Biol. Tech- (29) Picchioni, G. A.; Watada, A. E.; Conway, W. S.; Whitaker, B. D.; nol. 2003 , 29 , 29 -39. Sams, C. E. Postharvest calcium infiltration delays membrane lipid (14) Vick, B. A. A spectrophotometric assay for hydroperoxide lyase. catabolism in apple fruit. J. Agric. Food Chem. 1998 , 46 , 2452 -2457. Lipids 1991 , 26 , 315 -320. (15) Bradford, M. M. A rapid and sensitive method for the quantitation Received for review September 16, 2010. Revised manuscript received of microgram quantities of protein utilizing the principle of protein November 12, 2010. Accepted November 13, 2010. A. Ortiz is the dye binding. Anal. Biochem. 1976 , 72 , 248 -254. (16) Lara, I. Changes in flavour-related volatile production during post- recipient of an FPU grant from the Ministerio de Ciencia e harvest handling of apple and pear fruit. Fresh Produce 2010 , 4, Innovacio ´ n (MICINN ) of Spain. This work was supported through the 76 -84. AGL2006-00345/ALI project, financed by the Ministerio de Educacio ´ n (17) Buttery, R. G. Quantitative and sensory aspects of flavor of tomato y Ciencia (MEC ) of Spain. We are indebted to Mrs. P. Sopen ˜ a for and other vegetables and fruits. In Flavor Science: Sensible Principles technical assistance. Q R Q R H !"#$%" &'( ) $'( '"'"* +, - ./01 " " 2 "30# " " $ 20#4# "5 "6230# ! 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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 , G EMMA ECHEVERRI ´ A, J ORDI 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 O 2/2 kPa of CO 2), 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 CaCl 2-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 threshold of each individual emitted compound. Volatile produc- produce. In addition, calcium chloride (CaCl 2) has been widely 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: [email protected]; 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 4932 J. Agric. Food Chem., Vol. 57, No. 11, 2009 Ortiz et al. 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   before marketing and to assess possible interactions, CaCl - (storage period atmosphere calcium treatment) were weighed, placed 2 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. N 2 carrier gas, air, and H 2 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) CaCl 2 solution at ambient temperature for method of dynamic headspace as described in ref ( 10 ), with some 5 min. After treatment, CaCl 2-treated and untreated apples were stored at modifications. Briefly, intact fruits from each treatment were placed into ° -1 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 ) was passed ULO conditions (1 kPa of O 2/2 kPa of CO 2). The experimental chambers 3 through for 4 h. The effluent was then recovered in an adsorption tube (20 m ) at the UdL-IRTA research center were used for storage of fruit. (ORBO-32; Supelco, Bellefonte, PA) filled with 100 mg of activated The O 2 and CO 2 concentrations were monitored continuously and charcoal (20/40 mesh), from which volatile compounds were desorbed corrected automatically using N 2 from a tank and by scrubbing off excess by agitation for 40 min with 0.5 mL of diethyl ether. Identification and CO 2 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 H (32 mL min 1). The injector and detector were nol, hexyl acetate, 1-hexanol, and hexyl 2-methylbutanoate were obtained 2 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 Quı´mica, S.A. (Castellar del Vall es, 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 -1 Tokyo, Japan), and results were expressed as percent of sucrose in an was used as the carrier gas (42 cm s ), 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 D 65 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 -1 sing. Weight loss after lyophilization was consistently around 80%. One detector. Nitrogen was used as the carrier gas (24 cm s ), 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 Article J. Agric. Food Chem., Vol. 57, No. 11, 2009 4933 Table 1. Meaning of X, Y, and Z Values for the Generic Sample Labels Did Calcium-Treated Fruit Actually Incorporate Calcium? To check the actual incorporation of calcium into the tissues after 1 2 treatment and storage, flesh samples were analyzed by ICP-OES. a X 4 7 Dipping of apples in 2% (w/v) CaCl 2 resulted in significantly Y b air 1:2 increased concentrations of calcium in the pulp of fruit ( Table 3 ). Z c 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 CaCl 2 treatment was efficient in incorporating calcium into fruit c conditions (O 2/CO 2). Calcium treatment (% CaCl 2, 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 CaCl 2-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 CaCl 2 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, 4934 J. Agric. Food Chem., Vol. 57, No. 11, 2009 Ortiz et al. - Table 2. Emission of Aroma Volatile Compounds ( μg kg 1) by ‘Fuji Kiku-8’ Apples 0 (H) and 7 (H+7) Days after Harvest compound a RI b RI c OTH d He log OU f H+7 e log OU f code g 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 Compounds identified on the basis of a comparison of mass spectrometric data and retention indices with authentic reference compounds. b 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 f g same row followed by different lower case letters are significantly different at P e 0.05 (LSD test). Log 10 of odor unit value = log 10 (amount/OTH). 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 Storage a 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 CaCl 2 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. CaCl 4.40 Ab 5.89 Aa 2 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 CaCl 2-treated than in untreated butanoate, was higher in CaCl 2-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 CaCl 2-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, CaCl 2 The reduction in total volatile emission after long-term storage of Article J. Agric. Food Chem., Vol. 57, No. 11, 2009 4935 Table 4. Maturity and Quality Parameters of ‘Fuji Kiku-8’ Apples at Harvest and after 7 Days at 20 °C following Cold Storage a storage period 4 months 7 months at harvest atmosphere untreated CaCl 2 untreated CaCl 2 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 Storage a storage period 4 months 7 months atmosphere untreated CaCl 2 untreated CaCl 2 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 4936 J. Agric. Food Chem., Vol. 57, No. 11, 2009 Ortiz et al. Table 6. Specific Activities (U mg -1 protein) of Volatile-Related Enzymes in the Pulp of ‘Fuji Kiku-8’ Apples after 7 Days at 20 °C following Cold Storage a storage period 4 months 7 months atmosphere untreated CaCl 2 untreated CaCl 2 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 O 2 levels in fruit tissues. Previous work ( 29 ) has shown that gradients in O Figure 1. A B 2 Scores ( ) and loadings ( ) 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 CaCl , due to decreased O diffusivity in versus volatile-related enzyme activities ( variables) in ‘Fuji Kiku-8’ apple 2 2 X 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 CO compounds are labeled as indicated in Tables 1 and 2, respectively 2 levels in apples ( 30 , 31 ). Thus, dipping of fruit in a CaCl solution (AA, acetaldehyde). 2 could have lessened O 2 levels and caused CO 2 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 CaCl 2-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 CaCl 2 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 CaCl 2- pulp tissue, particularly for fruit treated with CaCl 2 and stored in treated fruit was also higher than in untreated apples ( Table 4 ). air. The plot also indicated higher volatile production for CaCl 2- 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 Article J. Agric. Food Chem., Vol. 57, No. 11, 2009 4937 - Table 7. Emission ( μg kg 1) of Alcohol Precursors for Volatile Ester 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 a Storage precursors. Anyhow, air-stored samples dipped in CaCl 2 emitted storage period higher levels of some volatile compounds than untreated fruit. 4 months 7 months After 7 months of cold storage, CaCl 2-treated apples stored in air were characterized by higher AAT activity in the pulp tissue atmosphere untreated CaCl 2 untreated CaCl 2 (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 CaCl 2-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. CaCl 2-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 CaCl -treated 2 LITERATURE CITED apples, particularly after 4 months of storage ( Table 5 ), despite lower LOX activity levels ( Table 6 ), suggests that applied calcium (1) Yuen, C. M. C. Calcium and fruit storage potential. Aust. Centre Int. might be exerting a protective role on membranes, which would Agric. Res. 1994 , 50 , 218–227. allow a better regulation of LOX activity. (2) Glenn, G. M.; Poovaiah, B. W. Calcium-mediated postharvest changes in texture and cell wall structure and composition in ‘Golden With regard to fruit stored for 7 months, no significant Delicious’ apples. J. Am. Soc. Hortic. Sci. 1990 , 115 , 962–968. influence of calcium treatment on PDC and ADH activities was (3) Conway, W. S.; Sams, C. E.; Abbott, J. A.; Bruton, B. D. 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Q R M 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 article info abstract 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 O 2:2 kPa CO 2) or ultra•low oxygen atmosphere (ULO; 1 kPa O 2:2 kPa CO 2) 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: [email protected] (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 D 65 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, CaCl 2•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. N 2 carrier gas, air and H 2 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 L 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 L 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 CaCl solution (2%, w/v, in 2 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, CaCl •treated and untreated apples were stored at 1 ◦C and 2 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 O :2 kPa CO ) or ultra•low oxygen atmosphere (ULO; 1 kPa 2 2 as the stationary phase, and a flame ionisation detector. Nitrogen O :2 kPa CO ). O and CO concentrations were monitored contin• 2 2 2 2 was used as the carrier gas (45 mL min −1), and operating conditions uously, and corrected automatically using N from a tank and by 2 were as follows: oven temperature 80 ◦C, injector temperature scrubbing off excess CO with a charcoal system. A humidifier was 2 180 ◦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 L 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•32 TM ; 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 m) capillary col• spectroscopy (ICP•OES) in a ‘Horiba Jobin Yvon ACTIVA’ spectrom• umn. The injection volume was 1 L 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• −1 expressed as mg 100 gFW . 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 −1 perature. Helium was used as the carrier gas at a flow rate of Calcium content (mg 100 gFW ) in the flesh of ‘Golden Reinders’ apples after 7 d at 20 ◦C following cold storage. 0.8 mL min −1, with a split ratio of 40:1, in the presence of air −1 −1 Storage atmosphere Treatment Storage period (400 mL min ) and H 2 (32 mL min ). The injector and detector ◦ 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• CaCl 2 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 CaCl 2 4.8 Aa 4.9 Aa the matrix to develop standard curves for each volatile analysed. A GC–MS system (Agilent Technologies 6890N•5973N) was used ULO Untreated 3.6 Ba 4.1 Ba CaCl 5.8 Aa 5.3 Aa for compound confirmation, in which the same capillary column 2 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 −1 gas (42 cm s ), 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 g 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 3 A). 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 3 B). 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) CaCl 2 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 CaCl 2 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 3 A). Although the 50 g 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 3 B). 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 A. Ortiz et al. / Postharvest Biology and Technology 57 (2010) 114–123 117 Table 2 Emission of aroma volatile compounds ( g kg −1) by ‘Golden Reinders’ apples at harvest. Volatile compound RI a OTH b Amount c log OAV d 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 ( g 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 Log 10 of odour activity value = log 10 (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 O 2 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 118 A. Ortiz et al. / Postharvest Biology and Technology 57 (2010) 114–123 Table 3 Emission ( g 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 CaCl 2 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 CaCl 2 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 CaCl 2 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. CaCl 2 *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 CaCl 2 *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. CaCl 2 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 CaCl 2 *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 CaCl 2 *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 CaCl 2 *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 CaCl 2 *281.6 Aa 38.4 Ab 9.4 Ac *296.9 Ba 52.0 Ab 10.4 Ac Ethyl hexanoate Untreated *1.5 Ba <0.5 n.d. *2.5 Aa 0.8 Ab n.d. CaCl 2 *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 CaCl 2 *913.7 Aa *190.3 Ab *47.8 Ac *942.7 Ba *207.5 Ab *40.0 Ac Propyl hexanoate Untreated 11.1 Ba 4.5 Ab 0.9 Ac 17.3 Aa 4.7 Ab 3.3 Ac CaCl 2 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 CaCl 2 *138.7 Aa *42.3 Ab *12.6 Ac *102.7 Ba *63.0 Ab *17.6 Ac Butyl hexanoate Untreated 226.4 Ba 58.4 Bb 39.4 Ab *395.4 Aa 80.0 Bb 49.3 Ac CaCl 2 *364.8 Aa 99.1 Ab 36.6 Ac *287.5 Ba 111.5 Ab 36.0 Ac Hexyl butanoate Untreated *411.9 Ba 101.6 Bb 58.2 Ab *838.1 Ba 170.2 Ab 61.8 Ac CaCl 2 *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 CaCl 2 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 CaCl 2 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 CaCl 2 219.3 Aa 110.0 Ab 32.4 Ac 254.2 Aa 140.7 Ab 39.2 Ac Butyl octanoate Untreated 12.1 Ba 9.6 Bb 3.7 Ac 22.4 Aa 12.3 Ab 3.7 Ac CaCl 2 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. CaCl 2 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 CaCl 2 3590.5 Aa 850.8 Ab 259.5 Ac 3876.7 Ba 1027.0 Ab 274.8 Ac B Tert •butyl propanoate Untreated 7.0 Aa 5.2 Ab 0.9 Ac 8.2 Aa 6.1 Ab 5.5 Ab CaCl 2 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 CaCl 2 *22.5 Aa *9.4 Ab 3.4 Ac *33.2 Ba *8.5 Ab 3.9 Ac Ethyl 2•methylbutanoate Untreated *10.9 Aa *7.7 Bb *7.3 Bb *4.0 a <0.5 *2.1 b CaCl 2 *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 CaCl 2 *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. A. Ortiz et al. / Postharvest Biology and Technology 57 (2010) 114–123 119 Table 3 ( Continued ) Volatile compound Treatment Storage period 19 weeks 31 weeks Air SCA ULO Air SCA ULO CaCl 2 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. CaCl 2 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 CaCl 2 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 CaCl 2 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 CaCl 2 *144.0 Aa *47.2 Ab 13.1 Ac *70.7 Ba *34.1 Ab 11.3 Ac 2•Methylbutyl 2•methylbutanoate Untreated 6.5 Ba 4.9 Bb 3.5 Ab 11.4 Aa 9.4 Ab 6.7 Ac CaCl 2 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 CaCl 2 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 CaCl 2 *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 CaCl 2 762.5 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 ( g 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 Air SCA ULO Air SCA ULO Ethanol Untreated 10.9 Ba 11.3 Ba 9.2 Aa 29.0 Aa 16.2 Ab 17.3 Aab CaCl 2 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 CaCl 2 6.8 Aa 2.7 Ab 1.0 Ac 10.7 Ba 2.4 Ab 1.6 Ac 2•Methyl•1•propanol Untreated 6.3 Ba 2.1 Bb 1.0 Ac 16.9 Ba 3.8 Bb 0.9 Ac CaCl 2 7.9 Aa 3.0 Ab 0.9 Ac 22.9 Aa 5.1 Ab 1.0 Ac 1•Butanol Untreated 66.8 Ba 10.3 Bb 5.2 Ac 304.3 Aa 18.4 Bb 5.2 Ac CaCl 2 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 CaCl 2 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 CaCl 2 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 CaCl 2 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 CaCl 2 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 CaCl 2 235.0 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). 120 A. Ortiz et al. / Postharvest Biology and Technology 57 (2010) 114–123 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 Air SCA ULO Air SCA ULO Skin tissue PDC Untreated 12.308 Ba 12.007 Ba 9.609 Ab 16.921 Aa 17.571 Ba 14.663 Bb CaCl 2 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 CaCl 2 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 CaCl 2 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 CaCl 2 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 CaCl 2 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 CaCl 2 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 CO 2 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 O 2 levels and caused CO 2 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 CaCl 2•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 O 2 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 O 2 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 Air SCA ULO Air SCA ULO Ethylene production ( L kg −1 h−1) Untreated 297.1 Aa 118.0 Ab 112.1 Ab 334.2 Aa 123.7 Ab 115.2 Ab CaCl 2 230.7 Ba 89.3 Ab 82.4 Ab 279.4 Ba 98.9 Ab 87.5 Ab Acetaldehyde content ( L L −1) Untreated 0.9 Ba 1.0 Ba 0.9 Ba 1.8 Aa 1.7 Aa 1.6 Aa CaCl 2 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 CaCl 2 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 CaCl 2 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 CaCl 2 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 CaCl 2 116.3 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). A. Ortiz et al. / Postharvest Biology and Technology 57 (2010) 114–123 121 Fig. 1. Correlation between the emission ( g 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• 122 A. Ortiz et al. / Postharvest Biology and Technology 57 (2010) 114–123 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. samples. Hewett, E.W., Thompson, C.J., 1992. Modification of internal carbon dioxide and oxygen levels in apple fruit by postharvest calcium application and modified In summary, postharvest calcium treatments have the potential atmospheres. Postharvest Biol. Technol. 1, 213–219. to improve flavour quality of ‘Golden Reinders’ apples after mid• Hoehn, E., Gasser, F., Guggenbühl, B., Künsch, U., 2003. Efficacy of instrumental term storage under air or, to a lesser extent, under SCA, through measurements for determination of minimum requirements of firmness, sol• 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. 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Boelens Aroma Chemical Information Service, Huizen, The Nether• expression and volatile ester emission during apple fruit maturation and ripen• lands. ing. Postharvest Biol. Technol. 49, 330–339. Q R !!" + +:M+* : :+ LWT - Food Science and Technology 42 (2009) 1520–1529 Contents lists available at ScienceDirect 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 Echeverrı´a, Jordi Graell, Isabel Lara * A`rea de Post-Collita, XaRTA, UdL-IRTA, Alcalde Rovira Roure 191, 25198 Lleida, Spain article info abstract 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% CO 2 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, Lo ´ pez, & 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 CO 2 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: [email protected] (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 (Segria`, data were parametrised as 0.56 (weaker perception) or 0.56 NE Spain) at commercial maturity according to the usual standards (stronger perception) according to Anzaldu ´ a-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 P 1, P 2 and P 3, 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 O 2: 10 kPa CO 2) 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 P 1 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 P 1 P2 P3 Period a 3 þ 1 15 þ 1 3 þ 1 15 þ 1 3 þ 1 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 þ1: 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 ( 20 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 þ1 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 þ1 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. 3 A), which explained alone 51% of To provide a general visualisation of all the information contained total variance. The loadings plot ( Fig. 3 B) 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. 3 B). 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, Gu ¨ nata, 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 C 6 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., 2006 ), and thus higher production of these compounds must have 1 2 3 a an influence on perception of the characteristic peach flavour by X P1 P2 P3 Yb Air 3:10 the consumer. Zc 3 15 Flavour perception was also related to SSC ( Fig. 3 B), consistent a with the relationship found between sensory acceptance and Picking date (P 1, P 2, P 3: 29th June, 3rd July and 6th July 2006, respectively). b Storage atmosphere conditions (O 2:CO 2). 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 A. Ortiz et al. / LWT - Food Science and Technology 42 (2009) 1520–1529 1523 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. 4 A), to this attribute ( Fig. 3 B). Interestingly, most volatile compounds illustrating the limited potential of peach fruit for post-harvest identified were associated to 15 þ1 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 þ1 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). 1524 A. Ortiz et al. / LWT - Food Science and Technology 42 (2009) 1520–1529 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. 4 B), 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 Code a Compound Code a 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 O or 30 kPa CO (Bonghi, Ramina, Ruperti, Vidrih, & Tonutti, 1999 ), 2 2 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 flavours ( Tonutti, Bonghi, Ramina, & Vidrih, 1998 ). In contrast, Pentyl acetate pa Linalool lin þ 2-Methylbutyl 2mb2mpr Hexyl hexanoate hh 15 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 the ‘‘peachy’’ aroma ( Horvat et al., 1990 ). Codes used for multivariate analysis. A. Ortiz et al. / LWT - Food Science and Technology 42 (2009) 1520–1529 1525 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. 5 B) shows that samples biosynthesis of aroma compounds ( Sanz, Olı´as, & Pe ´rez, 1997 ). AAT distributed preferentially according to picking date, P 2 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 P 2 fruit ( Fig. 4 ). The lowest values for AAT activity were ADH are also involved in the pathways to supply the required found for P 3 peaches, while the activity in the flesh tissue of P 1 precursors. Specifically, LOX is assumed to contribute to the samples was dependent upon storage atmosphere. These results breakdown of long-chain fatty acids to C 6 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. 5 A). Interestingly, all three cold storage of fruit ( Table 4 ). However, similar levels of AAT activity lactones contributing to the perception of peach flavour ( Fig. 3 B) were observed upon removal from storage regardless of storage were among the compounds seemingly most dependent on AAT atmosphere and period. For P 3 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 1526 A. Ortiz et al. / LWT - Food Science and Technology 42 (2009) 1520–1529 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 period a 3 þ 1 15 þ 1 3 þ 1 15 þ 1 Shelf life period b 03030 30 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 þ1: 15 days at 2 C þ 1 day at 7 C. b Days at 20 C following cold storage. A. Ortiz et al. / LWT - Food Science and Technology 42 (2009) 1520–1529 1527 which thus seems to be a major limiting factor determining the storage period, 15 þ1 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. 6 A), 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. P 2 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. 6 B). As increases in ADH activity were found uniquely for P 2 fruit, and only an example, the biplot of a PLSR model developed for ethanol ( X upon removal from storage regardless of period. Interestingly, P 2 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). 1528 A. Ortiz et al. / LWT - Food Science and Technology 42 (2009) 1520–1529 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. 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Lipids, 26 , Saftner, R. A., Abbott, J. A., & Conway, W. S. (2002). Instrumental and sensory quality 315–320. characteristics of ‘Gala’ apples in response to prestorage heat, controlled Visai, C., & Vanoli, M. (1997). Volatile compound production during growth and atmosphere, and air storage. Journal of the American Society for Horticultural ripening of peaches and nectarines. Scientia Horticulturae, 70 , 15–24. Science, 127 , 1006–1012. Yayaoui, F. E. L., Wongs-Aree, C., Latche ´, A., Hackett, R., Grierson, D., & Pech, J. C. Sanz, C., Olı´as, J. M., & Pe ´rez, A. G. (1997). Aroma biochemistry of fruits and vege- (2002). Molecular and biochemical characteristics of a gene encoding an tables. In F. A. Toma ´s-Barbera´n, & R. J. Robins (Eds.), Phytochemistry of fruit and alcohol acyl-transferase involved in the generation of aroma volatile esters vegetables (pp. 125–155). Oxford, UK: Clarendon Press. during melon ripening. European Journal of Biochemistry, 269 , 2359–2366. 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" !//0 + ++ > % Q3 R #%;(= !#! + B*,M?: + + Food Chemistry 123 (2010) 698–704 Contents lists available at ScienceDirect 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 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 article info abstract 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 nificant changes in the emission of some volatile esters, particularly of the straight-chain type. Some Controlled atmosphere 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 CO 2 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% O 2 + 10% CO 2 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: [email protected] (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 l À1. 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 s À1), 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 l À1. 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 min À1) 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 s À1), 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 min À1); 142–225 °C (5 °C min À1); 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 l À1 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 CO 2), 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 s À1), 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 kg À1. per treatment, kept in respiration jars and aerated continuously with humidified air at a rate of 5 l h À1. 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 N 2 as the carrier drogenase (ADH; EC 1.1.1.1) and alcohol o-acyltransferase (AAT; EC À1 gas, in the presence of air and H 2 (45, 400, and 45 ml min , 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 kg À1 hÀ1. (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 protein À1). 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- À1 À1 Ethylene production ( ll kg h ) 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 H a SL b 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 l À1) 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 l À1) 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). A. Ortiz et al. / Food Chemistry 123 (2010) 698–704 701 Table 3 Table 5 Emission of aroma volatile compounds ( lg kg À1) by ‘Tardibelle’ peach fruit at Methyl, ethyl, hexyl and acetate esters ( lg kg À1) emitted by untreated and 1-MCP- commercial harvest. treated ‘Tardibelle’ peach fruit after storage at 0 °C for 21 days. Compound KI a Amount b SL a Storage atmosphere Methyl acetate 834 2.69 ± 1.42 Air CA Ethyl acetate 901 13.0 ± 5.76 Untreated 1-MCP Untreated 1-MCP 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). 2-Methylbutyl 2-methylbutanoate 1293 1.50 ± 0.80 a Shelf life (days). Trans-2-hexenyl acetate 1348 n.d. Hexyl propanoate 1351 <0.5 1-Hexanol 1361 <0.5 a direct precursor for the synthesis of ethyl esters, by the specific Methyl octanoate 1405 <0.5 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 not shown). CA storage was likewise detrimental for the produc- d-Decalactone 2284 n.d. 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 kg À1) 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 SL a Storage atmosphere hyde to acetate and acetyl-CoA ( Kreuzwieser, Scheerer, & Air CA Rennenberg, 1999 ). Acetaldehyde can also be reduced to ethanol, Untreated 1-MCP Untreated 1-MCP 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 kg À1) 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 SL a 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 Shelf life (days). a Shelf life (days). 702 A. Ortiz et al. / Food Chemistry 123 (2010) 698–704 Table 7 AAT catalyses the esterification reaction between an alcohol À1 Flavour-related enzyme activities (U mg protein ) 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 SL a Storage atmosphere son, & Mattheis, 2000; Yahyaoui et al., 2002 ). When AAT activity Air CA levels were examined, no apparent relationship to the emissions Untreated 1-MCP Untreated 1-MCP of volatile esters was found. Furthermore, AAT gene expression has been reported to be ethylene-dependent in apple ( Defilippi, Skin tissue Kader, & Dandekar, 2005 ), while results do not support an ethylene LOX 0 3.37 a 1.48 b 2.50 ab 1.35 b 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- creased AAT activity in the flesh of 1-MCP-treated fruit was ob- HPL 0 89.2 c 56.3 d 182 a 150 b LSD = 26.874 7 72.9 b 40.8 c 161 a 172 a served, uniquely, one week after removal from storage under CA. 1-MCP-related inhibition of activity levels for this enzyme was also PDC 0 9.16 c 14.4 b 26.5 a 15.2 b 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, but activity recovered rapidly during the subsequent period at ADH 0 10.8 c 17.3 ab 19.9 a 15.9 b 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- À notes ( Herrmann, 1991 ) is reportedly 2 lg kg 1. 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 O 2 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 A. Ortiz et al. / Food Chemistry 123 (2010) 698–704 703 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 ). 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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. MATERIALS AND METHODS 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 th Proc. 6 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 A 235 , due to PL-mediated release of GalUA, was measured over -1 time. One unit of PL activity was defined as the increase in one unit of A 235 min . 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 (PAW sf ), 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), Na 2CO 3, 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, PAW sf 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 (NaCO 3-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 PAW sf yields, indicative of polymers solubilised in vivo. Higher yields of PAW sf , 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 KOH sf 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. Postharvest Biol. Technol. 43:307-318. Lara, I., García, P. and Vendrell, M. 2004. Modifications in cell wall composition after cold storage of calcium-treated strawberry ( Fragaria ananassa Duch.) fruit. Postharvest Biol. Technol. 34:331-339. 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. Siddiqui, S., Brackman, A., Streif, J. and Bangerth, F. 1996. Controlled atmosphere 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. Yoshioka, H., Aoba, K. and Kashimura, Y. 1992. Molecular weight and degree of methoxylation in cell wall polyuronide during softening in pear and apple fruit. J. Am. Soc. Hortic. Sci. 117:600-606. 1034 Tables Table 1. Cell wall-modifying enzyme activities in pulp tissue of ‘Golden Reinders’ apples during on-tree maturation a. -1 b Enzyme activity (U·mg protein Dafb ) 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 00.05 (LSD test). b Days after full bloom. Figures 110 a 100 a 90 b b 80 bc d cd d d 70 60 Firmness (N) Firmness 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 00.05 (LSD test) (dafb: days after full bloom). 1035 A B Fig. 2. 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L % # " # = ' E # =? @ == # ? *' ? # ? = 6 # = '# 6#=' #@ == # 0/31! 4E #=? @== #' ? # '%"# 6#=' #@== # 0/31! 4 $! ;"# 3 %4 # @ = #% 3J4 # ) Q*% 34 ' ? " 3 -4 # = = %# # Q;% C>%*0R ' "#6 )# # " # = / 3="# 4 3' ? " 4 ' > # = 6#=' # == #' ? # #%# =% 0/31! 4 /0 A B ml C D ml "! - #"#* '#*"''@ " 6 = %# 3 % 14 # ' '%"* 3 - @4 Q;% C>%*0R '> K. 34 # K 3 "4 " %@ 6 3"" " 4J `3" /. 3! ) '#" @ 3 %4 @6 '%# # ' @ 3 -4 ' # = = %# # Q;% C>%*0R ' "#6 )# # " # = ' > # = 6#=' # == #' ? # #%# =% 0/31! 4 %*0R ' "#6 )# #" # = ' > #=6#=' #== #' ? # #%# =% 0/31! 4 ;! !*<@ # #* *!*!*6%' # ' # = = %# # Q;%C>%*0R ' "#6 )# #" #= ' > #=6#=' #== #' ? # # %# =% 0/31! 4 1 @"% J -%'> 1 Q. B R ! "#$# /0 00M. Postharvest Biology and Technology 58 (2010) 88–92 Contents lists available at ScienceDirect 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 Departament de Química, Unitat de Postcollita•XaRTA, Universitat de Lleida, Rovira Roure 191, 25198 Lleida, Spain b School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK article info abstract 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: [email protected] (I. Lara). cultivar. 0925•5214/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi: 10.1016/j.postharvbio.2010.05.013 A. Ortiz et al. / Postharvest Biology and Technology 58 (2010) 88–92 89 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 Na 2CO 3, 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 Na 2CO 3•soluble fractions were hydrolysed with sulphuric acid for further analysis. For hydrolysis, 1 mL of 12 M H2SO 4 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, CDTA sf and Na 2CO 3sf 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 L an Effegi penetrometer equipped with an 8•mm diameter convex of ethyl acetate were added to each vial and aliquots of 1 L were tip. According to firmness values ( Fig. 1 A), 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). −1 −1 rate of 5 mL min , in the presence of air (450 mL min ) and H 2 (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 m). 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• •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; PAW sf ) 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 90 A. Ortiz et al. / Postharvest Biology and Technology 58 (2010) 88–92 a standard. One unit (U) of PG activity was defined as the liberation Table 1 of 1 mol of GalUA min −1. PME activity was measured according Yield (% CWM) of fractions isolated from cell wall materials of ‘Snow Queen’ fruit at each sampling date. to Hagerman and Austin (1986) . For the assay, the reaction mixture contained enzyme extract, apple pectin and bromothymol blue pre• Water sf CDTA sf Na 2CO 3sf KOH sf 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 −1 PME activity was defined as the decrease of one unit of A620 min . 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 −1 A235 min . 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 mol of glucose min −1. For the extraction of •galactosidase ( •Gal; EC 3.2.1.23) and a portion of these polysaccharides become water•soluble ( Dawson 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 ). •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 • and water ( Table 1 ), and indicating substantial changes in cell wall −1 Gal was defined as the liberation of 1 mol of p•nitrophenol min polysaccharides. This was confirmed by analysis of uronic acid con• d from p•nitrophenyl• • •galactopyranoside. One unit (U) of AFase tent in both the CDTA• and the Na CO •soluble fractions ( Table 2 ), −1 2 3 was defined as the release of 1 nmol of p•nitrophenol min from showing a significant decline during the melting phase, particularly p•nitrophenyl• ␣•l•arabinofuranoside. in the Na 2CO 3sf . 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• −1 activity (U mg protein ). 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 Na 2CO 3•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 CDTA sf (Table 2 ). These results sug• The softening pattern during on•tree ripening of ‘Snow Queen’ nectarines was representative of that corresponding to a melting flesh•type cultivar. Firmness loss was moderate until commercial Table 2 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. 1 A) 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. 1 B), in accordance with obser• Ara Xyl Man Gal Glc Uronic acids vations for other nectarine cultivars ( Dawson et al., 1992 ). Since CDTA sf 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 compositional and structural changes in the cell wall and flesh H8 2.42 c 0.11 d 0.28 a 1.10 a 3.56 a 7.97 d Na 2CO 3sf 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:PAW sf 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 Na 2CO 3 (Na 2CO 3sf ) and KOH (KOH sf ), 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 (CDTA sf ) 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). A. Ortiz et al. / Postharvest Biology and Technology 58 (2010) 88–92 91 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 Na 2CO 3sf (Table 2 ). gest some remobilisation of insoluble cell wall materials from the For some fruit including peach, pectin solubilisation has been Na 2CO 3• 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 CDTA sf 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 •Gal activity was insoluble and PAW•soluble cell wall materials ( Fig. 1 B) 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. 1 A), which is in accordance Depolymerisation of polyuronides from the pectin network, with the previous idea. However, the pattern of changes in •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 Na 2CO 3sf (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 TBG 4 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:PAW sf ratios ( Fig. 1 B) 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 CDTA sf Na 2CO 3sf PL •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). 92 A. Ortiz et al. / Postharvest Biology and Technology 58 (2010) 88–92 yields of the KOH sf (Table 1 ), which is an indicative of the hemicel• Bonghi, C., Ferrarese, L., Ruperti, B., Tonutti, P., Ramina, A., 1998. Endo• •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 during maturation, ripening and senescence of peach fruit. J. Exp. Bot. 55, KOH sf yields: although a significant decline was found already at the H2 stage ( Table 1 ), suggesting a role for this enzyme in the first 2029–2039. 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• is unclear. EGase activity and gene expression have been reported ering plants: consistency of molecular structure with the physical properties of 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. Lara, I., García, P., Vendrell, M., 2004. Modifications in cell wall composition after were reallocated transiently in the CDTA sf , suggesting the partici• × pation in the process of proteins involved in the rearrangement of cold storage of calcium•treated strawberry ( Fragaria ananassa Duch.) fruit. 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 during ethylene•induced ripening in banana: effect of 1•MCP, ABA and IAA. 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. A. Ortiz is the recipient of a FPU grant from the Ministerio de Moran, F., Nasuno, S., Starr, M.P., 1968. Extracellular and intracellular polygalactur• 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 Catalunya, and by Project AGL2006•00345/ALI, funded by MICINN. fruits. Phytochemistry 48, 249–255. 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. Sope na˜ 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 • 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. P v6B hryyEGh hD Q R ! "#$$ qv) %wsqpur! # % & &! FOCH 10943 No. of Pages 9, Model 5G 19 April 2011 Food Chemistry xxx (2011) xxx–xxx 1 Contents lists available at ScienceDirect 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 a b a,⇑ 4 Abel Ortiz , Jordi Graell , Isabel Lara Ayala 5 a Departament de Química, Unitat de Postcollita-XaRTA, Universitat de Lleida, Rovira Roure 191, 25198 Lleida, Spain 6 b Departament de Tecnologia d’Aliments, Unitat de Postcollita-XaRTA, Universitat de Lleida, Rovira Roure 191, 25198 Lleida, Spain 7 8 article info abstract 21 60 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 CO 2) 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 firmness. ULO-storage and calcium dips were effective for firmness preservation, seemingly due to 34 a-L-Arabinofuranosidase 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: [email protected] (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 FOCH 10943 No. of Pages 9, Model 5G 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 4000 g 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 4000 g 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; PAW sf ) 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 Na CO , and 170 111 Apple ( Malus  domestica Borkh., cv. Golden Reinders) fruit 2 3 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 À À respectively. Each fraction was dialysed (mol wt. cut-off 7000) 174 115 (NE Spain). Ethylene production at harvest was 1.4 ll kg 1 h 1; 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 CaCl 2 solution (2%, w/v, in and Na CO -soluble fractions were pre-hydrolysed in 1 ml of 178 119 deionised water) at ambient temperature for 5 min. Subsequently, 2 3 12 M H 2SO 4 for 1 h at 37 °C, prior to dilution in 11 ml distilled 179 120 CaCl 2-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 CO 2). O 2 and CO 2 concentrations were monitored continu- od ( Blumenkrantz & Asboe-Hansen, 1973 ), using galacturonic acid 182 123 ously, and corrected automatically using N 2 from a tank and by as a standard, and results were expressed as % (w/w). 183 124 scrubbing off excess CO 2 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 À ° 132 nitrogen, freeze-dried, powdered, and kept at 80 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 ° À 134 muffle furnace at 500 C for 2 h. Ashes were digested thereafter min 1. 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 À1 was defined as the decrease of one unit of A 620 min . 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 À1 141 spectrometer, and results expressed as mg 100 g FW . À1 as the increase of one unit of A 235 min . 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 À 144 firmness measurements were carried out individually on 15 fruit was defined as the release of 1 lmol of glucose min 1. 206 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 FOCH 10943 No. of Pages 9, Model 5G 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 À1 214 of 1 lmol of p-nitrophenol min 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 À 3.2.1.55) activity, the crude extract was obtained after mixing Calcium content (mg 100 g FW 1) 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 221 according to Wei et al., 2010 . One unit (U) of AFase was defined 19 Weeks 31 Weeks À 222 as the liberation of 1 nmol of p-nitrophenol min 1 from p-nitro- Air Untreated 2.8 b 2.9 b CaCl 3.4 a 5.2 a 223 phenyl- a-L-arabinofuranoside. All assays were done in triplicate, 2 ULO Untreated 3.6 b 4.1 b 224 and total protein content in the extracts was determined with CaCl 2 5.8 a 5.3 a 225 the Bradford method (1976) , using BSA as a standard, and results À 226 were expressed as specific activity (U mg protein 1). 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 À 231 1.5 l h 1. 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. N 2 carrier gas, air and H 2 flows were 45, 400 and À 236 45 ml min 1, respectively. The injector and detector were held at 237 120 °C and 180 °C, correspondingly. Results were expressed as ll À À 238 ethylene kg 1 h 1. 239 2.7. Microscopy observations 240 Small pieces (1 mm 3) 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 LSD 0.05 . 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 256 (LSD) Fisher’s test at P 6 0.05 with the SAS programme package 19 Weeks 31 Weeks 257 (SAS Institute, Cary, NC, USA, 1988). For these analyses, a multi-fac- 0 d 7 d 14 d 0 d 7 d 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 CaCl 2 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 CaCl 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 2 262 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). 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 FOCH 10943 No. of Pages 9, Model 5G 19 April 2011 4 A. Ortiz et al. / Food Chemistry xxx (2011) xxx–xxx 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 0 d 7 d 14 d 0 d 7 d 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) CaCl 2 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 CaCl 2 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) CaCl 2 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 CaCl 2 1.18 b 1.56 b 2.16 b 2.26 b 2.90 b 4.37 b CDTA sf Air Untreated 35.41 b 31.35 b 28.51 b 30.39 c 25.16 d 18.32 d (LSD = 2.87) CaCl 2 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 CaCl 2 30.79 c 31.36 b 27.84 b 40.52 a 38.74 a 38.99 a Na 2CO 3sf Air Untreated 18.03 b 17.65 b 16.67 c 19.17 b 19.72 b 16.60 b (LSD = 1.43) CaCl 2 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 CaCl 2 21.45 a 21.42 a 20.48 a 20.43 a 20.10 a 18.20 a KOH sf Air Untreated 9.74 a 7.90 a 4.02 b 5.07 a 5.23 a 5.95 a (LSD = 1.62) CaCl 2 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 CaCl 2 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. 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 FOCH 10943 No. of Pages 9, Model 5G 19 April 2011 A. Ortiz et al. / Food Chemistry xxx (2011) xxx–xxx 5 284 14 days at 20 °C. Calcium-treated fruit stored in air also remained Higher yields of Na 2CO 3sf , 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 CDTA sf 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 PAW sf yields (data not shown). Yet nei- sidered to contain the middle lamella pectin. This is consistent 319 298 ther CWM nor PAW sf 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 Na 2- 323 302 2004; Peña & Carpita, 2004 ) have pointed out that modifications CO 3sf 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. 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 FOCH 10943 No. of Pages 9, Model 5G 19 April 2011 6 A. Ortiz et al. / Food Chemistry xxx (2011) xxx–xxx 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. 3 A) or 31 ( Fig. 3 B) 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 PAW sf and W sf , 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 protein À1) 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 0 d 7 d 14 d 0 d 7 d 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) CaCl 2 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 CaCl 2 102.11 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) CaCl 2 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 CaCl 2 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) CaCl 2 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 CaCl 2 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) CaCl 2 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 CaCl 2 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) CaCl 2 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 CaCl 2 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) CaCl 2 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 CaCl 2 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). 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 FOCH 10943 No. of Pages 9, Model 5G 19 April 2011 A. Ortiz et al. / Food Chemistry xxx (2011) xxx–xxx 7 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 0 d 7 d 14 d 0 d 7 d 14 d CDTA sf Air Untreated 10.28 b 9.15 b 9.72 b 10.27 a 8.27 c 8.75 c (LSD = 1.39) CaCl 2 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 CaCl 2 8.79 c 12.74 a 12.95 a 9.42 a 13.29 a 13.13 b Na 2CO 3sf Air Untreated 65.65 b 63.36 b 61.23 c 64.52 b 61.01 c 58.43 c (LSD = 1.94) CaCl 2 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 CaCl 2 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 MdPL 1 clusters 422 373 these enzyme activities and Na 2CO 3sf 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 Na 2CO 3sf (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 CDTA sf 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 CDTA sf 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 KOH sf 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 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 FOCH 10943 No. of Pages 9, Model 5G 19 April 2011 8 A. Ortiz et al. / Food Chemistry xxx (2011) xxx–xxx 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 440–444. 542 471 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 544 545 473 associated to cell wall expansion and reported to decrease as fruit Biology and Technology, 50 , 70–78. Hayama, H., Shimada, T., Fujii, H., Ito, A., & Kashimura, Y. (2006). Ethylene regulation 546 474 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. 548 549 476 delayed fruit softening after storage of ‘Golden Reinders’ apples Hwang, J., Pyun, Y. R., & Kokini, J. L. (1993). Sidechains of pectins: Some thoughts on 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 softening by antisense expression of a pectate lyase gene. Plant Physiology, 128 , 558 483 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 (Malus domestica ) fruit: A review. New Zealand Journal of Crop and Horticultural 561 Science, 30 , 145–160. 562 485 Acknowledgements Klein, J. D., Hanzon, J., Irwin, P. L., Ben Shalom, N., & Lurie, S. (1995). Pectin esterase 563 activity and pectin methyl esterification in heated golden delicious apples. 564 Phytochemistry, 39 , 491–494. 565 486 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 567 568 488 through the AGL2006-00345/ALI project, financed by the Ministe- Agriculture, 76 , 421–426. Lara, I., Echeverría, G., Graell, J., & López, M. L. (2007). Volatile emission after 569 489 rio de Educación y Ciencia (MEC) of Spain. The authors are indebted controlled atmosphere storage of ‘Mondial Gala’ apples ( Malus  domestica ): 570 490 to P. Sopeña and E. Comabella for technical assistance and to X. Cal- Relationship to some involved enzyme activities. Journal of Agricultural and Food 571 572 491 omarde for microscopy observations. 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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 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 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 , pectin, firmness, pectolytic enzymes, calcium Abstract In this work, ‘Fuji Kiku 8’ apples were treated with 2% (w/v) CaCl 2 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 CaCl 2 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 CaCl 2 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. MATERIALS AND METHODS ‘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) CaCl 2 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 O 2 : 2 kPa CO 2) and ultra low oxygen (ULO; 1 kPa O 2 : 2 kPa CO 2). 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., -1 Madrid, Spain) spectrometer. Results were expressed as mg 100 g FM . 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. III rd IC Postharvest Unlimited 2008 Ed: W.B. Herppich 213 Acta Hort. 858, ISHS 2010 (Ortiz and Lara, 2008). !-Xyl and !-Gal activities were measured according to Cleemput -1 et al. (1997) and Vicente et al. (2005). Results were expressed as units mg prot . 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, -1 filtered, lyophilised and weighed to determine yield of CWM (g 100 g FM ). 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), Na 2CO 3, and KOH, in order to fractionate soluble, non covalently-bound, covalently-bound and matrix glycans, respectively. Each fraction was filtered, dialysed, lyophilised and -1 weighed. Yields were expressed as g 100 g CMW . 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. RESULTS AND DISCUSSION Increases of fruit firmness due to CaCl 2 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 Na 2CO 3-soluble fraction (Na 2CO 3sf), which suggests this fraction may be a key factor for preserving firmness in apples. Accordingly, ULO-stored apples were characterised by higher levels of Na 2CO 3sf 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 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. 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. Postharvest Biol. Technol. 34:331-339. 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 kiwifruit ( Actinidia deliciosa ). Plant Physiol. 98:71-81. 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. 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. Postharvest Biol. Technol. 38:213-222. 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 AIR 1 LO ULO Untreated 55.3 Bb 2 72.8 Aa 72.4 Aa CaCl 2 (2%, w/v) 63.1 Ab 72.0 Aa 72.3 Aa 1 AIR: 21 kPa O 2 + 0.03 kPa CO 2; LO: 3 kPa O 2 + 2 kPa CO 2; ULO: 1 kPa O 2 + 2 kPa CO 2 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. Figures e 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 ORTIZ 1, MIQUEL VENDRELL 2 and ISABEL LARA 1* 1Departament de Química, Unitat de Postcollita-XaRTA, Universitat de Lleida, Alcalde Rovira Roure 191, 25198 Lleida, Spain 2Departament de Genètica Molecular de Plantes, Consorci CSIC-IRTA, Jordi Girona 18-26, 08034 Barcelona, Spain (e-mail: [email protected]) (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 RBatsch) fruit during the post-harvest period lead to chemicals could provide an alternative method to delay major losses throughout the marketing chain due to 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 CO 2 (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 (Na 2CO 3sf ), and matrix glycans (hemicelluloses; KOH sf ), treated with 1 µl l –1 1-MCP (SmartFresh TM ; 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 O 2 : 10 kPa CO 2), 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- -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 ϫ 3 Germany) as described previously (Pathak and Sanwall, mm). Analyses were conducted isothermally at 100ºC, 1998), using galacturonic acid (GalUA) as a standard. –1 with N 2 as the carrier gas (at 45 ml min ), in the One Unit of PG activity was defined as that causing the –1 presence of air and H 2 at flow rates of 400 and 45 ml liberation of 1.0 µmol GalUA min . 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 –1 –1 expressed as µl C 2H4 kg FW h . 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 –1 Fruit firmness (in N) was measured on opposite sides decrease of 1.0 A 620 units min . 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 –1 defined as that causing an increase of 1.0 A 235 units min . 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 ϫ 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 -galactosidase ( -Gal; EC Redgwell et al . (1992), with some modifications. Samples 3.2.1.23) and ␣-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 ϫ 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). -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 -Gal or AFase activity was defined as that resulting in the dialysate was centrifuged at 4,000 ϫ 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- -D-galactopyranoside or p-nitrophenyl- ␣-L- supernatant (henceforth termed the PAW-soluble arabinofuranoside, respectively. fraction; PAW sf ) 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 Na 2CO 3, 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 (water sf ), loosely- Institute, Cary, NC, USA). Means were separated using bound pectin (CDTA sf ), covalently-bound pectin Fisher’s LSD test at P ≤ 0.05. A. O RTIZ , M. V ENDRELL and I. L ARA 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 PAW sf (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 PAW sf 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 PAW sf yields also decreased. Similarly,untreated when combined with CA storage, which contradicted samples stored in a CA showed lower PAW sf 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 Na 2CO 3sf values upon removal from cold storage. TABLE III Cell wall solubilisation (% FW) § in ‘Tardibelle’ peach fruit after cold storage for 21 d at 0ºC Air storage CA storage 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 PAW sf 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 PAW sf at harvest were 2.0 and 0.71% (w/w), respectively. ‡Days at 20ºC following cold storage. §Fraction yields were recorded as g 100 g –1 fresh weight (FW). CWM, insoluble cell wall material; PAW sf , fraction soluble in 2:1:1 (w/v/v) phenol:acetic acid:water. 178 Cell wall disassembly in peach fruit 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 Air storage CA storage Fraction § At harvest Shelf-life ‡ Untreated 1-MCP-treated Untreated 1-MCP-treated † Water sf 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 CDTA sf 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 Na 2CO 3sf 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 KOH sf 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 †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). ‡Days at 20ºC following cold storage. §Fraction yields were recorded as g 100 g –1 insoluble cell wall materials (CWM). Water sf , fraction soluble in water; CDTA sf , fraction soluble in 0.05 M cyclohexane- trans -1,2-diamine tetra-acetate; Na 2CO 3sf , fraction soluble in 0.05 M Na 2CO 3; KOH sf , 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 Na 2CO 3sf also relationship between KOH sf 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 KOH sf 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 Na 2CO 3sf 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 Na2CO 3- 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 ␣- 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 -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 CDTA sf 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). -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 A. O RTIZ , M. V ENDRELL and I. L ARA 179 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 Air storage CA storage 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 -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 †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 enzyme activity are significantly different at P ≤ 0.05 (LSD test). ‡Days at 20ºC following cold storage. §PME, pectin methylesterase; PG, polygalacturonase; PL, pectate lyase; -Gal, -galactosidase; AFase, ␣-L-arabinofuranosidase; EGase, endo -1,4- - 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 Na 2CO 3sf during the softening process. Partial inhibition peaches stored in air than in CA-stored samples (Table of -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 ϫ domestica Borkh.) fruit has been enhancement in PG and PL activities relative to the shown to be more closely related to -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, -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.) -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, - 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, 180 Cell wall disassembly in peach fruit PL, -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 6 th 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. 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ZILIOTTO , F., B EGHELDO , M., R ASORI , A., B ONGHI , C. and T ONUTTI , Changes and postharvest regulation of activity and gene expres- P. (2008). Trascriptome profiling of ripening nectarine ( Prunus sion of enzymes related to cell wall degradation in ripening persica L. Batsch ) fruit treated with 1-MCP. Journal of apple fruit. Postharvest Biology and Technology , 56, 147–154. Experimental Botany , 59, 2781–2791. 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 O 2 : 10 kPa CO 2) 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, ααα-L-arabinofuranosidase and βββ-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 CO 2 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. MATERIALS AND METHODS 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 O 2: 10 kPa CO 2) 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 Na 2CO 3, 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 mol 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 1 A620 min . 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 1 A235 min . 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 mol 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 mol 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). Statistical Analysis 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. RESULTS AND DISCUSSION 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 Na 2CO 3sf , 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 201 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 A. Ortiz is the recipient of a FPU grant from the Ministerio de Ciencia e 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. 202 Capítulo XV 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. 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 kiwifruit ( Actinidia deliciosa ). Plant Physiol. 98:71 81. 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. 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. Postharvest Biol. Technol. 38:213 222. 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 Xa Air 3:10 Yb 3+1 15+1 Zc 0 3 a Storage atmosphere conditions (O 2:CO 2). b Storage period at 2ºC (days) + 1 day at 7ºC. c Shelf life period (days at 20ºC). 203 Capítulo XV Table 2. Firmness (N) of ‘Rich Lady’ peaches after storage at 2ºC. Atmosphere Storage period + days at 20ºC 3+1 a 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+1 a 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 PAW sf 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 CDTA sf 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 Na 2CO 3sf 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 KOH sf 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 3+1: 3 days at 2ºC + 1 day at 7ºC; 15+1: 15 days at 2ºC + 1 day at 7ºC. 204 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+1 a 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 3+1: 3 days at 2ºC + 1 day at 7ºC; 15+1: 15 days at 2ºC + 1 day at 7ºC. 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. 205 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 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 , 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 th Proc. 6 International Postharvest Symposium Eds.: M. Erkan and U. Aksoy 799 Acta Hort. 877, ISHS 2010 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. MATERIALS AND METHODS 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) CaCl 2 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. Statistical Analysis 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 00.05. 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. RESULTS AND DISCUSSION 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 CaCl 2 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 CaCl 2 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 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. P. Sopeña and A. Latorre are acknowledged for technical assistance. Literature Cited Abbott, J.A., Klein, J.D., Campbell, T.A., Conway, W.S. and Sams, C.E. 2000. Sensory and firmness measurements of calcium- and heat-treated apples. J. Texture Stud. 31:109-121. CAMO ASA. 1997. Unscrambler Users Guide, version 7.6. Program package for multivariate calibration. Trondheim, Norway: CAMO ASA. Chardonnet, C., Charron, C., Sams, C. and Conway, W. 2003. Chemical changes in the cortical tissue and cell walls of calcium-infiltrated ‘Golden Delicious’ apples during storage. Postharvest Biol. Technol. 28:97-111. De Smedt, V., Pauwel, E., De Baerdemaeker, J. and Nicolaï, B. 1998. Microscopic observation of mealiness in apples: a quantitative approach. Postharvest Biol. Technol. 14:151-158. De Smedt, V., Barreiro, P., Verlinden, B.E., Veraverbeke, E.A., De Baerdemaeker, J. and Nicolaï, B.M. 2002. A mathematical model for the development of mealiness in apples. Postharvest Biol. Technol. 25:273-291. Echeverría, G., Graell, J., Lara, I. and López, M.L. 2008a. Physicochemical measurements in ‘Mondial Gala ®’ apples stored at different atmospheres: Influence on consumer acceptability. Postharvest Biol. Technol. 50:135-144. Echeverría, G., Graell, J., Lara, I., López, M.L. and Puy, J. 2008b. Panel consonance in the sensory evaluation of apple attributes: Influence of mealiness on sweetness perception. J. Sens. Stud. 23:656-670. Glenn, G.M. and Poovaiah, B.W. 1990. Calcium-mediated postharvest changes in texture and cell wall structure and composition in ‘Golden Delicious’ apples. J. Amer. Soc. Hort. Sci. 115:962-968. Harker, F.R., Kupferman, E.M., Marin, A.B., Gunson, A.B. and Triggs, C.M. 2008. Eating quality of apples based on consumer preferences. Postharvest Biol. Technol. 50:70-78. Péneau, S., Hoehn, E., Roth, H.R., Escher, F. and Nuessli, J. 2006. Importance and consumer perception of freshness of apples. Food Qual. Pref. 17:9-19. Péneau, S., Brockhoff, P.B., Hoehn, E., Escher, F. and Nuessli, J. 2007. Relating consumer evaluation of apple freshness to sensory and physico-chemical measurements. J. Sens. Stud. 22:313-335. SAS Institute Inc. 1988. SAS/STAT Guide for Personal Computers, 6 th ed. SAS Institute Inc., Cary, NC, USA. Wyllie, S.G. 2008. Flavour quality of fruit and vegetables: are we on the brink of major advances? p.3-10. In: S.G. Brückner and S.G. Wyllie (eds.), Fruit and vegetable flavour. Recent advances and future prospects. CRC Press: Boca Raton, USA. 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 (% CaCl 2, w/v) Table 2. Calcium content a (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 CaCl 2-treated untreated CaCl 2-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 00.05 (LSD test). Table 3. Standard quality parameters a 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 untreated CaCl 2-treated untreated CaCl 2-treated 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 0.05 (LSD test). 803 Table 4. Sensory attributes a 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 untreated CaCl 2-treated untreated CaCl 2-treated 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 00.05 (LSD test). 804 Figures A B 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 I AD index (index of absorbance difference = A 670 A720 ) based on Vis spectroscopy. Fruit in the second maturity class defined based on I AD 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. MATERIALS AND METHODS 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 I AD 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 I AD (M1: I AD 0.17 0.15; M2: I AD 0.14 0.12; M3: I AD 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 Statistical Analysis 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. RESULTS AND DISCUSSION Standard Quality Parameters at Harvest ® At harvest, ‘Big Top ’ nectarines graded according to I AD 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 I AD 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 A. Ortiz is the recipient of a FPU grant from the Ministerio de Ciencia e 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 I AD (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 I AD 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 fruits stored 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 91011 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 a a 20 ºC 7 ab a a ab b b b b a b b b a 6 a a 5 4 (hedonic scale: 1-9) scale: (hedonic acceptance Sensory 3 2 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. 229 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 230 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 O 2 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 CO 2 (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 231 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 232 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 ( g 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). 233 Discusión general 2. PRODUCCIÓN DE COMPUESTOS VOLÁTILES AROMÁTICOS EN RESPUESTA A LA MANIPULACIÓN POST-COSECHA. 2.1. Manzana (Malus × domestica Borkh, cv. ‘Golden Reinders’ y ‘Fuji Kiku-8’). 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 234 Discusión general cloruro cálcico sobre el aroma en manzanas conservadas a 1 ºC. La utilización de la tecnología ULO (en este caso 1 kPa O 2 : 2 kPa CO 2) 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 235 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 O 2 : 2 kPa CO 2), 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 236 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 237 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 CO 2. 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 O 2: 10 kPa CO 2). Se estudiaron dos variedades en 238 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 239 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 240 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 241 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. 242 Discusión general 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. 3.1. Manzana (Malus × domestica Borkh, cv. ‘Golden Reinders’ y ‘Fuji Kiku-8’). 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 243 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 Na 2CO 3, 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 Na 2CO 3, 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 244 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 245 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 βββ-galactosidasa βββ-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 246 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). 247 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. 248 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. (LSD firmness = 5.2; LSDCDTA = 3.4; LSD Na 2CO 3 = 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. (LSD firmness = 4.9; LSD Wsf = 4.1; LSD CDTA = 2.3; LSD Na 2CO 3 = 1.7; LSD KOH = 2.4). 249 Discusión general Las modificaciones observadas en los rendimientos de las fracciones solubles en Na 2CO 3 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 Na 2CO 3 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 Na 2CO 3 (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 Na 2CO 3, 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. 250 Discusión general 4. METABOLISMO DE LAS PAREDES CELULARES Y PÉRDIDA DE FIRMEZA EN RESPUESTA A LA MANIPULACIÓN POST-COSECHA. 4.1. Manzana (Malus × domestica Borkh, cv. ‘Golden Reinders’ y ‘Fuji Kiku-8’). 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 O 2 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. 251 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 CaCl 2 1.856 b 1.929 a ULO Control 1.675 c 1.763 b CaCl 2 1.960 a 1.706 b Agua fs FN Control 3.491 a 3.474 a CaCl 2 2.280 b 3.224 ab ULO Control 1.889 b 2.580 b CaCl 2 1.747 b 3.674 a CDTA fs FN Control 28.066 c 26.248 c CaCl 2 34.248 a 32.336 b ULO Control 32.269 ab 38.994 a CaCl 2 31.943 b 40.617 a Na 2CO 3fs FN Control 17.594 c 16.526 b CaCl 2 19.314 b 16.682 b ULO Control 19.125 b 18.331 a CaCl 2 20.846 a 18.299 a KOH fs FN Control 6.981 b 5.586 a CaCl 2 7.488 b 6.542 a ULO Control 8.756 a 6.178 a CaCl 2 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 Na 2CO 3 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). 254 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 Na 2CO 3 y de los niveles de firmeza respecto a los frutos conservados en frío normal (cap. XV, Tabla 3) en melocotones ‘Rich Lady’. 256 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 Na 2CO 3 (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 Na 2CO 3 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. 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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 ( γγγ- 266 Conclusiones octalactona, δδδ-decalactona y γγγ-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 βββ-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, βββ-Gal y βββ-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 βββ-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, βββ-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 βββ-Gal y βββ-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, βββ-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