'Scarlett Spur Red Delicious' Apple Volatile Production Accompanying

Article pubs.acs.org/JAFC

‘Scarlett Spur Red Delicious’ Apple Volatile Production Accompanying Physiological Disorder Development during Low pO2 Controlled Atmosphere Storage Christie Lumpkin,†,§ John K. Fellman,† David R. Rudell,‡ and James Mattheis*,‡ †

Department of Horticulture, Washington State University, Pullman, Washington 99164, United States USDA-ARS Tree Fruit Research Laboratory, Wenatchee, Washington 98801, United States

‡

ABSTRACT: Apple (Malus domestica Borkh.) fruit volatile production is regulated by a variety of factors including low oxygen storage conditions. This study examined the impact of low pO2 controlled atmospheres on ‘Scarlett Spur Red Delicious’ apple volatile production and disorder development. Accumulation of apple volatile compounds was characterized during long-term cold storage at 0.5 °C in air or low pO2 (0.3, 0.8, or 1.5 kPa) with 1 kPa CO2. Volatile accumulation differed quantitatively with pO2 as acetaldehyde, ethanol, and ethyl ester accumulation increased with decreased pO2 during the first weeks in storage. Differences in volatile accumulation among atmospheres were evident through 6 months. The rate of ethanol accumulation increased with decreased pO2 and could potentially be used to monitor low O2 stress. Incidence of low oxygen disorders after 9 months was highest in fruit held at the lowest pO2. The sesquiterpene α-farnesene was not detected throughout the storage period. KEYWORDS: anaerobic metabolism, low pO2 stress, alcohols, esters, fruit quality, physiological disorders

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INTRODUCTION Apple fruit are routinely stored commercially for months in controlled atmosphere (CA) conditions, typically with 1−3 kPa O2, 1−3 kPa CO2, at 0.5−2 °C.1 The low pO2 and high pCO2 limit ethylene action2 and aerobic respiration, resulting in decreased metabolic activity and extended storage life.3 Reduced ethylene action impedes normal volatile production by limiting ester biosynthesis, lipoxygenase activity, and β-oxidation.4,5 When CA gas composition is outside the range tolerated by apple fruit, additional changes in volatile production occur, and internal and external disorders may develop. As oxygen levels decrease below the anaerobic compensation point, O2 dependent processesincluding fatty acid synthesis/degradation and the esterification of alcoholsare increasingly inhibited as fermentative metabolism occurs.6,7 The activity of alcohol acyltransferase, the terminal enzyme in volatile ester biosynthesis, and enzymes catalyzing ethylene synthesis is also suppressed, resulting in delays in ripening and volatile synthesis.8,9 Fruit stored previously in ultralow oxygen inducing anaerobic metabolism produce low amounts of straight-carbon chain compounds, esters (including 2-methylbutyrate), aldehydes, and ketones,10−12 and increased amounts of ethanol, acetaldehyde, and ethanol-derived ethyl esters (especially ethyl acetate)7 compared to fruit stored in air. The increase in ethanol provides increased substrate for ester production resulting in an increase in ethyl esters but a decrease in butyl and hexyl esters.10,13 Ultralow pO2 storage, where O2 is maintained at less than 1 kPa, can minimize or prevent the oxidative apple peel disorder superficial scald in susceptible apple cultivars.14−19 However, postharvest disorders, including internal browning, off-flavor development, and changes in peel color, can also occur in low pO2 storage due to prolonged periods of anaerobiosis.20,21,7 Susceptibility to development of postharvest disorders, including © XXXX American Chemical Society

superficial scald, is influenced by pre- and postharvest factors including cultivar, growing season, production region, harvest maturity, storage temperature, storage duration, and storage atmosphere.16,22,23 Rudell et al. (2009)24 monitored metabolic changes relating to apple superficial scald development and reported metabolic divergence among fruit that will and will not develop the disorder occurs around 4 weeks after harvest, roughly 2 months before symptom development. Assuming low pO2 injury is also preceded by metabolic divergence, certain volatiles may also be linked to low pO2 injury development. Additionally, while it is known that volatile production changes once fruit are removed from storage,25,26 dynamics of volatile production during storage have not been extensively investigated.27 The objectives of this study were to characterize apple volatile production during long-term cold storage in air or low pO2 controlled atmospheres and to identify what, if any, relationships exist between volatile accumulation during storage and disorders induced by low pO2 stress.

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MATERIALS AND METHODS

‘Scarlett Spur Red Delicious’ (Malus domestica Borkh.) apple fruit were harvested at commercial maturity (168 days after full bloom) in a commercial orchard near Monitor, WA, U.S.A. Fruit maturity and quality were defined by assessment of internal ethylene content (IEC), weight, firmness, starch hydrolysis, soluble solids content (SSC), and titratable acidity (TA) of 18 fruit. Ethylene in a 0.5 mL gas sample removed from the fruit core was analyzed using a 5880A gas chromatography instrument with flame ionization detection (GC-FID) Received: November 22, 2013 Revised: January 21, 2014 Accepted: January 23, 2014

A

dx.doi.org/10.1021/jf405267b | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

Table 1. Abbreviation, Source, and Mass Spectral Taga volatile compound

abbreviation

mass spectral tag (retention index, target ion)

1-butanol 1-hexanol 1-pentanol 1-propanol 2-ethyl-1-hexanol 2-methyl-1-butanol 2-methyl-1-propanol 2-methylbutyl acetate 2-methylbutyl butanoate 2-methylbutyl propanoate 2-methylpropyl acetate 4-allylanisole 6-methyl-5-hepten-2-ol 6-methyl-5-hepten-2-one acetaldehyde acetone benzaldehyde butyl 2-methylbutyrate butyl acetate butyl butyrate butyl hexanoate butyl propanoate butyric acid decanal ethanol ethyl 2-methylbutyrate

1-butanol 1-hexanol 1-pentanol 1-propanol 2-eth 1-hex 2-meth 1-but 2-meth 1-pro 2-MB ace 2-MB but 2-MB Prop 2-MP ace estragole 6M5H2Ol 6M5H2One acet acetone benz but 2-MB but ace but but but hex but pro butyric decanal ethanol eth 2-MB

661.1, 56 877.3, 56 760.5, 55 559.6, 31 1027.2, 57 743.2, 57 622.9, 43 881.8, 43 1056.5, 71 971.1, 57 775.1, 56 1198.8, 148 992, 95 983.6, 108 462.4, 29 508.8, 43 963.1, 106 1039.8, 103 818.2, 43 993.8, 71 1186.1, 117 908, 57 837.9, 60 1203.2, 57 516.7, 31 852.8, 102

a

standard source Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich J.T. Baker Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich synthesized synthesized Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Decon Sigma-Aldrich

volatile compound ethyl acetate ethyl butyrate ethyl hexanoate ethyl octanoate ethyl pentanoate ethyl propionate heptanal hexanal hexyl 2-methylbutyrate hexyl acetate hexyl butyrate hexyl propanoate methyl 2-methylbutyrate methyl acetate methyl butyrate methyl hexanoate nonanal octanal pentanal pentyl acetate propyl 2-methylbutanoate propyl acetate propyl butyrate propyl hexanoate propyl propanoate

abbreviation eth ace eth but eth hex eth oct eth pen eth pro heptanal hexanal hex 2-MB hex ace hex but hex pro meth 2-MB meth ace meth but meth hex nonanal octanal pentanal pen ace prop 2-MB pro ace pro but pro hex pro pro

mass spectral tag (retention index, target ion)

standard source

613.9, 43 805.2, 71 996, 88 1190.8, 88 900.4, 101 711.9, 57 899.6, 70 799.6, 56 1231.6, 103 1011, 56 1187.9, 89 1100.6, 57 777.2, 88 531.6, 74 721.8, 74 924, 74 1102.3, 57 1000, 84 701.1, 44 913.8, 43 945.8, 103 714, 61 898.1, 71 1091.1, 99 811.9, 57

Fischer Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich synthesized Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich synthesized Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich

Synthesized volatiles produced using modification of methods in Fischer et al., 1895.31

Figure 1. Principal Components Analysis plot of headspace volatile compounds collected during storage of ‘Scarlett Spur Red Delicious’ apple fruit. Gas samples collected from 3 replicate storage chambers for each atmosphere were analyzed by GC-MS. Red: 0.3 kPa O2. Green: 0.8 kPa O2. Blue: 1.6 kPa O2. Orange: air. CO2 in all low O2 atmospheres was 1 kPa. All fruit stored at 0.5 °C. (Hewlett-Packard, Avondale, PA) equipped with a 50 cm long × 0.32 cm internal diameter glass column packed with 80−100 mesh Porapak Q (Supelco, Bellafonte, PA). The injector, oven, and detector temperatures were 100 °C, 130 °C, and 200 °C, respectively. Gas flows for air, N2 carrier, and H2 were 5.0, 0.5, and 0.5 mL s−1, respectively. Firmness was analyzed with a Mohr Digitest 1.25 penetrometer

(Mohr and Associates, Richland, WA) equipped with an 11 mm tip. Starch hydrolysis was visually assessed on a full width tissue slice cut from the fruit equator using a 1−6 scale (1 = no hydrolysis, all tissue black, 6 = hydrolysis complete, tissue unstained) after staining with a 0.024 M I−KI solution.28 Fresh juice prepared using a Champion juicer (Plastaket Mfg., Lodi, CA) was used to measure SSC with a B



Article

Figure 2. Heatmap of ‘Scarlett Spur Red Delicious’ volatile accumulation during cold storage. Gas samples collected from three replicate storage chambers for each atmosphere were analyzed by GC-MS. Storage temperature was 0.5 °C and storage chamber atmospheres were air or 0.3, 0.8, or 1.6 kPa O2 with 1 kPa CO2. Volatile compounds were hierarchically clustered using Pearson’s correlation and Ward’s linkage algorithm. Atmosphere treatment indicated on the left y-axis by colored bars, storage duration (days) indicated on the right y-axis. Compound concentration increases with blue to red color change. Compound abbreviations presented in Table 1. hand-held refractometer (ATAGO, Tokyo), and TA by titrating 10 mL juice with 0.1 M KOH to pH 8.2 using an autotitrator (TIM850, Radiometer Analytical, Copenhagen, Denmark). Fruit (18 fruit/pressed paper tray) were stored in air for 3 days at 0.5 °C before being weighed and placed into 0.145 m3 stainless steel CA chambers with Plexiglas doors, four trays per chamber.

CA chamber atmospheres were established over two days using compressed air and CO2 plus N2 from a membrane generator (Permea, St. Louis, MO). Chamber gas composition was analyzed every 90 min and automatically corrected as necessary (Techni-Systems, Chelan, WA); therefore, chamber atmospheres were static except when adjustments were conducted.29 Fruit chlorophyll fluorescence measured with the C


D

ethanol

ethyl acetate

methyl 2-methylbutyrate

methyl acetate

methyl butyrate

1-propanol

ethyl butyrate

ethyl pentanoate

ethyl hexanoate

ethyl 2-methylbutyrate

ethyl propanoate

group 1 compd.

0.3 0.8 1.6 air 0.3 0.8 1.6 air 0.3 0.8 1.6 air 0.3 0.8 1.6 air 0.3 0.8 1.6 air 0.3 0.8 1.6 air 0.3 0.8 1.6 air 0.3 0.8 1.6 air 0.3 0.8 1.6 air 0.3 0.8 1.6 air 0.3 0.8

atm 0.082D 0.060D 0.015 0.050B 0.127D 0.093C 0.031 0.090ABC 0.008aD 0.004abC 0.002b 0.003abB 0.000C 0.000D 0.000 0.001 0.122E 0.099C 0.041 0.103C 0.021 0.017 0.008C 0.010 0.003 0.004B 0.001 0.004B 0.001C 0.001B 0.000 0.000B 0.003B 0.004B 0.001 0.004BC 0.276E 0.346 0.051 0.191AB 0.671C 0.945B

0 0.512aD 0.270abCD 0.054b 0.029bB 0.496aCD 0.244bC 0.064b 0.063bBC 0.028aCD 0.011bC 0.005b 0.002bB 0.001aBC 0.001bD 0.000b 0.000b 0.517aCDE 0.280abABC 0.088b 0.074bC 0.058 0.034 0.026BC 0.022 0.008 0.008AB 0.003 0.002B 0.006C 0.008AB 0.000 0.000B 0.010B 0.009B 0.003 0.004BC 2.23aE 0.867b 0.202bc 0.099cB 9.64aBC 5.04bAB

4 0.774aD 0.530aCD 0.095b 0.040bB 0.703aCD 0.385bBC 0.087c 0.074cBC 0.041aBCD 0.017bBC 0.006b 0.002bB 0.002aBC 0.001bCD 0.000bc 0.000c 0.726aBCD 0.471aABC 0.124b 0.083bC 0.000c 0.062a 0.025bcC 0.031b 0.012 0.015AB 0.004 0.003B 0.012C 0.011AB 0.000 0.000B 0.014B 0.013B 0.004 0.005BC 3.04aDE 0.963b 0.265bc 0.183cAB 9.23aBC 8.93aA

7 1.28aCD 1.03aB 0.268b 0.015bB 1.11aBC 0.729bAB 0.189c 0.032cC 0.066aABCD 0.030bABC 0.013b 0.002bB 0.004aAB 0.002abBC 0.001bc 0.000c 1.03aBC 0.743aAB 0.259b 0.039bC 0.261 0.000 0.038BC 0.059 0.021a 0.019aAB 0.005ab 0.001bB 0.038C 0.008AB 0.001 0.001B 0.026aB 0.023aAB 0.006b 0.000bC 6.00aCD 2.08b 0.515c 0.086cB 17.2aABC 9.17bA

14 2.21aC 1.54aA 0.402b 0.090bB 1.57aAB 1.06aA 0.270b 0.110bABC 0.094aAB 0.048abA 0.020b 0.012bB 0.006aA 0.003abA 0.001b 0.001b 1.23aAB 0.922aA 0.336b 0.111bC 0.232 0.106 0.049BC 0.022 0.026 0.035A 0.008 0.006B 0.073aBC 0.023bAB 0.004b 0.003bAB 0.055aAB 0.044aA 0.009b 0.003bBC 9.81aAB 2.47b 0.445b 0.359bAB 22.9aAB 6.19bAB

25

46 3.67aA 1.68bA 0.429bc 0.096cB 2.28aA 0.944bA 0.282bc 0.101cABC 0.106aA 0.042abAB 0.023b 0.005bB 0.006aA 0.002bAB 0.001b 0.000b 1.63aA 0.855bA 0.321bc 0.142cC 0.559 0.095 0.124AB 0.104 0.397 0.025AB 0.011 0.016AB 0.195aABC 0.014bAB 0.021b 0.010bAB 0.054aAB 0.023bAB 0.009b 0.006bABC 13.1aA 2.03b 0.557b 0.232bAB 31.5aA 5.70bAB

days in storage 3.65aA 0.760bBC 0.211b 0.159bB 1.70aAB 0.374bBC 0.172b 0.093bABC 0.088aABC 0.024bABC 0.014b 0.008bB 0.004aAB 0.001bCD 0.001b 0.001b 1.71aA 0.425bABC 0.169b 0.375bC 0.449 0.133 0.162A 0.262 0.053ab 0.016bcAB 0.009c 0.064aAB 0.266aAB 0.043bA 0.021b 0.039bA 0.048aAB 0.010bB 0.006b 0.012bABC 8.83aBC 1.67b 0.378b 0.327bAB 32.1aA 3.73bAB

82

Table 2. Group 1 ‘Scarlett Spur Red Delicious’ Volatile Accumulation (ηmol L−1) During Controlled Atmosphere or Air Storagea 110 2.64aAB 0.263bCD 0.054b 0.160bB 1.21aBC 0.204bC 0.078b 0.108bABC 0.037aBCD 0.011bC 0.006b 0.011bB 0.002aBC 0.001bcD 0.000c 0.001ab 1.06aBC 0.200cBC 0.059c 0.626bBC 0.136 0.037 0.056BC 0.210 0.039a 0.008bAB 0.003b 0.047aAB 0.144aABC 0.015bAB 0.007b 0.028bAB 0.044aAB 0.006bB 0.003b 0.009bABC 6.87aBC 0.906b 0.121b 0.331bAB 23.8aAB 3.65bAB

194 0.811aD 0.034bD 0.013b 0.574aA 0.972aBC 0.105cC 0.078c 0.325bA 0.029CD 0.023ABC 0.016 0.041A 0.001BC 0.000D 0.000 0.001 0.460bDE 0.170bcBC 0.072c 1.49aA 0.078 0.008 0.021C 0.202 0.039b 0.009bAB 0.011b 0.104aA 0.340aA 0.017bAB 0.007b 0.029bAB 0.103aA 0.010bB 0.021b 0.019bAB 6.86aBC 0.360b 0.045b 1.54bAB 29.1aA 2.41bB

250 0.048D 0.137D 0.049 0.199B 0.195D 0.125C 0.107 0.294AB 0.007bD 0.018bBC 0.012b 0.050aA 0.000aC 0.000abD 0.000b 0.001a 0.052E 0.419ABC 0.053 1.18AB 0.000 0.039 0.000C 0.238 0.005 0.025AB 0.005 0.102A 0.041C 0.012AB 0.028 0.016AB 0.019B 0.026AB 0.017 0.024A 2.98DE 1.70 1.45 2.16A 18.3ABC 4.07AB

Journal of Agricultural and Food Chemistry Article


acetaldehyde

HarvestWatch system (Satlantic Inc., Halifax, NS) was used to determine the lowest pO2 set-point based on changes in fruit chlorophyll fluorescence as pO2 decreased.30 Fruit were stored at 0.5 °C in air or CA (0.3, 0.8, or 1.6 kPa O2, 1 kPa CO2) with four chambers for each atmosphere: three for sampling volatile compounds (volatile chambers) and one from which fruit were periodically removed and assessed for quality (quality chambers). After 1, 2, 4, and 8 months in storage, one tray was removed from each quality chamber. Fruit were examined for external disorders and then held at 20 °C in air for 7 days. On day 7, the rate of ethylene evolution was determined. Evolved ethylene was analyzed in gas samples collected from 4 replicates, 4 fruit each, from each storage atmosphere placed into 3.79 L glass jars with Teflon lids (Berghof/ America, Coral Springs, FL) fitted with Tygon and Teflon tubing. Compressed air was passed through Purafil (Purafil Inc., Atlanta), calcium hydroxide, a carbon filter (Agilent Technologies, Santa Clara, CA), and Tenax TA (80−100 mesh, Alltech Associates, Deerfield, IL) before flowing through the jars. The purified air was passed through the jars for 60 min, then a 1 mL gas sample was collected at the outlet from each jar and 0.5 mL injected into the 5880A GC-FID (HewlettPackard, Avondale, PA) described previously. The incidence of internal and external disorders (superficial scald, decay, internal browning) was also recorded. Chamber ethylene concentration was determined by collecting a gas sample from each storage chamber into a 1 L Tedlar bag (SKC Inc., Eighty Four, PA). Ethylene in a 0.5 mL sample from each bag was analyzed using the 5880A GC-FID previously described. After 9 months storage plus 7 days at 20 °C, apples from the volatile chambers were assessed for internal and external disorders. Volatile compounds were collected directly from each storage chamber 0, 4, 7, 14, 25, 46, 82, 110, 194, and 250 d after chambers were sealed. Tygon tubing was attached to an outlet gas port on the side of each chamber and gas samples (0.25−1 L) were drawn through a 17.8 cm Tenax TA Glass TD Tube (Supelco, Bellefonte, PA) containing 60 mm/180 mg Tenax using a pump and volume was measured using a digital flow meter. Traps were thermally desorbed (TDS3 and TDSA2, Gerstel, Linthicum, MD) using a temperature program of 20 °C for 30 s, increased at 1 °C s−1 to 250 °C and held for 360 s. Desorbed compounds were cryogenically trapped at −130 °C (CIS-3, Gerstel) using the Gerstel Maestro Global Analytical System. Following the transfer of desorbed compounds from the TDS, the cryotrap temperature was increased 10 °C s−1 to 250 °C and held for 120 s as compounds were transferred to a Hewlett-Packard 5890A5971A GC-MSD system (Hewlett-Packard, Palo Alto, CA) equipped with a DB-5MS column (Agilent Technologies, Santa Clara, CA) 30 m long × 0.25 mm internal diameter with 0.25 μm film thickness. The oven initial temperature (35 °C) was held 600 s, increased at 0.17 °C s−1 to 250 °C, then held 180 s. Linear gas velocity of the He carrier gas was 33.9 cm s−1. Mass spectra were recorded with a Hewlett-Packard Enhanced Chemstation (G1701BA Version B.01.00; Agilent Technologies, Santa Clara, CA). Using MSD Chemstation Data Analysis (Agilent Technologies, Santa Clara, CA), AMDIS GC/MS Analysis, and NIST MS Search 2.0 (NIST, Gaithersburg, MD), compounds were identified by comparing sample retention times and spectra with those of standards (Table 1). Sample compounds were quantified by comparing base peak abundances of selected ions with those for standards of known concentration. Standards were obtained from Sigma-Aldrich (Milwaukee, WI) except 1-propanol (J.T. Baker, Center Valley, PA), ethyl acetate (Fischer Scientific, Pittsburgh, PA), and ethanol (Decon Laboratories, King of Prussia, PA). Esters synthesized31 included 2-methylbutyl butyrate, 2-methylbutyl propanoate, ethyl pentanoate, and propyl 2-methylbutryate. For each compound, synthesis was performed using 5 g of the organic acid and excess alcohol (at least two times the molar equivalent of the organic acid) refluxed for 1 h with 3 mL of concentrated sulfuric acid at the boiling point of the alcohol. The resulting mixture was partitioned against 50 mL water three times; then, the nonaqueous phase was purified by distillation. Purity was assessed using the GC-MS method previously described. Chromatographic data was mean centered and scaled according to the standard deviation. MetaboAnalyst32 was used to generate

Headspace gas samples collected from 3 replicate storage chambers for each atmosphere were analyzed by GC-MS. Chambers were maintained at 0.3, 0.8, or 1.6 kPa O2 with 1 kPa CO2 or air. All fruit was stored at 0.5 °C. Storage duration and atmosphere differences are denoted by upper and lowercase letters, respectively, and values with the same letter are not significantly different (P ≤ 0.05, Tukey’s HSD).

250

Article

a

0.768b 3.13bAB 2.52aA 0.000bC 0.338b 0.000bD

194 110

0.678b 0.281bB 0.965aBC 0.000bC 0.000b 0.000bD 1.13b 0.267bB 1.61aABC 0.156bC 0.000b 0.000bD

82 46

1.03b 0.282bB 2.10aAB 0.313bBC 0.123b 0.076bBCD 0.976b 0.261bB 1.28aABC 0.263bBC 0.155b 0.023bD 3.46c 0.205 dB 1.96aAB 0.695bA 0.185c 0.034cCD 2.76b 0.358cB 1.58aABC 0.545bAB 0.159bc 0.096cBCD 0.550 0.284B 0.227C 0.199BC 0.081 0.156AB 1.6 air 0.3 0.8 1.6 air

1.60c 0.220cB 1.02aBC 0.247bBC 0.094b 0.148bABC

days in storage

25 14 7 4 0 atm group 1 compd.

Table 2. continued

12.6 7.31A 0.503C 0.288BC 0.281 0.238A


E



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Figure 3. ‘Scarlett Spur Red Delicious’ apple firmness (A), and titratable acidity (B) of fruit previously stored in a controlled atmosphere (CA) or air at 0.5 °C. CA kPa O2 concentrations were 0.3, 0.8, or 1.6, all with 1 kPa CO2. Apples were removed from storage and analyzed after 7 days at 20 °C. Values are means of 18 individual fruit. Storage duration and atmosphere differences are denoted by upper and lowercase letters, respectively, and values with the same letter are not significantly different (P ≤ 0.05, Tukey’s HSD). heatmaps from the normalized data analyzed by hierarchical clustering analysis using Pearson’s correlation and Ward’s linkage algorithm. Principal Components Analyses were performed using UnscramblerX 10.1 (CAMO Software, Woodbridge, NJ). The predictive models were created using cross-validation and outliers were removed. Statistical analyses were conducted using SAS 9.3 (SAS Institute, Cary, NC). Fruit quality data was analyzed by ANOVA, and means were separated using Tukey’s HSD (P < 0.05). Non-normalized volatile compound data was analyzed using a GLM with storage duration as a repeated measure, and means were separated using Tukey’s HSD (P < 0.05).

accompanied by decreased butyl and hexyl ester content, consistent with previous reports.10,13 Day 14 ethanol concentrations were significantly different among atmospheres, and ethanol increased with decreased pO2. Ethyl esters also accumulated in chambers held at 0.8 kPa O2, but concentrations in these chambers were significantly lower (ethyl propanoate, days 46−194; ethyl acetate, days 4−194; ethyl 2-methylbutyrate, days 4−14 and 46−194; ethyl hexanoate, days 4−14, 82 and 110; ethyl pentanoate, days 4,7, and 46−110; ethyl butyrate, days 46−110) compared with chambers held at 0.3 kPa O2. Several volatiles began accumulating in significantly higher concentrations in the air chambers during the latter storage period (days 84−250) including ethyl hexanoate, ethyl pentanoate, ethyl butyrate, and methyl butyrate. The air stored fruit was riper compared with CA fruit during this period and had begun to senesce based on firmness, titratable acidity, and internal breakdown incidence in air stored fruit (Figures 3, 4) resulting in the production of volatiles typical of overripe fruit.35,36

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RESULTS AND DISCUSSION Values for fruit maturity and quality at harvest were weight 223 ± 7 g, firmness 70.3 ± 0.9 N, internal ethylene 0.91 ± 0.33 μL L−1, starch index 2.2 ± 0.2, SSC 11.9 ± 0.2%, and TA 0.278 ± 0.007%. Principal Components Analysis (PCA) generated a model with principal components accounting for 25, 21, 17, and 10% (total: 72%) of the variance (Figure 1). The model identified storage duration and storage atmosphere effects on sample variation, with atmospheres diverging during the first 2 weeks storage. Volatile compound accumulation among storage atmospheres was the most similar during the first two and final 20 weeks storage. Atmosphere treatment differences were greatest on days 25, 46, and 82. The lack of significant atmosphere treatment effects at the end of storage suggests fruit volatile production was similar metabolically, possibly due to decreased metabolic rates and/or a lack of substrate for volatile production.11,33 At 250 days, chamber volatile content was lower compared to previous dates for all atmospheres (Figure 2) indicating analysis of volatile accumulation in storage after 6 months may not be useful for monitoring anaerobic oxygen conditions imposed early in storage. The hierarchical clustering procedure identified four main groups of volatile compounds. Volatiles in group 1, including many that are products of fermentation, were significantly highest from day 4 through day 194 in chambers where O2 was 0.3 kPa (Table 2). The PCA loading plot (not shown) also associated fermentative volatiles with the 0.3 kPa O2 atmosphere. The increase in fermentative compounds indicates the 0.3 kPa O2 atmosphere induced oxygen stress and anaerobic metabolism.7 Ethanol was the most abundant compound accumulating at 0.3 kPa O2 followed by ethyl acetate. Ethanol accumulation was higher in 0.3 and 0.8 kPa O2 chambers compared with 1.6 kPa O2 and air chambers. The increase in ethanol likely provided substrate for ethyl ester production34 and was

Figure 4. ‘Scarlett Spur Red Delicious’ apple physiological disorders after 9 months storage in a controlled atmosphere (CA) or air at 0.5 °C. CA kPa O2 concentrations were 0.3, 0.8, or 1.6, all with 1 kPa CO2. Fruit were evaluated after removal from cold storage plus 7 days at 20 °C. Values are means of 216 fruit for each storage treatment. Within each disorder, columns with the same letters are not significantly different (P ≤ 0.05, Tukey’s HSD).

Comparing days within storage atmospheres, most volatiles in group 1 were significantly highest during days 25−110. Ethanol concentration, however, did not significantly decrease in chambers held at 0.3 kPa O2. This lack of change may indicate that the rates of fruit ethanol production and its metabolism to F


G

estragole

ethyl octanoate

hexyl 2-methylbutyrate

hexyl butyrate

butyl hexanoate

hexyl propanoate

propyl hexanoate

hexanal

heptanal

pentanal

butyric acid

group 2 compd.


atm

0.010abA 0.000b 0.013aA 0.000b 0.006A 0.008A 0.006A 0.008A 0.003AB 0.003A 0.003A 0.003A 0.030B 0.052B 0.045A 0.052A 0.003B 0.003D 0.003BC 0.002B 0.003AB 0.003C 0.002BC 0.002AB 0.007aAB 0.004bBCD 0.005abB 0.001cB 0.001aAB 0.000bcCD 0.001abDE 0.000cB 0.008aAB 0.004bCD 0.005bEF 0.001cB 0.000 0.000 0.000B 0.000 0.000 0.000

0 0.011abA 0.003b 0.013aA 0.003b 0.006A 0.006A 0.003AB 0.004B 0.004aA 0.003abA 0.003abA 0.002bAB 0.051AB 0.046B 0.041AB 0.034AB 0.004B 0.003CD 0.003BC 0.003AB 0.003AB 0.003BC 0.003ABC 0.003AB 0.008aAB 0.005bBC 0.006abB 0.002cAB 0.001aAB 0.001bBC 0.001abCDE 0.000cB 0.010aAB 0.006bBC 0.008abCD 0.002cAB 0.000 0.000 0.000B 0.000 0.000 0.000

4 0.009aA 0.003b 0.001bB 0.003ab 0.001B 0.005AB 0.002B 0.003B 0.004A 0.003A 0.003A 0.002AB 0.048AB 0.045B 0.035B 0.030AB 0.004B 0.004CD 0.003AB 0.002B 0.003AB 0.003ABC 0.003ABC 0.002AB 0.007aAB 0.004bCD 0.006aB 0.001bB 0.001aAB 0.000bCD 0.001aCD 0.000bB 0.012aAB 0.005bBCD 0.009aBC 0.001bB 0.000 0.000 0.000B 0.000 0.000 0.000

7 0.009A 0.002 0.004AB 0.003 0.000B 0.002BC 0.002B 0.001B 0.002aBC 0.002abB 0.002abB 0.001bBC 0.234A 0.000B 0.024C 0.013BC 0.005AB 0.005B 0.005AB 0.003AB 0.003AB 0.003ABC 0.004ABC 0.003AB 0.010aAB 0.006bAB 0.007abA 0.002cAB 0.002aAB 0.001bB 0.001abB 0.000cAB 0.016aAB 0.008bAB 0.012abAB 0.003cAB 0.000 0.000 0.000B 0.000 0.000 0.000

14 0.000B 0.000 0.000B 0.000 0.000B 0.000C 0.000B 0.000B 0.001CD 0.000C 0.000C 0.000C 0.000bB 0.431aA 0.008bD 0.006bC 0.007A 0.007A 0.006A 0.007A 0.009A 0.004A 0.005A 0.006A 0.015A 0.008A 0.008A 0.007A 0.005A 0.002A 0.002A 0.002cA 0.037A 0.011A 0.014A 0.013A 0.000 0.000 0.000A 0.000 0.000 0.000

25

46 0.000B 0.000 0.000B 0.000 0.000B 0.000C 0.000B 0.000B 0.000D 0.000C 0.000C 0.000C 0.000B 0.000B 0.000D 0.000C 0.005AB 0.005B 0.004AB 0.003AB 0.003AB 0.003ABC 0.003ABC 0.002AB 0.004aB 0.002bCDE 0.003abC 0.001cB 0.001aAB 0.001bBC 0.001aCDE 0.000cB 0.008aAB 0.004bBCD 0.006abDE 0.002cAB 0.000 0.000 0.000B 0.000 0.000 0.000

days in storage 0.000B 0.000 0.000B 0.000 0.000B 0.000C 0.000B 0.000B 0.000D 0.000C 0.000C 0.000C 0.000B 0.000B 0.000D 0.000C 0.004B 0.004BC 0.004AB 0.002B 0.004abAB 0.004aAB 0.004aA 0.002bAB 0.003aB 0.002bDE 0.003aC 0.000cB 0.001aAB 0.001bBC 0.001aBC 0.000cB 0.010aAB 0.004bBCD 0.008aCD 0.001cB 0.000 0.000 0.000B 0.000 0.000 0.000

82

Table 3. Group 2 ‘Scarlett Spur Red Delicious’ Volatile Accumulation (ηmol L−1) During Controlled Atmosphere or Air Storagea 110 0.000B 0.000 0.000B 0.000 0.000B 0.000C 0.000B 0.000B 0.000D 0.000C 0.000C 0.000C 0.000B 0.000B 0.000D 0.000C 0.003B 0.003DE 0.002BC 0.002B 0.004aAB 0.003abC 0.003abABC 0.001bB 0.002aB 0.000bE 0.001bD 0.000bB 0.001aAB 0.000bCD 0.000bEF 0.000bB 0.006aAB 0.001bD 0.003bFG 0.000bB 0.000 0.000 0.000B 0.000 0.000 0.000

194 0.000B 0.000 0.000B 0.000 0.000B 0.000C 0.000B 0.000B 0.000D 0.000C 0.000C 0.000C 0.000B 0.000B 0.002D 0.000C 0.004aB 0.002bE 0.003abBC 0.001bB 0.006aAB 0.002abC 0.004bcAB 0.001cB 0.002aB 0.000bE 0.001bD 0.000bB 0.001aAB 0.000bCD 0.001bDE 0.000bB 0.007aAB 0.001bD 0.003bG 0.000bB 0.000 0.000 0.000B 0.000 0.000 0.000

250 0.000B 0.000 0.000B 0.000 0.000B 0.000C 0.000B 0.000B 0.000D 0.000C 0.000C 0.000C 0.004B 0.003B 0.003D 0.000C 0.000C 0.001F 0.001C 0.001B 0.001B 0.001D 0.002C 0.000B 0.000B 0.000E 0.000D 0.000B 0.000B 0.000D 0.000F 0.000B 0.001B 0.000D 0.001G 0.000B 0.000 0.000 0.000B 0.000 0.000 0.000



0

0.000 0.000 0.001aAB 0.001aA 0.001abA 0.000bAB 0.002BC 0.002 0.002 0.002A 0.001 0.001AB 0.001AB 0.001 0.001 0.001 0.001 0.001AB

atm

1.6 air 0.3 0.8 1.6 air 0.3 0.8 1.6 air 0.3 0.8 1.6 air 0.3 0.8 1.6 air

0.000 0.000 0.001AB 0.001A 0.001A 0.000AB 0.002BC 0.002 0.002 0.002A 0.001 0.000AB 0.001AB 0.001 0.001a 0.001ab 0.001ab 0.001bAB

4 0.000 0.000 0.001aAB 0.001abA 0.001abA 0.001bAB 0.002BC 0.002 0.002 0.001A 0.001 0.001AB 0.001AB 0.001 0.001a 0.001ab 0.001a 0.001bAB

7 0.000 0.000 0.001aAB 0.001abA 0.001abA 0.001bAB 0.002aABC 0.001b 0.002ab 0.002abA 0.001 0.001AB 0.001AB 0.001 0.001a 0.001ab 0.001ab 0.001bAB

14 0.000 0.000 0.001A 0.001A 0.001A 0.001A 0.002AB 0.002 0.003 0.002A 0.001 0.001A 0.001AB 0.001 0.001a 0.001b 0.001b 0.001bAB

46 0.000 0.000 0.001AB 0.000AB 0.000AB 0.001AB 0.002BC 0.001 0.002 0.002A 0.001 0.001AB 0.001AB 0.001 0.001 0.001 0.001 0.001AB

days in storage 25

82 0.000 0.000 0.001AB 0.000B 0.000B 0.000B 0.001BC 0.001 0.001 0.000B 0.002 0.001AB 0.000B 0.001 0.003 0.003 0.002 0.000B

110 0.000 0.000 0.000B 0.000B 0.000B 0.000B 0.001C 0.001 0.001 0.001A 0.001 0.000B 0.000B 0.001 0.000 0.000 0.000 0.000B

194 0.000 0.000 0.001A 0.001AB 0.001AB 0.000B 0.003A 0.001 0.003 0.001AB 0.002 0.001A 0.001A 0.001 0.002a 0.001b 0.001ab 0.001bAB

250 0.000 0.000 0.001AB 0.000B 0.000B 0.000B 0.002aAB 0.002ab 0.002ab 0.001bA 0.001 0.001AB 0.001AB 0.001 0.001 0.001 0.001 0.001A


a

benzaldehyde

nonanal

2-ethyl 1-hexanol

octanal

group 2 compd.

Table 3. continued


H



2-methylbutyl propanoate

I

butyl acetate

2-methylpropyl acetate

propyl butyrate

pentyl acetate

butyl propanoate

butyl butyrate

propyl acetate

propyl propanoate

propyl 2-methylbutyrate

2-methylbutyl butyrate

atm

group 3 compd. 0.002AB 0.002BCD 0.002BC 0.002 0.002AB 0.002BC 0.001BC 0.002AB 0.032AB 0.027CD 0.019C 0.032 0.012CD 0.010DE 0.008C 0.012B 0.054DE 0.046C 0.039AB 0.056B 0.015AB 0.018AB 0.014CD 0.017B 0.012AB 0.012AB 0.010BCD 0.014C 0.009DE 0.009C 0.007C 0.008B 0.024A 0.020B 0.015B 0.027C 0.017 0.020 0.014 0.025B 0.091ABC 0.091

0 0.002AB 0.002BC 0.003BC 0.003 0.002AB 0.002BC 0.002BC 0.002AB 0.047A 0.038CD 0.031BC 0.036 0.013CD 0.016CDE 0.018BC 0.016B 0.116CD 0.081BC 0.065AB 0.056B 0.014AB 0.017AB 0.015BCD 0.025AB 0.011AB 0.013AB 0.015ABCD 0.020C 0.016BCD 0.014BC 0.012C 0.011B 0.025A 0.027B 0.028B 0.040C 0.023 0.024 0.019 0.027B 0.116A 0.111

4 0.002AB 0.003BC 0.003ABC 0.004 0.002AB 0.002BC 0.002ABC 0.003AB 0.045A 0.045C 0.037ABC 0.046 0.012CD 0.021CD 0.026ABC 0.024AB 0.137CD 0.101ABC 0.076AB 0.083B 0.014AB 0.019AB 0.016BCD 0.031AB 0.009AB 0.014AB 0.017ABCD 0.027BC 0.019BCD 0.018ABC 0.014BC 0.015B 0.023AB 0.033AB 0.037B 0.058C 0.021 0.025 0.019 0.035B 0.112A 0.117

7 0.002AB 0.003B 0.005AB 0.005 0.002AB 0.003ABC 0.003AB 0.004AB 0.044A 0.050BC 0.061ABC 0.050 0.015C 0.026C 0.055ABC 0.024AB 0.244aAB 0.135abABC 0.122abAB 0.074bB 0.015bAB 0.024abAB 0.021abABC 0.055aAB 0.008AB 0.014AB 0.024ABC 0.036BC 0.023AB 0.022ABC 0.021ABC 0.025B 0.025A 0.035AB 0.068AB 0.079BC 0.018 0.020 0.023 0.030B 0.098bAB 0.100b

14 0.004A 0.004A 0.007A 0.007 0.004A 0.003A 0.003A 0.007A 0.052A 0.074AB 0.094A 0.061 0.032A 0.053B 0.106A 0.035AB 0.325A 0.196AB 0.217A 0.144B 0.034bA 0.035bAB 0.030bAB 0.110aAB 0.022A 0.020AB 0.037A 0.061ABC 0.030A 0.028ABC 0.032AB 0.057AB 0.041A 0.054AB 0.122A 0.123ABC 0.023 0.022 0.032 0.049AB 0.118bA 0.129b

25

46 0.003AB 0.005A 0.005AB 0.004 0.002AB 0.003AB 0.003AB 0.003AB 0.044A 0.086A 0.082AB 0.076 0.027AB 0.072A 0.100AB 0.041AB 0.285A 0.202A 0.229A 0.304AB 0.020bAB 0.046abAB 0.033abA 0.104aAB 0.011AB 0.025A 0.032AB 0.072ABC 0.020BC 0.032AB 0.038A 0.076AB 0.040A 0.095A 0.147A 0.179ABC 0.012 0.028 0.031 0.088AB 0.060CD 0.627

days in storage 0.003AB 0.003B 0.004ABC 0.004 0.001AB 0.002BC 0.002ABC 0.002AB 0.031bAB 0.042abC 0.051abABC 0.084a 0.017bBC 0.022abCD 0.028abABC 0.055aAB 0.152bBC 0.124bABC 0.157bAB 0.664aA 0.022bAB 0.056abA 0.031bA 0.129aA 0.011bAB 0.029bA 0.022bABCD 0.135aA 0.017bBCD 0.037bA 0.038bA 0.118aA 0.036bA 0.076bAB 0.070bAB 0.328aAB 0.010b 0.028b 0.032b 0.126aA 0.064bBCD 0.197b

82

Table 4. Group 3 ‘Scarlett Spur Red Delicious’ Volatile Accumulation (ηmol L−1) during Controlled Atmosphere or Air Storagea 110 0.002AB 0.002CD 0.002BC 0.003 0.001B 0.002C 0.001BC 0.002AB 0.025bAB 0.027bCD 0.031bBC 0.094a 0.015bC 0.012bCDE 0.011bC 0.068aA 0.139bCD 0.056bC 0.072bAB 0.527aA 0.022bAB 0.051abA 0.024bABC 0.108aAB 0.009bAB 0.025bAB 0.012bBCD 0.119aAB 0.012bCD 0.026abABC 0.020bABC 0.092aAB 0.029bA 0.070bAB 0.038bB 0.360aA 0.007b 0.016b 0.017b 0.082aAB 0.044bDE 0.128b

194 0.002AB 0.001DE 0.002BC 0.002 0.002AB 0.001CD 0.002BC 0.001B 0.044abA 0.025bCD 0.055abABC 0.090a 0.004bDE 0.005bE 0.006bC 0.046aAB 0.093bCDE 0.026cC 0.036bcAB 0.403aAB 0.027AB 0.036AB 0.023ABC 0.058AB 0.006bAB 0.010bAB 0.006bCD 0.042aBC 0.011bCDE 0.020bABC 0.013bBC 0.065aAB 0.035bA 0.056bAB 0.034bB 0.249aABC 0.012b 0.011b 0.024b 0.104aAB 0.033bDE 0.072b

250 0.000B 0.000E 0.000C 0.000 0.000B 0.000D 0.001C 0.001B 0.007AB 0.015D 0.011C 0.036 0.000E 0.003E 0.000C 0.007B 0.010E 0.065C 0.009B 0.116B 0.003bB 0.007bB 0.005bD 0.023aB 0.000bB 0.002abB 0.001bD 0.006aC 0.002bE 0.008abC 0.003bC 0.029aAB 0.004bB 0.024abB 0.007bB 0.084aBC 0.006 0.023 0.007 0.044AB 0.009E 0.150



hexyl acetate

2-methylbutyl acetate

esters and other compounds was similar during this period in storage. Acetaldehyde, also a product of fermentative metabolism, is metabolized first to ethanol and then to ethyl esters.10,37 Low oxygen CA storage reduces fruit metabolic rate including activity of the terminal enzyme in ester synthesis alcohol acyltransferase8 such that ester production decreases as storage duration increases.5,11,12,25,26,28,29,37 Methyl acetate, methyl 2-methylbutyrate, and acetaldehyde were all significantly highest at day 194. Although methanol was not detected, the presence of methyl esters indicates methanol was likely present as a substrate for ester synthesis.34 The group 1 volatiles associated with fermentative metabolism in ‘Delicious’ may be useful indicators for monitoring the occurrence of anaerobic conditions during CA storage. An ideal indicator would have a marked concentration change soon after low oxygen conditions occur, and many of the group 1 volatiles are significantly highest in the 0.3 kPa O2 chambers by day 4. The increase in ethanol concentration during the days after 0.3 kPa O2 was established may indicate its utility as an early indicator of low O2 stress due to the higher rate of increase between ultralow oxygen and more typical (1.6 kPa) O2 CA conditions. The increase in ethanol at 0.8 kPa O2 for which low oxygen injury development was similar to that occurring in fruit stored at 1.6 kPa O2 or in air indicates a threshold concentration associated with injury risk may need to be identified to enable ethanol monitoring in storage room headspace as an indicator of low O2 injury risk. Amounts of the group 2 compounds butyl hexanoate, hexyl butyrate, and hexyl 2-methylbutyrate were significantly highest in 0.3 and 1.6 kPa O2 through day 82 and at 0.3 kPa O2 on days 110 and 194 (Table 3). On the same dates, the concentrations of these compounds were lowest in air chambers. Hexyl propanoate (days 82−194), benzaldehyde (days 4−25, 194), and heptanal (days 4, 14) were highest in 0.3 kPa O2 and lowest in air. Amounts of butyric acid, pentanal, hexanal, ethyl octanoate, estragole, octanal, 2-ethyl-1-hexanol, nonanal, and propyl hexanoate were unaffected by storage conditions. Within atmospheres, butyric acid, pentanal, heptanal, and hexanal accumulated during the first 7 days of storage then significantly decreased during days 14−25. Aldehydes present in preclimacteric fruit are responsible for the ‘green/unripe’ aroma and, as ripening occurs, the volatile profile shifts to esters and a ‘fruity’ aroma.34,38 Many group 2 volatiles were significantly highest on day 25 and significantly decreased by day 82 including propyl hexanoate, hexyl propanoate, butyl hexanoate, hexyl butyrate, hexyl 2-methylbutyrate, and octanal. This pattern indicates high production of these compounds occurred relatively early in the ripening process. Amounts of ethyl octanoate, estragole, 2-ethyl 1-hexanol, nonanal, and benzaldehyde did not change with storage duration. Lack of atmosphere treatment differences for these compounds indicates production was unaffected by storage atmospheres and ripening metabolism, while volatiles unaffected by storage duration were unrelated to the progression of ripening. Group 3 volatiles accumulated in significantly higher concentrations in air chambers, while fruit stored in 0.3 kPa O2 produced the lowest amounts of these compounds (Table 4). These volatiles, including propyl acetate, butyl butyrate, butyl propanoate, pentyl acetate, propyl butyrate, 2-methylpropyl acetate, butyl acetate, and 2-methylbutyl acetate, are compounds produced during typical climacteric ripening of ‘Delicious’ apples.10 Due to fermentative metabolism in the 0.3 and 0.8 kPa O2 stored fruit, ethanol accumulation provided substrate to favor


250

Article

a

0.038b 0.632aAB 0.065bB 0.076bD 0.137abC 0.244a 0.256A 0.292ABC 0.219B 0.295AB

194 110

0.080b 1.27aAB 0.076bB 0.149bBCD 0.204bABC 0.537a 0.210AB 0.366AB 0.221AB 0.408AB 0.168b 1.50aAB 0.115bB 0.278bAB 0.349abAB 0.641a 0.218AB 0.438A 0.316A 0.532A

82 46

0.747 2.05A 0.186A 0.384A 0.379A 0.626 0.134BC 0.221BCD 0.196BC 0.309AB 0.181b 0.279aAB 0.246A 0.253ABC 0.228ABC 0.281 0.137abBC 0.124bCD 0.119bCD 0.253aAB 0.110b 0.193aB 0.223A 0.159BCD 0.229ABC 0.235 0.081CD 0.068D 0.046DE 0.076AB 0.095 0.170B 0.231A 0.232ABCD 0.225ABC 0.231 0.059CD 0.045D 0.039DE 0.031B 0.075 0.092B 0.188A 0.226ABCD 0.160BC 0.157 0.028D 0.024D 0.021E 0.017B 1.6 air 0.3 0.8 1.6 air 0.3 0.8 1.6 air

0.097 0.121B 0.250A 0.213ABCD 0.223ABC 0.203 0.050aCD 0.036abD 0.032abDE 0.025bB

days in storage


Table 4. continued

0.014 0.300AB 0.047B 0.101CD 0.070C 0.141 0.043bCD 0.082bD 0.075bDE 0.206aAB


J


K

2-methyl 1-butanol

2-methyl 1-propanol

6-methyl-5-hepten-2-ol

6-methyl-5-hepten-2-one

decanal

butyl 2-methylbutyrate

1-hexanol

acetone

1-butanol

1-pentanol

methyl hexanoate

group 4 compd.


atm 0.000B 0.000D 0.000B 0.000C 0.007abB 0.008a 0.006bC 0.008aBC 0.190ABC 0.195 0.160BC 0.236 0.088AB 0.098AB 0.067 0.120AB 0.013B 0.012B 0.013B 0.014B 0.002B 0.002B 0.002D 0.001B 0.000B 0.000B 0.000 0.000 0.002abB 0.002ab 0.002aB 0.002b 0.000B 0.000B 0.000C 0.000 0.000 0.000 0.000 0.000 0.005abD 0.005ab

0 0.000AB 0.000CD 0.000B 0.000C 0.008B 0.008 0.007C 0.007BC 0.232AB 0.207 0.171BC 0.207 0.165A 0.142AB 0.051 0.095B 0.019B 0.017B 0.013B 0.019B 0.002B 0.002B 0.002CD 0.002B 0.000B 0.000B 0.000 0.001 0.003B 0.003 0.003B 0.002 0.000B 0.000AB 0.000C 0.000 0.000 0.000 0.000 0.000 0.008D 0.007

4 0.001aAB 0.000abCD 0.000bB 0.000bC 0.009B 0.009 0.008C 0.009BC 0.233AB 0.222 0.175BC 0.246 0.000bC 0.133aAB 0.112ab 0.091abB 0.022B 0.023B 0.014B 0.016B 0.002B 0.002B 0.002CD 0.002B 0.001B 0.000B 0.000 0.001 0.003B 0.003 0.003B 0.002 0.000B 0.000AB 0.000C 0.000 0.000 0.000 0.000 0.000 0.012D 0.010

7 0.001aAB 0.001abBCD 0.000bB 0.000bC 0.007B 0.008 0.008C 0.011BC 0.254A 0.199 0.183BC 0.355 0.000bC 0.041abB 0.112a 0.085abB 0.041B 0.036B 0.026B 0.032B 0.002B 0.002B 0.003BCD 0.002B 0.000B 0.000B 0.000 0.001 0.005aB 0.003ab 0.003abB 0.003b 0.000B 0.000AB 0.000C 0.000 0.000 0.000 0.000 0.015 0.020D 0.016

14 0.002A 0.001ABCD 0.000B 0.001C 0.009B 0.014 0.018C 0.020BC 0.124BC 0.162 0.195BC 0.719 0.007C 0.051B 0.051 0.047B 0.065B 0.093B 0.105B 0.228B 0.002B 0.002B 0.003BCD 0.002B 0.001B 0.000B 0.001 0.001 0.004B 0.003 0.003B 0.003 0.000B 0.000AB 0.000C 0.000 0.000 0.000 0.028 0.023 0.060CD 0.070

46

days in storage 0.002aA 0.001abABC 0.000bB 0.000abC 0.008B 0.007 0.010C 0.018C 0.190abABC 0.135b 0.125bC 0.370a 0.041BC 0.046B 0.062 0.051B 0.084B 0.077B 0.046B 0.129B 0.006B 0.003B 0.003BC 0.004B 0.001B 0.000B 0.000 0.001 0.005B 0.004 0.004B 0.005 0.000B 0.000A 0.000C 0.000 0.000 0.000 0.017 0.015 0.047CD 0.038

25 0.002abA 0.001abABC 0.001bB 0.002aABC 0.021AB 0.024 0.046B 0.039AB 0.150bABC 0.213b 0.368bA 1.26a 0.000bC 0.038abB 0.044ab 0.115aAB 0.075B 0.103B 0.157B 0.104B 0.003abB 0.003aB 0.003aBCD 0.001bB 0.001B 0.001B 0.001 0.000 0.003B 0.004 0.004B 0.005 0.000B 0.000A 0.000C 0.000 0.000 0.000 0.020 0.009 0.087bBC 0.160ab

82

Table 5. Group 4 ‘Scarlett Spur Red Delicious’ Volatile Accumulation (ηmol L−1) During Controlled Atmosphere or Air Storagea 110 0.001bAB 0.001bcABCD 0.000cB 0.002aBC 0.018AB 0.022 0.043B 0.048ABC 0.122bBC 0.174ab 0.285abAB 1.39a 0.012bBC 0.050bB 0.062b 0.292aAB 0.330aB 0.312aB 0.227abB 0.130bB 0.003aB 0.002abB 0.002abCD 0.001bB 0.000B 0.000B 0.000 0.001 0.004B 0.004 0.003B 0.004 0.000B 0.000AB 0.000C 0.000 0.000 0.000 0.015 0.010 0.090BC 0.155

194 0.002bA 0.001bAB 0.001bA 0.004aAB 0.033A 0.031 0.078A 0.115A 0.224bAB 0.234b 0.278bAB 2.08a 0.048bBC 0.080bAB 0.074b 0.278aAB 1.07A 1.02A 1.22A 0.876A 0.020A 0.014A 0.017A 0.020A 0.002aA 0.001abA 0.001b 0.001b 0.013aA 0.004b 0.009abA 0.003b 0.004aA 0.001bA 0.003abA 0.000b 0.000 0.039 0.176 0.012 0.338bA 0.302bc

250 0.001bAB 0.002abA 0.001bAB 0.005aA 0.007B 0.019 0.012C 0.130ABC 0.104C 0.442 0.083C 3.21 0.044BC 0.156A 0.103 0.353A 0.030bB 0.067bB 0.063bB 0.302aB 0.002bB 0.004abB 0.004abB 0.010aAB 0.001AB 0.001B 0.001 0.001 0.002B 0.002 0.003B 0.003 0.001B 0.001B 0.002B 0.001 0.013 0.054 0.019 0.000 0.133B 0.327



ethyl-ester synthesis and decreased butyl and hexyl ester production, consistent with previous reports.10,13 Amounts of these compounds increased regardless of storage atmosphere from 25 to 82 days, however, these compounds continued to accumulate in air chambers until 194 days storage and had significantly higher levels compared with the CA chambers from 82 days until the end of storage. Amounts of propyl 2-methylbutyrate and propyl propanoate were significantly higher in air chambers compared with some or all CA treatments from 82 to 194 days storage. Within atmospheres, all group 3 volatiles were significantly higher on day 25 and/or day 46 compared to other sampling dates. High amounts of group 3 compounds in the air chambers and relatively lower levels in CA chambers indicates reduced ester production occurs during as well as following CA storage compared to fruit stored in air.39,11,4 Group 4 compounds methyl hexanoate, 1-butanol, and acetone were significantly higher in air compared to CA chambers (Table 5), and amounts increased for the entire storage duration. 2-Methyl-1-butanol concentration was significantly higher in chambers held at 1.6 kPa O2 on days 82 and 194 compared with amounts in air or 0.3 kPa O2 chambers. Accumulation of 2-methyl-1-butanol may have been lower in air storage due to its metabolism to 2-methylbutyl acetate.40 No consistent atmosphere treatment effects were observed for 1-pentanol, 1-hexanol, butyl 2-methylbutyrate, 2-methyl-1-propanol, decanal, 6-methyl-5hepten-2-one, and 6-methyl-5-hepten-2-ol. Amounts of all group 4 compounds except 1-butanol were higher on day 250 and/or day 194 compared with most/all other days for most atmospheres. As stated previously, alcohols are substrates for ester production and are actively converted to esters. However, by the end of storage, ester production decreases as ripening progresses and alcohols that could serve as substrates for ester synthesis (1-propanol, 1-butanol, 2-methyl-1-butanol, and 1-pentanol) accumulate. Atmosphere−day interactions were significant for 31 of 51 volatiles detected. 1-Propanol, methyl butyrate, pentanal, propyl hexanoate, hexyl propanoate, butyl hexanoate, hexyl butanoate, hexyl 2-methylbutyrate, estragole, octanal, 2-ethyl-1hexanol, nonanal, benzaldehyde, 2-methylbutyl propanoate, 2-methylbutyl butyrate, propyl 2-methylbutyrate, hexyl acetate, 1-hexanol, butyl 2-methylbutyrate, and decanal did not have a significant atmosphere−day interaction. Several of these volatiles (estragole, octanal, 2-ethyl-1-hexanol, nonanal, benzaldehyde, 1-hexanol, butyl 2-methylbutyrate, and decanal) were not affected by storage conditions, implying that their production is also unrelated to the progression of fruit ripening. Dynamics of apple fruit volatile production following storage have been characterized25,26 but information regarding production dynamics during storage is lacking. Argenta et al. (2004)7 reported ‘Fuji’ apple volatile production 24 h after removal from 8 months in 0.5 kPa O2 storage was less than that of fruit previously stored in air. Production of 1-butanol, 1-pentanol, and 1-hexanol and the corresponding esters for each alcohol was reduced following low oxygen storage, while amounts of ethanol, ethyl acetate, and ethyl esters increased. Higher oxygen CA storage also results in reduced poststorage alcohol, hexyl- and butyl ester emission with increased ethanol, ethyl acetate and acetaldehyde compared with fruit stored in air.11,41 Diminution of post-storage volatile emission also increases with increasing storage duration and decreased CA oxygen content.11 Imposition of anaerobic storage conditions following 4−5 months CA storage also results in enhanced


250

Article

a

0.830aA 0.139c

194 110

0.228BC 0.084 0.241aBC 0.092b

82 46

0.106C 0.104 0.043C 0.061 0.017C 0.028 0.009C 0.015 0.004bC 0.007a 1.6 air

0.007C 0.010

days in storage


Table 5. continued

0.423B 0.173


L



Article

Figure 5. ‘Scarlett Spur Red Delicious’ fruit internal ethylene (A) and storage chamber ethylene concentration (B). All fruit stored at 0.5 °C. Controlled atmosphere kPa O2 concentrations were 0.3, 0.8, or 1.6, all with 1 kPa CO2. Internal ethylene values are means (n = 4) of apples (∼ 1 kg) for each storage atmosphere, measured after removal from cold storage plus 7 days at 20 °C. Storage chamber ethylene was measured in gas samples removed from storage chambers (n = 3 per atmosphere) containing 72 fruit each. Time and treatment differences are denoted by upper or lowercase letters, respectively. Within each month or treatment, columns with the same letter are not significantly different (P < 0.05, Tukey’s HSD).

indicates that those fruit had become senescent. Consistent with previous studies, superficial scald levels were significantly higher in 1.6 kPa O2 than in 0.3 or 0.8 kPa O2 stored fruit. This indicates ultralow O2 storage was able to reduce scald incidence.15−17 However, 0.3 kPa O2 stored fruit was negatively affected by the anaerobic conditions, resulting in significantly higher levels of stem browning and fermentative volatiles. In summary, storage duration and O2 concentration quantitatively impacted volatile production during long-term storage. Differences due to storage atmosphere were greatest during the first 6 months, and volatile production measured during CA storage was similar to measuring production after storage. Butyl and propyl ester production was inhibited by CA storage conditions and anaerobic oxygen storage promoted fermentative volatile production. CA storage prolonged storage life by reducing ethylene production, disorder incidence, and losses in firmness, and titratable acidity. The rate of ethanol production increased with decreasing kPa O2 and could be useful in monitoring for low O2 stress during storage. Monitoring production of other fermentative volatiles such as acetaldehyde and ethyl acetate could also have potential as stress indicators. Fermentative volatile production was significantly different among atmospheres as early as day 4, so monitoring can begin early in storage.

acetaldehyde, ethanol, and ethyl ester production with reduced hexyl and butyl ester production.10 The current results indicate accumulation of many alcohols and esters is also reduced during low oxygen CA storage compared to fruit stored in air. Hexyl ester accumulation, however, was not affected by low O2 storage conditions in this study. Fermentative volatile accumulation during storage was similar to that reported for fruit held in air after removal from CA. Fruit ethylene production after storage increased relative to values at harvest regardless of storage atmosphere (Figure 5), and fruit stored in air consistently had the highest ethylene production. Ethylene production by fruit previously stored in 0.3 kPa O2 did not change with storage duration but fruit stored in air, 0.8 kPa O2, or 1.6 kPa O2 significantly increased by months 4 and/or 8 compared to month 2. Ethylene content was significantly higher in storage chambers maintained with air compared to chambers with 0.3 or 0.8 kPa O2 at 6, 7, and 8 months and 1.6 kPa O2 chambers at 6 and 8 months. However, within atmospheres, storage duration did not significantly impact ethylene content with only 1.6 kPa O2 having significant differences in ethylene content between 5 and 8 months. The increase in ethylene production from harvest to month 1 indicates climacteric ripening was ongoing. Apple volatile production during storage increased in the first 3 months, also consistent with apple ripening. CA gas composition appeared to have no effect on the timing of ripening but did impact the amounts of ethylene and volatile production. Volatile accumulation among atmospheres was the most different during 1−3 months storage, confirming that gas composition had a large effect on the metabolic rates of ripening processes that impact volatile production. In addition, anaerobic conditions caused further changes in volatile production during the first 3 months in storage as indicated by the increased distance between the 0.3 kPa O2 and the 0.8 and 1.6 kPa O2 results in Figure 1. As expected, the rise in ethylene and volatile production was also accompanied by a gradual decrease in firmness and titratable acidity42 (Figure 3). The lower ethylene levels in all CA treatments accompanying decreased losses in firmness and titratable acidity reflects the requirement for ethylene to promote ripening during cold storage. At 9 months, internal breakdown incidence was significantly higher in air than in all CA treatments (Figure 4). The higher cortex browning and internal breakdown in air stored apples

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 509-664-2280. E-mail: [email protected]. Present Address §

(C.L.) Western Colorado Research Center, Colorado State University, Grand Junction, CO 81503-9621, United States Funding

Supplemental funding for this research was received from the Washington Tree Fruit Research Commission. Mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Notes

The authors declare no competing financial interest. M



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Article

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ACKNOWLEDGMENTS We thank David Buchanan, Janie Countryman, and Rachel Leisso for excellent technical assistance.

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