Temperature of hypoxic treatment alters volatile composition of juice from ‘Fuji’ and ‘Royal...

Postharvest Biology and Technology 22 (2001) 71–83

Temperature of hypoxic treatment alters volatilecomposition of juice from ‘Fuji’ and ‘Royal Gala’ apples

Jonathan Dixon, Errol W. Hewett *College of Sciences, Institute of Natural Resources, Massey Uni6ersity, Pri6ate Bag 11 222, Palmerston North, New Zealand

Received 7 March 2000; accepted 24 August 2000

Abstract

Exposure of apples to hypoxia induces changes in volatile concentrations in extracted juice, potentially affectingaroma quality, but there is little information on how the temperature of hypoxic treatment may affect volatileconcentration. ‘Fuji’ (FU) and ‘Royal Gala’ (RG) apples were exposed to hypoxia, 100% CO2 for 24 h, at 10°C, 15,20 or 25°C and maintained at treatment temperature for up to 14 days. CO2 and ethylene production and firmnessof intact fruit were proportional to temperature but were unaffected by exposure to hypoxia. Ethyl esters fromextracted juice were enhanced at all temperatures at differential rates according to cultivar. Four patterns of totalvolatile concentration change with temperature after exposure to hypoxia were identified which may be related tochanges in rates of volatilisation and volatile production. Apples treated and maintained at 10°C had the greatestoverall enhancement of ethyl esters and least decrease in other esters in juice compared to apples treated andmaintained at 15, 20 and 25°C. This enhancement in volatiles persisted for up to 10 days after removal from hypoxia.Maintenance of apple quality after treatment with hypoxia was better at low temperatures suggesting that applestreated with hypoxia and maintained below 15°C would have enhanced volatile concentrations compared withuntreated apples and apples maintained above 15°C. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Malus domestica Borkh.; Aroma; Flavour; Temperature; Quality; Solvent extraction; CO2 treatment

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1. Introduction

Apple aroma contains more than 300 volatilecompounds consisting mainly of esters, alcoholsand aldehydes, with their production influencedby temperature. Ester and alcohol concentrationsand production increased as temperature in-

creased in ‘Jonathan’ apples at temperatures from−1°C to 10°C for up to 12 weeks (Wills andMcGlasson, 1971). Ester concentration was maxi-mum at 22°C for ‘Red Delicious’ apples, anddecreased at 32°C, while at 46°C aroma produc-tion was inhibited (Guadagni et al., 1971). Heattreatment of ‘Golden Delicious’ apples of 38°Cfor 4 days reduced volatile production comparedto fruit at 22°C (Fallik et al., 1997).

Exposure of fruit to hypoxic atmospheres forseveral days is a possible non-chemical insect dis-

* Corresponding author. Tel.: +64-6-3569099,ext.5231; fax:+64-6-3505679.

E-mail address: [email protected] (E.W. Hewett).

0925-5214/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.

PII: S 0925 -5214 (00 )00159 -9

J. Dixon, E.W. Hewett / Posthar6est Biology and Technology 22 (2001) 71–8372

infestation treatment (Lay-Yee and Whiting,1996; Whiting et al., 1996). Hypoxic atmo-spheres kill insects faster at 20°C or above thanat B5°C; at higher temperatures the target in-sect has increased metabolic activity and rate offumigant uptake (Ke and Kader, 1992; Paulland Armstrong, 1994). Exposure of apples tohypoxia induces substantial qualitative andquantitative changes in volatile concentration(Ampun, 1997) and generally reduces softening,CO2 and ethylene production, probably due tothe inhibitory effects that high concentrations ofCO2, acetaldehyde, ethanol or low O2 concentra-tions have on enzyme systems within fruit.Treating a range of fruit with atmospheres upto 100% CO2 or N2 at −1.1°C to 32°C for afew hours up to 6 weeks enhanced maintenanceof quality during long term storage (Eaves etal., 1968), acclimatised fruit to low O2 storageconditions (Little et al., 1982), allowed for non-damaging disinfestation treatments (Ke et al.,1991; Yahia and Vazquez-Moreno, 1993) andenhanced aroma/flavour volatile concentration(Shaw et al., 1992; Dourtoglou et al., 1994; Am-pun, 1997; Dixon and Hewett, 2000).

Little is known about the interaction of tem-perature and hypoxia on aroma volatile produc-tion in apples, although it is expected that itwould be reduced at low temperatures in linewith a general reduction in metabolism. At 20°Cthere is a large increase in acetaldehyde, etha-nol, ethyl esters and some alcohols while thereis a corresponding decrease in acetate esters andno change in concentration of aldehydes (Am-pun, 1997; Dixon, 1999). After exposure to hy-poxia, the magnitude of the increases inacetaldehyde, ethanol and ethyl esters should bedecreased at reduced temperatures while raisingfruit temperature should increase volatile pro-duction.

If hypoxic treatments are to be used as disin-festation treatments, consideration must be givenas to when such treatments should be applied inthe coolchain. Apple ripening rate is propor-tional to temperature, the higher the tempera-ture the faster the ripening (Wills et al., 1997).To maximise preservation of postharvest qualityapples should be cooled as soon as possible af-

ter harvest. By exposing apples to hypoxia attemperatures of 20°C and above, unacceptablelosses in quality attributes such as firmness andbackground colour may occur. As an example,rate of loss in background colour of ‘Cox’s Or-ange Pippin’ apples maintained for 1 day at20°C is estimated to be the same as for fruitkept at 3°C for 9 days (Dixon and Hewett,1998). In this experiment ‘Fuji’ (FU) and ‘RoyalGala’ (RG) apples were exposed to hypoxic con-ditions and subsequently stored at temperaturesranging from 10°C to 25°C to evaluate the ef-fect of post-hypoxia temperature on aromavolatile production.

2. Materials and methods

2.1. Fruit supply

FU and RG apples (Malus domestica Borkh.)were harvested at mid commercial harvest, 7May 1995 for FU and 31 March 1995 for RG,from Hawke’s Bay, New Zealand. Fruits weregraded to export standard, packed to count 125(148-g average fresh weight), transported unre-frigerated to Massey University by road andplaced at 0 9 1°C within 3 days of harvest. Be-fore treatment, fruits were removed from cool-store and equilibrated overnight to treatmenttemperatures of 10, 15, 20 and 25 C9 1°C.Fruits were divided into two groups, one wasexposed to hypoxia, the other was maintained inair as a control. For each treatment and sampletime there were four replicates each of three ap-ples.

2.2. Hypoxic treatment

Prior to CO2 treatment fruit was stored at0 9 1°C. This was because the size of the ex-periment and number of samples involved meantthat not all treatment samples could be ex-tracted and analysed at the same time; hence thesampling procedure was staggered in such a wayas to minimise storage effects. FU apples werestored for 52 days for treatments at 10°C and25°C and 82 days for treatments at 15 and20°C. RG apples were stored for 75 days for

J. Dixon, E.W. Hewett / Posthar6est Biology and Technology 22 (2001) 71–83 73

treatments at 10°C and 25°C and 109 days fortreatments at 15 and 20°C. Fruits were removedfrom 0°C and equilibrated for 12 h to achieve thetreatment temperature. Fruits were placed into24.1 l perspex chambers connected to a manifold,in parallel, leading from a high-pressure cylinderof pure CO2 (BOC Gases New Zealand, NewZealand). CO2 was humidified by bubblingthrough water before passing into the manifold.During treatment, chambers were purged continu-ously with CO2 for 24 h. Outlets from each cham-ber were joined and the outflow bubbled throughwater to create sufficient backpressure to min-imise CO2 flow rate differences between chambers.Initial CO2 flow rate in each chamber was 2.5l min−1 for 2–3 h (6.2 volume changes h−1) afterwhich it was reduced to 1 l min−1 (2.5 volumechanges h−1). Excess gas was vented to outsideair. The CO2 and O2 contents of chambers weremonitored by gas chromatography, every fewminutes initially and every few hours thereafter. Aconcentration of B0.5% O2 was reached within 4h of commencing purging and remained constantuntil chamber opening after 24 h. After treatment,fruit were maintained at treatment temperaturesfor 14 days.

2.3. Fruit assessment

Fruit were assessed the day before treatment(day −1), on removal from hypoxia (day 0) andat 2-day intervals following treatment duringripening at treatment temperatures for CO2 andethylene, firmness, mass, volatile concentration ofjuice and fermentation volatiles in the juiceheadspace. This allowed for differences in volatileconcentration due to fruit ripening to be ac-counted for in the data analysis.

2.4. Firmness

Firmness was measured using a hand held Ef-fegi penetrometer (model FT327) with a 11.1 mmmeasuring head on paired surfaces on opposingsides of fruit at the equator. The average of thetwo measurements was multiplied by 9.81 to con-vert the instrument reading to Newtons.

2.5. CO2 and ethylene production

Each replicate of 3 apples, from each treatment,was weighed and, depending on size of fruit,placed into 1.6 l or 1.8 l glass preserving jars. 1 mlof gas was removed from each jar 15–30 minafter sealing using a plastic syringe (1 cc Gradu-ated Monoject® syringe with detachable needle,25 gauge × 5/8%%, Sherwood Medical, MO, USA).CO2 was analysed using a thermal conductivitydetector at 60°C and 90 mA current in a Shi-madzu 8A gas chromatograph (GC) equippedwith an Alltech CTR I column (Alltech cat. no.8700) at 30°C with hydrogen at a flow rate of 30ml min−1 as carrier gas. Ethylene was measuredusing a flame ionization detector at 150°C,column 110°C and injector 190°C, in a Shimadzu4B-PTF GC equipped with a F-1 grade, 80/100mesh activated alumina 1.83 m × 3.18 mmcolumn (Alltech cat. no. 80072). Nitrogen at aflow rate of 30 ml min−1 was the carrier gas. Theflame was maintained with hydrogen at 30ml min−1 and air at 300 ml min−1. CO2 andethylene production was calculated according toBanks et al. (1995).

2.6. Juice preparation

Each group of three unpeeled apples from eachtreatment was ground in a domestic juicer at 20°C(Kenwood Centrifuga, model JE500, KenwoodAppliances, New Zealand). Juice was collectedand left at ambient temperature (20 9 3°C) for atleast 30 min before volatile extraction and analy-sis (Dixon, 1999).

2.7. Headspace 6olatiles

Acetaldehyde, ethyl acetate and ethanol in theheadspace of apple juice were analysed. 30 ml ofapple juice was placed into 50 ml glass Erlenmyerflasks sealed with Suba-Seal® (No. 33) rubberstoppers, maintained at 30°C in a water bath.After at least 15 min a 1-ml gas sample, takenfrom the flask headspace, was measured using aflame ionization detector at 180°C, column 45°Cand injector 110°C in a PYE UNICAM GC fittedwith a 1.83 m × 3.18 mm stainless steel column


(Supelco cat. no. 1-2212) containing a 10% Car-bowax 20M coating on 80/100 ChromosorbWAW support. Nitrogen at a flow rate of 30ml min−1 was the carrier gas. The flame wasmaintained with hydrogen at 30 ml min−1 and airat 300 ml min−1.

2.8. Juice 6olatiles

Volatile compounds were extracted from applejuice using a diethyl ether:n-pentane solvent mix-ture (2:1 v/v, Analar BDH) (Larsen and Poll,1990). Two 10-ml aliquots of juice were eachplaced into separate 20 ml scintillation vials(Wheaton Scientific, NJ, USA) fitted with a metalfoil liner cap. The internal standard (IS), consist-ing of 0.2 ml octyl acetate (Aldrich Chemical, WI,USA), was added to each juice aliquot before 10ml of solvent mixture. Vials were capped tightlyand mixed with a vortex stirrer for 3–5 s beforestorage at −18°C until the aqueous phase wasfrozen. The unfrozen solvent phase was decantedfrom the aqueous phase that was discarded. Thediethyl ether:n-pentane solvent extract was con-centrated approximately 50 fold by drying from10 ml to about 200 ml using N2 stream. The liquidwas placed into a 250 ml flat bottom glass insert(Sun International Trading cat. no. 200 232) in a1.5 ml glass screw top autosampler vial (SunInternational Trading cat. no. 200 250) suitablefor a Hewlett Packard 5890 series II plus GLCautosampler (Hewlett Packard 7673 Controllerand Injector and Model 185968 100 samplecarousel). Vials were sealed with a plastic septum(Sun International Trading cat. no. 200 368) be-fore placement in the carousel. One microlitresamples were injected into the GLC. Quantifica-tion of volatiles in the solvent extract was bycomparison with authentic compounds made to aconcentration of 200 ml l−1 in the solvent mixture.

One microlitre of solvent extract was measuredby capillary gas chromatography using a HewlettPackard 5890 Series II Plus GC connected to anIBM compatible personal computer equippedwith Hewlett Packard ChemStation software (ver-sion B.02.04). The capillary column was a J&W30 m × 0.32 mm (i.d.) fused silica, DBWAX, 0.5mm film thickness (Alltech cat. no. 93526). Injec-

tor and detector temperatures were 150°C and250°C respectively. Oven temperature was held at40°C for 5 min, then programmed to 120°C at5°C min−1 then to 190°C at 20°C min−1 with noholding time making a total run time of 24.5 min.Hydrogen was used as the carrier gas with a linearflow rate of 30 ml min−1. A split injection modewas used with a split flow rate of 100 ml min−1

and split ratio of 15:1. Septum purge flow ratewas 5–6 ml min−1. Air and hydrogen flow ratesto the detector were 400 and 30 ml min−1,respectively.

2.9. Data analysis

Each experiment was conducted as a completelyrandom design with sampling during storage asrepeated measures. Means and standard errors ofthe means for each cultivar and treatment temper-ature for headspace and juice volatiles weregraphed using an Origin graphics package v. 5(Microcal Software, USA). Data were subjectedto analysis of variance using SAS v. 6.12 (SASInstitute, Cary, USA). Significant main mean ef-fects were separated by Duncan’s multiple rangetest at the 5% level of significance.

3. Results

3.1. CO2 and ethylene production and firmness

At each temperature FU and RG fruit hadsimilar rates of CO2 production (Fig. 1). For bothcultivars CO2 production was proportional totemperature, increasing by about 50nmol kg−1 s−1 per 5°C (Fig. 1). CO2 productionremained unchanged for 14 days at 10 and 15°Cbut slowly declined at 20°C and 25°C. Exposureto hypoxia did not affect CO2 production for FUand RG fruit except for RG fruit exposed tohypoxia and stored at 25°C where CO2 produc-tion increased slightly from 4 to 8 days.

In FU and RG fruit exposed to hypoxia at 15,20 and 25°C, ethylene production decreased 2days after removal, before increasing at 4 days tobe similar to untreated control fruit (Fig. 2). ForFU and RG fruit at 10°C exposed to hypoxia,


ethylene production was reduced only at 4 days.Rate of ethylene production increased with tem-perature to a maximum at 20°C, but declined at25°C to rates similar to those at 15°C (Fig. 2).Pattern of ethylene production was different be-tween cultivars; in FU fruit ethylene productionsteadily increased during 14 days at 10 and 15°Cbut declined at 20 and 25°C, while in RG fruitethylene production increased to a peak then de-clined. The higher the temperature the soonermaximum ethylene production occurred (Fig. 2).

There was no difference in firmness of FU orRG apples after exposure to hypoxia at any tem-perature or sampling time (data not shown).

Fig. 2. Ethylene production of ‘Fuji’ () and ‘Royal Gala’(�) apples stored in air (—) or exposed to hypoxia (·······),100% CO2 for 24 h at (a) 25°C; (b) 20°C; (c) 15°C and (d)10°C and maintained at treatment temperature for up to 14days. Day −1 is the day before treatment, day 0 is onremoval from treatment. Average and standard errors of 4replicates.

Fig. 1. CO2 production of ‘Fuji’ () and ‘Royal Gala’ (�)apples stored in air (—) or exposed to hypoxia (·······), 100%CO2 for 24 h at (a) 25°C; (b) 20°C; (c) 15°C and (d) 10°C andmaintained at treatment temperature for up to 14 days. Day−1 is the day before treatment, day 0 is on removal fromtreatment. Average and standard errors of 4 replicates.

3.2. Hypoxic effects on ester concentration

Detailed changes in concentration of ethyl bu-tanoate and butyl acetate are presented as generalexamples of ethyl esters that increased after expo-sure to hypoxia, and acetate esters that decreasedafter exposure to hypoxia (Fig. 3). In FU fruit,ethyl butanoate concentration was enhanced afterexposure to hypoxia, reaching a maximum 2–4days after removal from hypoxia and declining toconcentrations similar to controls after 10–14days. The magnitude of the enhancement of ethylbutanoate after exposure to hypoxia in FU fruitwas 39.5, 16, 18.3 and 8 fold at 10, 15, 20 and25°C, respectively (Fig. 3(a)). This contrasted with


control fruit where ethyl butanoate concentrationsincreased only slightly as temperature increased

(Fig. 3(a)). Only in RG fruit exposed to hypoxiaat 20°C were ethyl butanoate concentrations en-

Fig. 3. Ethyl butanoate and butyl acetate concentration in apple juice from (a), (c) FU and (b), (d) RG apples treated with air (—)or exposed to hypoxia (·······), 100% CO2 for 24 h and maintained at treatment temperature at 10°C (), 15°C (�), 20°C (�) and25°C (") for up to 14 days. Day −1 is the day before treatment, day 0 is on removal from treatment. Average and standard errorsof 4 replicates.


hanced from 0 to 4 days (Fig. 3(b)), there beingno increase at 10, 15 and 25°C. At 10°C controlFU fruit had about the same concentrations ofethyl butanoate as RG fruit, but two to threetimes more than control RG fruit at 15, 20 and25°C. Patterns of change in concentration of ethylbutanoate in RG were different at 15 and 20°Cthan at 10 and 25°C. It is possible that the formerfruits were riper than fruits at 10 and 25°C as theywere held 4 weeks longer at 0°C before treatment.

The highest concentration of butyl acetate inFU and RG fruit occurred at 10°C and the lowestat 25°C (Fig. 3(c), (d)). As fruit ripened at eachtemperature, butyl acetate concentration declined.Exposure to hypoxia reduced butyl acetate con-centration in FU fruit by about 50% in fruittreated at 10 and 15°C (Fig. 3(c)). The decrease inbutyl acetate concentration was less as tempera-ture increased, and the return to control concen-trations occurred earlier, being 4, 6 and 12 days at25, 20 and 15°C respectively with no recovery at10°C (Fig. 3(c)). After exposure of RG fruit tohypoxia at 10°C butyl acetate concentration wasunchanged compared to control fruit (Fig. 3(d)).At 15°C butyl acetate concentrations were initiallyless than for controls but were similar from 4days. At 20°C butyl acetate was initially less thancontrol fruit, increasing briefly to control levelsbefore decreasing from 6 through 14 days. At25°C butyl acetate decreased initially through 2days then increased slightly from 4 days.

3.3. Total 6olatile concentration during 14 daysripening

Total volatile production (Fig. 4) was calcu-lated as area under the curve of plots of volatileconcentration with time, for volatiles regarded ashaving important sensory characteristics. In gen-eral, pattern of total volatile concentration withtemperature was the same in hypoxic-treated fruitas in control fruit. These patterns of enhancementor decrease in total volatile concentration com-pared to control fruit in FU or RG apples ex-posed to hypoxia were: no change in butan-1-ol,hexan-1-ol, hexanal and pentyl acetate for FU, orin ethyl propionate, ethyl butanoate, hexanal andpentyl acetate for RG; no change in concentration

at 10 and 25°C and a decrease in concentration at15 and 20°C of trans-2-hexenal for FU andhexan-1-ol and hexyl acetate for RG.

For FU fruit exposed to hypoxia compared tocontrol fruit, total volatile concentration was en-hanced with increasing temperature and the pat-tern was the same as for control fruit, althoughthe magnitude of enhancement decreased as tem-perature increased for ethyl propionate and ethyl2-methyl butanoate (Fig. 4); the magnitude ofenhancement of ethyl butanoate decreased withincreasing temperature (Fig. 4); hexyl acetate wasless than control at 10, 15 and 20°C and was notdifferent at 25°C, the magnitude of decrease be-coming less as temperature increased; acetalde-hyde was enhanced at all temperatures but themagnitude of enhancement declined from 10 to15°C, was the same for 15 and 20°C and increasedat 25°C.

When RG fruit exposed to hypoxia were com-pared to control fruit there was no difference inbutan-1-ol concentration at 10, 15 and 20°C andan increase at 25°C and there was a decrease intrans-2-hexenal concentration at all temperatures.Ethyl 2-methyl butanoate was not detected in thejuice of control or hypoxic-treated RG apples.

3.4. Volatile changes after exposure to hypoxia

Concentrations of all volatiles were analysed 2days after removal from hypoxia when enhance-ment of ethyl esters was the greatest, and after 10days when volatile concentrations had returned tocontrol concentrations (Tables 1 and 2). Generallyvolatile concentration decreased as temperaturesincreased, although the effect of temperaturevaried for specific volatiles.

Two days after removal from hypoxia, FU andRG fruit produced enhanced concentrations ofacetaldehyde, ethanol and ethyl acetate at all tem-peratures. FU apples had consistent enhance-ments of ethyl propionate, ethyl butanoate, ethylhexanoate and ethyl 2-methyl butanoate at 10through 25°C. Production of ethyl hexanoate andethyl 2-methyl butanoate only occurred in fruitexposed to hypoxia. Concentration of specificethyl esters varied with temperature. For example,ethyl propionate concentrations at 15–25°C were


Fig. 4. Total volatile concentration over 14 days, as area under the curve, of selected volatiles from ‘Fuji’ () and ‘Royal Gala’ (�)apples stored in air (—) or exposed to hypoxia (········), 100% CO2 for 24 h at 25°C, 20°C, 15°C and 10°C and maintained attreatment temperature for up to 14 days. Day −1 is the day before treatment, day 0 is on removal from treatment. Average andstandard errors of 4 replicates.

similar but almost double those at 10°C (Table 1);ethyl butanoate concentrations were similar at 10,15 and 20°C but were much lower at 25°C. Inaddition, most acetate esters decreased following

hypoxic treatment at 10, 15 and 20°C, but per-sisted only for pentyl and hexyl acetates at 25°C.In contrast, RG fruit had no enhancement ofethyl ester or decrease in acetate ester concentra-


Table 1Concentration of volatiles in juice from ‘Fuji’ and ‘Royal Gala’ apples stored in air at different temperatures 2 days after removal from 100%CO2. Mean of 4 replicatesa

Temperature (°C)

2010 2515

C T C T CT TC

FujiAlcohols (mmol l−1)

80 2633 80 47937954 1126Ethanol 211100484 618Propan-1-ol 785194 790 1186 7722895215 4120 4346 49363169 3728Butan-1-ol 24572999

NDND 55 62 49 83 73 42Pentan-1-ol1006 1420 833 1348 698Hexan-1-ol 461621 911ND ND ND ND 85ND 1293ND2MBb

Acetate esters (mmol l−1)15 148 10 64 11 53 15 161Ethyl acetate33 31 87 98 81 61 59 33Propyl acetate

531 309 306 191274 211569 100Butyl acetateND21 29 28 ND 34 ND NDPentyl acetate

190 131 124 69 85Hexyl acetate 30187 99387 90 138 59 236239 735192MBAb

Ethyl esters (mmol l−1)ND 199 50 388 43 384 ND 364Ethyl propionate

107728 89 1427 61 1123 58 486Ethyl butanoate65 56 67 57ND NDEthyl pentanoate ND37

543ND ND 420 ND 207 ND 78Ethyl hexanoateE2MBb NDND 280 ND 316 ND 203145

Aldehydes (mmol l−1)7 69Acetaldehyde 70 111 94 649295231 230 140Hexanal 209136 194 140117823 769 783 696 785 524528trans-2-Hexenal 794

Royal GalaAlcohols (mmol l−1)

0 3719 0 32113005 65Ethanol 1154813188Propan-1-ol 10855 168 329 331 3181014300 4477 7159 106714152 61693931 6182Butan-1-ol

ND40 42 42 72 111 78 70Pentan-1-ol714 630Hexan-1-ol 1188548 1393 857 690578ND 165 154 ND 594170 4222MBb 129

Acetate esters (mmol l−1)8 85 9 81 9 61 10 96Ethyl acetate

16 19Propyl acetate ND35 ND 54 2945556 556 623 736969 510926 277Butyl acetate

ND17 13 15 ND ND ND NDPentyl acetate306 335 226 202 279 225 174 74Hexyl acetate

25 24 51 39 5437 ND252MBAb

Ethyl esters (mmol l−1)32 35 ND ND ND ND ND NDEthyl propionate

21 21Ethyl butanoate 2863 45 72 8181ND ND ND ND32 3826 36Ethyl pentanoate

NDND ND 9 ND ND ND NDEthyl hexanoateE2MBb ND ND ND ND ND ND NDND

Aldehydes (mmol l−1)0 76Acetaldehyde 00 63 0 6730

117 131 142 140 162 178 173 102Hexanal501 501 529 552 669 609 713 333trans-2-hexenal

a Treatment: C = stored in air; T = exposed to hypoxia. Numbers in bold for a given compound within a row for a given temperatureare significantly different at P = 0.05. ND = not detected, concentration below 0.1 ml l−1.

b 2MB = 2 and 3 methyl butan-1-ol; 2MBA = 2 methyl butyl acetate; E2MB = ethyl-2-methyl butanoate.


Table 2Concentration of volatiles in juice from FU and RG apples stored in air and different temperatures at 10 days after removal from 100% CO2.Mean of 4 replicatesa

Temperature (°C)

1510 20 25

T C T CC T C T

FujiAlcohols (mmol l−1)

4231 322 704Ethanol 7045874 1487 5985 4879598 1330 1263 3344ND 2926Propan-1-ol 10795 10686

3632Butan-1-ol 3488 4568 3516 3753 4339 6517 788751 51 48 79 87Pentan-1-ol 134ND 165905 790 618 451751 417Hexan-1-ol 599 780

2MBb 1603ND 3513 2503 ND ND ND 180Acetate esters (mmol l−1)

25 13 14Ethyl acetate 1424 14 44 2945 89 82 16452 127Propyl acetate 286 268

445Butyl acetate 280 255 151 178 151 262 275ND 30 28 36 37Pentyl acetate NDND ND107 90 44 51186 39Hexyl acetate 66 68

2MBAb 247418 110 48 51 47 724 619

Ethyl esters (mmol l−1)129 59 110 130Ethyl propionate 165ND 318 384503 141 199 13738 188Ethyl butanoate 240 309

NDEthyl pentanoate ND 63 46 78 41 60 3539 14 14 23 18Ethyl hexanoate NDND 22129 45 107 110ND 153E2MBb 380 381

Aldehydes (mmol l−1)47 28 57 719 71Acetaldehyde 296 162

Hexanal 147125 200 158 210 222 424 646595 448 421 667 663 1276 1839483trans-2-hexenal

Royal GalaAlcohols (mmol l−1)

518 0 440 1765 222 415Ethanol 10350126 580 678 1110119 471Propan-1-ol 1020 1080

3887Butan-1-ol 4081 12359 13182 9424 7958 3422 6089ND 85 88 66 89 ND 60Pentan-1-ol ND467 1431 1172 837471 708Hexan-1-ol 162 277

2MBb 184217 270 239 311 196 216 221

Acetate esters (mmol l−1)19 5Ethyl acetate 155 12 7 6 822 86 89 10125 68Propyl acetate 49 44

894Butyl acetate 869 1308 1222 684 454 194 282ND 13 ND ND 27 ND NDPentyl acetate ND253 355 269 139302 95Hexyl acetate 31 39

2MBAb 2839 94 110 102 59 73 65

Ethyl esters (mmol l−1)ND 31Ethyl propionate NDND ND 45 ND ND26 65 54 4427 49Ethyl butanoate 26 26

NDEthyl pentanoate ND 63 53 51 38 ND NDND ND ND NDEthyl hexanoate NDND ND NDND ND ND NDND NDE2MBb ND ND

Aldehydes (mmol l−1)44 0 24 140 5Acetaldehyde 0 1585 239Hexanal 25894 194 218 76 97280 894 903 753346 727trans-2-hexenal 357 392

a Treatment: C = stored in air; T = exposed to hypoxia. Numbers in bold within a row for a particular compound for a given temperatureare significantly different at P = 0.05. ND = not detected, concentration below 0.1 ml l−1.

b 2MB = 2 and 3 methyl butan-1-ol; 2MBA = 2 methyl butyl acetate; E2MB = ethyl-2-methyl butanoate.


tion after exposure to hypoxia at 10, 15 and 20,but at 25°C butyl acetate, hexyl acetate, 2-methylbutyl acetate, hexanal and trans-2-hexenal haddecreased (Table 1).

Ethyl esters were enhanced in FU fruit exposedto hypoxia after 10 days at 10°C in contrast tofruit at 20°C and 25°C, where there was no differ-ence between control and hypoxic-treated fruit. 10days after hypoxic treatment, FU fruit at 10°Chad increased concentrations of ethyl propionate,ethyl butanoate, ethyl hexanoate, ethyl 2-methylbutanoate and some alcohols (propan-1-ol, pen-tan-1-ol, 2 methyl butan-1-ol) and acetaldehyde.FU fruit at 15°C had increased concentrations ofethanol, ethyl propionate, ethyl 2-methyl bu-tanoate and acetaldehyde with decreased concen-trations of butyl acetate and hexyl acetate 10 daysafter exposure to hypoxia. RG fruit at 10 and15°C had increased concentrations of ethanol andacetaldehyde 10 days after exposure to hypoxia.No volatile enhancement occurred in either culti-var at 20 and 25°C 10 days after treatment.

The acetate esters, butyl acetate, hexyl acetateand 2 methyl butyl acetate tended to decreasewith increasing temperature in control fruit. Noconsistent changes occurred in alcohol concentra-tions with temperature. Detectable concentrationsof some volatiles were found only at specifictemperatures. For example, 2 methyl butan-1-olat 25°C was detected in FU fruit 2 days aftertreatment, ethyl propionate at 10°C, ethyl pen-tanoate at 10°C and 25°C and pentyl acetate at10°C and 15°C in RG fruit 2 days after treatment(Table 1).

4. Discussion

In general, the magnitude of enhancement ofethyl ester concentrations in hypoxic-treated fruitdecreased as temperature increased. Volatile con-centrations of control fruit increased with temper-ature in agreement with previous research, wherevolatiles increased with temperature to a maxi-mum at about 20–25°C (Guadagni et al., 1971;Wills and McGlasson, 1971). In ‘Red Delicious’apples maintained at 10, 19 or 28°C while exposedto an atmosphere of 130 mM ethanol per kg of

apple, ethyl ester accumulation decreased at 28°Cbut was enhanced at 10°C compared to controlfruit (Berger et al., 1992). Since the respirationrate of apples increases as temperature increases(Fidler and North, 1971) it would be expectedthat volatile production rates from fruit wouldalso increase with temperature thereby increasingconcentrations in extracted juice. Such an as-sumption may not be true for all volatiles, asprecursors that arise from b-oxidation ortransamination may have different temperatureoptima. Differential changes in specific volatileconcentrations at each temperature could alsoreflect differences in degree of partitioning ofvolatiles into liquid or lipophilic portions of thecell, or to different rates of volatilisation fromtissue (Jennings, 1965; Carelli et al., 1991) ratherthan changes in rates of volatile biosynthesis(Wills and McGlasson, 1969). This may explainthe different patterns of total volatile concentra-tion change over 14 days for each temperature.Concentration increased with temperature for cer-tain compounds (acetaldehyde, ethyl propionateand ethyl-2-methyl butanoate) suggesting thattheir rate of biosynthesis increased faster than lossfrom the tissue. As direct emission of volatiles wasnot measured from either intact fruit or juice, it isnot possible to determine what proportion ofvolatiles was lost through volatilisation. Volatileconcentration might decrease with temperaturefor other compounds (such as ethyl butanoateand hexyl acetate) where volatile biosynthesis isinsufficient to compensate for increased rates ofloss. Concentrations of hexanal and trans-2-hexe-nal, products of tissue disruption, did not changeas temperature increased. A peak in butan-1-ol,hexan-1-ol and pentyl acetate concentration at 15or 20°C suggests that these temperatures may beoptimum for their biosynthesis.

A close relationship exists between volatile andethylene emissions in pre-climacteric fruit (Songand Bangerth, 1996) and between volatiles andtissue sensitivity to ethylene in post-climactericapples (Bangerth et al., 1998). In the present studyethylene production increased to a peak at 20°Cfor FU and RG apples before declining at 25°C.This production pattern did not occur for any ofthe volatiles analysed; rather our results suggest


that changes in volatile concentration may berelated to an indirect co-ordinating effect of ethyl-ene on metabolism rather than by a direct effect ofethylene on the biosynthetic pathway of volatiles.

In general, exposing FU and RG apples tohypoxia at each temperature enhanced ethyl esters,fermentation volatiles and decreased acetate estersin agreement with previous results (Ampun, 1997;Dixon, 1999). Specific changes in volatile concen-trations of hypoxic-treated fruit depended on tem-perature and cultivar, but generally following thesame patterns of change with temperature as didcontrol fruit. The greatest increase in volatile con-centration in juice from fruit maintained at 10°Cduring and after exposure to hypoxia and thisincrease lasted for up to 10 days. As the tempera-ture optimum for the ester-forming enzyme, alco-hol acylCoA transferase, is about 30°C (Sanz et al.,1997), then rates of volatile synthesis will be tem-perature-dependent. However, our results suggestthat other factors such as decreased partitioning,increased volatile evaporation at warmer tempera-tures, and/or limited synthesis of acylCoAs andalcohols from fatty acids at low temperatures (Sanzet al., 1997), may limit volatile accumulation inapple tissue. In banana fruit, different biosyntheticpathways for volatiles appear to have varyingthermal sensitivities resulting in different mixturesof volatile compounds being produced at differenttemperatures (Mattei, 1975).

One example of an enzyme system that may havedifferential temperature effects on volatile concen-tration is the fermentation pathway where ethanolis produced by reduction of acetaldehyde by alco-hol dehydrogenase (ADH). ADH of ‘Cox’s OrangePippin’ apples has different substrate specificitiesfor aldehydes (Bartley and Hindley, 1980) andcould potentially have different temperature op-tima for each aldehyde. Ethanol accumulation atdifferent temperatures may depend on specificanaerobic respiration rates that differ among fruitand cultivars (Ke et al., 1991). The fact that ethanolconcentrations in juice from control and from FUapples were very much greater than in juice fromRG apples 10 days after exposure to hypoxia,would suggest that ADH in FU apples is alwayshigher than in RG apples after hypoxia has beenremoved.

Further evidence for differential temperatureeffects on volatile concentration is the presence orabsence of specific volatiles at some temperaturesbut not others. In FU apples maintained at 25°C,2 methyl butan-1-ol was only present 2 days afterexposure to hypoxia. Ethyl propionate was foundonly in RG apples 2 days after exposure to hypoxiaat 10°C. 2 days after exposure to hypoxia pentylacetate was absent in FU apples at 25°C and in RGapples at 20 and 25°C. Such differences in volatilecomposition at different temperatures suggest thatrates of synthesis of some volatiles are very temper-ature-dependent. To date, temperature optima andsubstrate specificity of the enzymes involved involatile formation have not been determined inapple tissue.

It has been shown that as postharvest tempera-ture of apples increased, the enhancement ofvolatile concentrations in juice from fruit exposedto hypoxia was reduced. It is likely that this was dueto altered rates of volatile production, increasedpartitioning in the cell and/or increased rates ofvolatilisation. A temperature of 10°C resulted inmaximum accumulation of volatiles in juice of FUand RG apples exposed to hypoxia, with lesserconcentrations occurring in apples maintained at15°C, 20°C or 25°C. The minimum decrease inacetate esters occurred at 10°C and increases inethyl esters were only slightly less than at warmertemperatures. These results suggest that applestreated with hypoxia and maintained above 15°Cfor more than a few days would have reducedvolatile concentrations and more rapid deteriora-tion of quality than fruit exposed to hypoxia at atemperature of 10°C or less. This could haveimplications on overall quality of apples exposed todisinfestation treatments utilising high tempera-tures for sustained periods.

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