factors affecting apple aroma flavour volatile concentration

New Zealand Journal of Crop and Horticultural Science, 2000, Vol. 28: 155-1730014-0671/00/2803-0155 $7.00 © The Royal Society of New Zealand 2000

155

Review

Factors affecting apple aroma/flavour volatile concentration:a review

JONATHAN DIXON

ERROL W. HEWETTInstitute of Natural ResourcesCollege of SciencesMassey UniversityPrivate Bag 11 222Palmerston North, New Zealandemail: [email protected]

Abstract Typical apple {Malus domestica Borkh.)flavour develops during ripening. Maximum endog-enous volatile concentration occurs at the climactericpeak but it is not known whether the volatilebiosynthetic enzymes are constitutive or induced dur-ing the climacteric. Exposing apples to hypoxic con-ditions induces accumulation of high concentrationsof acetaldehyde and ethanol; after return to air ethylesters are enhanced and non-ethyl esters decrease.There are differences in degree of ethyl ester enhance-ment among cultivars. These may be because of: dif-ferential activity or synthesis of alcohol acyl CoAtransferase (AAT) or alcohol dehydrogenase (ADH);separate iso-forms of AAT and ADH each with theirown substrate specificity; variation in alcohol precur-sors in different cultivars; or a combination of all three.Volatile production is greater at higher temperaturesin the range from 0 to 30°C but exposure to low tem-peratures (<3°C) for more than 3 months decreasesproduction. Temperature influences the production ofspecific volatiles with some compounds only beingproduced at certain temperatures. It is not known howtemperature will affect volatile production after expo-sure to hypoxia. It is suggested that the enhanced vola-tile production that occurs in apples following anhypoxic treatment might overcome or reverse the de-creases that are induced by low temperatures and con-trolled atmosphere (CA) storage. The use of hypoxiato enhance volatile concentrations may be a beneficial

H00004Received 11 February 2000; accepted 6 August 2000

side effect when such treatments are used for disinfes-tation purposes. It is possible that given equal efficacy,hypoxia could be either preferred or used as an adjunctto heat treatments to eradicate insects. In additionhypoxic treatment of fresh fruit could induce signifi-cant increases in volatile concentrations that could beused in production of high quality essences from ap-ple juice.

Keywords Malus domestica Borkh,; hypoxia;temperature; maturity; volatile biosynthesis; ethylesters; flavour enhancement; disinfestation; storage;fruit quality; apple juice

INTRODUCTION

Fresh apple (Malus domestica Borkh.) exports fromNew Zealand comprise c. 7% of world trade in thisfruit (Steele 1995). Although New Zealand applesare considered to be of premium quality in overseasmarkets, consumers are increasingly demanding thatstored apples more closely match the appearance,taste, and texture of freshly harvested fruit. Thisrepresents a particular challenge for New Zealandapple exporters because of long distances to principalmarkets in Europe and North America. Controlledatmosphere (CA) storage has attracted considerableuse worldwide for apple storage for bettermaintenance of fruit quality. One drawback of bothCA and long-term air storage is the loss of acceptableapple flavour and aroma (Bangerth & Streif 1987).

Until the late 1970s most research on aroma andflavours of apple fruit concentrated on identifyingvolatiles produced by ripening fruit (Tressl et al.1975). Recent reviews have discussed the biochemi-cal origin of aroma volatiles and improvements inmethods for separation and identification of volatilecompounds, often in trace amounts of a few parts permillion (Dimick & Hoskin 1983; Yahia 1994; Sanzet al. 1997). These new methods have allowed re-searchers to examine in more detail biosyntheticpathways and control mechanisms in the synthesisand subsequent accumulation and release of volatiles

156 New Zealand Journal of Crop and Horticultural Science, 2000, Vol. 28

from apples. This is resulting in a better understand-ing of how biochemical and environmental factorsinfluence aroma and flavour of apple fruit (Yahia1994). Increased interest in non-chemical pre-treat-ments to preserve or improve apple fruit quality, andas disinfestation treatments, has highlighted deficien-cies in our knowledge of factors that affectpostharvest apple flavour development. The follow-ing review summarises knowledge of the composi-tion of apple volatiles, how they are synthesised andhow postharvest factors and exposure to hypoxiatogether affect volatile concentration.

Apple volatilesFruit aroma is a complex mixture of a large numberof volatile compounds that contribute to the overallsensory quality of fruit specific to species andcultivar (Sanz et al. 1997). Over 300 volatile com-pounds have been measured in the aroma profile ofapples. These compounds include alcohols, alde-hydes, carboxylic esters, ketones, and ethers (Dimick& Hoskin 1983). About 20 of these chemicals are"character impact" compounds (Table 1\ Such com-pounds have a range of aroma thresholds (Table 2).Some are present in very low concentrations andcontribute potent aroma characteristics typical ofapple aroma/flavour (e.g., ethyl-2-methyl butanoate(Flath et al. 1967)). Others contribute to aroma in-tensity (e.g., /ra«5'-2-hexenal) or are related to aromaquality (e.g., ethanol) (Durr & Schobinger 1981).

Volatile compounds identified in apple aroma

Extensive lists of volatiles extracted from apples andapple essences have been compiled (Dimick &Hoskin 1983; Paillard 1990). Although there is agreat range of compounds in the volatile profile ofapples, the majority are esters (78-92%) andalcohols (6-16%) (Paillard 1990). The most abun-dant compounds are even numbered carbon chainsincluding combinations of acetic, butanoic, andhexanoic acids with ethyl, butyl, and hexyl alcohols(Paillard 1990). Higher molecular weight volatiles,often with one or two hydrophobic aliphatic chains,are likely to be trapped by skin waxes and are gen-erally not found in the headspace (Paillard 1990).

Apple aroma in different cultivarsMost aroma compounds, in variable proportions, arepresent in volatile emissions from most applecultivars but there appear to be no key characteris-tic compound for any given cultivar (Cunninghamet al. 1986; Paillard 1990). Notwithstanding this,large sensory differences in flavour and aroma exist

among cultivars (Poll 1981; Cunningham et al.1986). A taste panel assessment of pasteurised ap-ple juice from 18 apple cultivars for aroma and tasteafter 6 months of storage at 10°C showed that panel-lists preferred cultivars with strong aroma and "char-acteristic" apple taste rather than juices with weakaroma and "uncharacteristic" apple taste (Poll 1981).

Apple volatile production has been categorisedaccording to: type and quantity of esters or alcohols(Dirinck & Schamp 1989; Paillard 1990), aroma pro-duction pattern (Dirinck & Schamp 1989), skin col-our (Paillard 1979), or C6 aldehydes (Paillard 1990).Ester type cultivars are categorised according to typesof esters: acetate ester types ('Calville Blanc', 'GoldenDelicious'), butanoate ester types ('Belle de Boskoop','Canada Blanc', 'Richared'), propanoate ester types('Reinette du Mans', 'Richared', 'Starking'), andethanolic ester types ('Starking') (Paillard 1990).Yellow-skinned cultivars have been reported to pro-duce mainly acetic acid esters and red-skinnedcultivars mostly butyric acid esters (Paillard 1979).High concentrations of hexyl acetate and butyl acetatewere considered to characterise 'Cox's Orange Pip-pin', 'Elstar', 'Golden Delicious', 'Jonagold' and'Jublie Delbar', with 'Granny Smith', 'Nico','Paulared', and 'Summerred' being characterised byhigh concentrations of ethyl butanoate and hexan-1-ol and 'Boskoop' and 'Jacques Lebel' characterisedby a-famesene and hexyl 2-methyl butanoate (Dirinck& Schamp 1989). Concentration of C6 aldehydes for'Cox's Orange Pippin' and 'Jonathan' apples was 4—5 times that of' Golden Delicious' for hexanal and 100-fold more for /ra/w-2-hexenal (Paillard 1990). Applecultivars also differ in concentrations of other volatilessuch as 4-methoxyllylbenzene (a spice-like aromacompound, according to an English sensory panel)which can constitute up to 0.27% of headspacevolatiles in some cultivars (Williams et al. 1977).

Biogenesis of volatilesAs volatiles are comprised of at least five chemicalclasses there are several pathways involved in volatilesynthesis. These have not been fully described butappear to be common for different fruits. Volatilesimportant for aroma and flavour are synthesised fromamino acids, membrane lipids and carbohydrates (Sanzet al. 1997). In apple aroma, the majority of volatilesare esters, the formation of which is dependent onavailability of C2-C8 acids and alcohol (Paillard 1979;De Pooter et al. 1981; Knee & Hatfield 1981). Sitesof volatile biosynthesis within cells are not known,although lipoxygenase occurs at a membrane site intomatoes (Riley et al. 1996).

Dixon & Hewett—Apple aroma/flavour volatile concentration

Table 1 Important apple (Mains domestica) volatile compounds and their sensory descriptions.

157

Compound

Aldehydesacetaldehyde/ra/w-2-hexenal

hexanal

Alcoholsbutan-1-ol

hexan-1-ol/ram--2-hexenol

Estersbutyl acetate

pentyl acetate

hexyl acetate

2 methyl butyl acetate

ethyl butanoate

ethyl-2-methyl butanoate

4-methoxyallyl benzenemethyl-2-methyl butanoatepropyl-2-methyl butanoatebutyl-2-methyl butanoatehexyl-2-methyl butanoatebutyl hexanoatehexyl propanoatebutyl butanoatebutyl propanoatehexyl butanoatehexyl hexanoate

Sensory description

green/sharpgreen/sharpoverall intensitygreen appleharmonious, fruitygreen/sharp, earthyoverall intensitygood, green applegrass like

overall flavour, aroma,sweet aroma

earthy, unpleasantharmonious, fruity

red apple aromaCox-like aromaharmoniousnail polishbanana likeapple, fruityGalared apple aromacharacteristic appleCox-like aromaripe Golden Delicioussweet fruity, appleGala, ripe, pearoverall aroma,characteristic apple

solventbanana likefruity, esteryharmonious, fruityfruityapple likesweet strawberryspicy, aniseedsweet fruityvery sweet, strawberryfruity, appleapple, grapefruitgreen appleapplerotten apple, cheesyfruity, appleappleapple

Cultivar

Golden DeliciousGolden DeliciousMclntoshDeliciousmanyGolden DeliciousMclntoshDeliciousmany

Royal Gala, GoldenDelicious

Golden Deliciousmany

Royal GalaCox's Orange PippinmanyGalaCox's Orange PippinGolden DeliciousGalaRoyal Gala

Cox's Orange PippinGolden Delicious

Royal Gala

GalaCox's Orange PippinGolden DeliciousmanyGolden DeliciousDeliciousGalamanyGalaGalaGalaGalaGalaGalaGalaGalaGalaGala

Reference

Rizzoloetal. (1989)Rizzoloet al. (1989)Panasiuketal. (1980)Flathetal. (1969)Duerr(1979)Rizzoloetal. (1989)Panasiuketal. (1980)Flathetal. (1969)Duerr(1979)

Young etal. (1996);Rizzoloetal. (1989)Rizzoloetal. (1989)Duerr(1979)

Young etal. (1996)Williams & Knee (1977)Duerr(1979)Plotto(1998)Williams & Knee (1977)Rizzoloetal. (1989)Plotto(1998)Young etal. (1996)

Williams & Knee (1977)Rizzoloetal. (1989)

Plotto(1998)Young etal. (1996)

Plotto(1998)Williams & Knee (1977)Rizzoloetal. (1989)Deurr(1979)Rizzoloetal. (1989)Flathetal. (1967)Plotto(1998)Williams etal. (1977)Plotto(1998)Plotto(1998)Plotto(1998)Plotto(1998)Plotto(1998)Plotto(1998)Plotto(1998)Plotto(1998)Plotto(1998)Plotto(1998)


Fatty acidsFatty acids are major precursors of aroma volatilesin most fruit (Sanz et al. 1997). The biosyntheticpathways involved include P-oxidation, hydroxyacidcleavage (leading to lactones), and lipoxygenase toform aldehydes, ketones, acids, alcohols, lactones,and esters from lipids (Heath & Reineccius 1986).Aroma volatiles in intact fruit are formed via the (3-oxidation biosynthetic pathway, whereas when fruittissue is disrupted, volatiles are formed via thelipoxygenase pathway (Schreier 1984).

Flavour and aroma characteristics of apples developduring ripening after harvest (Tressl & Drawert 1973;Tressl et al. 1975). As apples ripen, rates of lipid syn-thesis and membrane fluidity increase (Bartley 1985)as does lipoxygenase activity in disrupted tissue ofriper fruit (Wooltorton et al. 1965). Pulp of 'Cox'sOrange Pippin' apples contains mostly phospho-,galacto, and steryl lipids that are composed mostly oflinoleic and linolenic acids (c. 50% and 10-25% oftotal lipid respectively) (Galliard 1968). The propor-tion of linolenic acid in lipids of post-climacteric applesis lower than in pre-climacteric apples. Lower linolenicacid concentrations are associated with plastid struc-tures, and result from decreased concentrations of

monogalactosyl diglyceride, digalactosyl diglyceride,and phosphatidal glycerol and not a change to the fattyacid distribution of individual lipids (Galliard 1968).Decreases in chlorophyll concentration were observedto occur with decreases in lipids, which agrees withthe observation that during apple ripening chloroplastsbreak down (Galliard 1968). Chloroplast breakdowncould therefore provide the major source of linoleic andlinolenic fatty acids for volatile biosynthesis in fruit.

/3-oxidation

Studies using radio-labelled substrates and precur-sors with pears (Jennings 1967), bananas (Tressl &Drawert 1973), and apples (Paillard 1979; Bartleyet al. 1985), have established that P-oxidation of fattyacids is the primary biosynthetic process providingalcohols and acyl co-enzyme A (CoA) for ester for-mation (Sanz et al. 1997). Substrate feedingexperiments with 'Golden Delicious' apples usingC1-C6 aldehydes, or C2-C6 carboxylic acid vapours(De Pooter et al. 1983; De Pooter et al. 1987), in-duced increases in esters typical of those expectedfor p-oxidation of the added compounds (De Pooteretal. 1981, 1982). Similar experiments with 'Cox'sOrange Pippin' apples, using methyl esters of short

Table 2 Aroma threshold values of important volatile compounds in apples (Malus domestica).

Compound Threshold (ml litre"1) Reference

Aldehydesacetaldehdyehexanalmm?-2-hexenal

Alcoholsethanolpropan-1 -olbutan-1-olhexan-1-ol2 methyl butan-1 -ol

Estersethyl acetatepropyl acetatebutyl acetatepentyl acetatehexyl acetateethyl butanoateethyl-2-methyl butanoateethyl propionateethyl pentanoateethyl hexanoatepropyl butanoate2 methyl butyl acetate

0.015-0.12 Flath etal. (1967)0.005 Paillard (1990)

0.001-0.017 Flath etal. (1967); Hatanaka (1993)

100-900 Teranishi et al. (1987)40-9 Flath etal. (1967)

0.5 Flath etal. (1967)0.15-0.5 Flath etal. (1967)

0.25 Buttery etal. (1973)

13.5-0.005 Takeoka et al. (1996); Teranishi et al. (1987)2.0 Takeoka etal. (1996)

0.066 Takeoka et al. (1996); Teranishi et al. (1987)0.043-0.005 Takeoka et al. (1996); Teranishi et al. (1987)0.115-0.002 Takeoka et al. (1996); Teranishi et al. (1987)

0.001 Takeoka et al. (1995); Teranishi et al. (1987)0.0001-0.000006 Flath et al. (1967); Takeoka et al. (1995); Teranishi et al. (1987)

0.01 Takeoka et al. (1995); Teranishi et al. (1987)0.0015-0.005 Takeoka et al. (1995); Teranishi et al. (1987)

0.001 Takeoka etal. (1995); Teranishi et al. (1987)0.018 Teranishi etal. (1987)

0.011-0.005 Teranishi et al. (1987)

Dixon & Hewett—Apple aroma/flavour volatile concentration 159

chain C4-C8 fatty acids, resulted in esters with analkyl group of Cn-2, Cn-4 confirming the presenceof an active p-oxidation pathway in whole fruit(Bartley et al. 1985). Perdeuterated linoleic acid fedto 'Red Delicious' apples produced only C6metabolites, implying that saturated ester volatilesarise by P-oxidation, rather than peroxidation, offatty acid precursors (Rowan et al. 1997). Substratesfor ester biosynthesis may also be formed via oc-oxi-dation (Tressl & Drawert 1973; Rowan et al. 1997).Feeding dueterated cw-3-hexenal and trans-2-hexenal to 'Red Delicious' apples resulted in a rangeof labelled volatiles, including ethyl pentanoate andpentyl acetate that could only result from a-oxida-tion (Rowan et al. 1997).

Fatty acid acyl-CoA derivatives are converted toshorter chain acyl-CoAs by losing two carbons inevery round of the p-oxidation cycle, requiring flavinadenine dinucleotide (FAD), nicotinamide adeninedinucleotide (NAD), and free CoA. Acyl CoAs arereduced by acyl CoA reductase to aldehyde that inturn is reduced by alcohol dehydrogenase (ADH) toalcohol for use by alcohol acyl CoA transferase(AAT) to produce esters (Bartley et al. 1985).Bartley et al. (1985) proposed that varietaldifferences in volatile composition of apples dependon the specific activities of P-oxidation enzymes, thatwill influence for example, the rate of transformationof butanoate to acetate (Paillard 1979).

Lipoxygenase (LOX)

When fruit are homogenised, linoleic and linolenicacid are oxidised to various C6 and C9 aldehydes(Drawert 1975; Galliard & Matthew 1977; Lea 1995).

These volatiles reach maximum concentration in thefirst 10-30 min after homogenisation (Drawert et al.1986). Such C6 aldehydes are responsible for the"green" odour notes in plant aroma (Hatanaka 1993).In intact fruit, enzymes in the lipoxygenase (LOX)biosynthetic pathway and their substrates havedifferent subcellular locations, preventing formationof volatile compounds (Sanz et al. 1997). Duringripening, cell walls and membranes may become morepermeable, allowing the LOX pathway to becomeactive without tissue disruption (Sanz et al. 1997).Lipoxygenase activity of'Schone van Boskoop' appleswas greatest during the climacteric peak. 'GoldenDelicious' apples metabolised linolenic acid morereadily than linoleic acid (Kim & Grosch 1979).'Golden Delicious' apples treated with hexanal andhexanoic acid vapours had increased hexyl, butyl, andethyl esters (De Pooter et al. 1983). Therefore, the LOXbiosynthetic pathway has the potential to providesubstrates for ester production (De Pooter et al. 1983).If the LOX biosynthetic pathway were active duringripening, it would act as an alternative to P-oxidationof fatty acids.

Amino acids

Branched chain alcohols, carbonyls, and esters areproduced by metabolism of the amino acids valine,leucine, iso-leucine, alanine, and aspartic acid (Heath& Reineccius 1986; Sanz et al. 1997). Varyingconcentrations of free amino acids could account fordifferent concentrations of branched chain volatiles infruit; for example, during ripening of banana fruit, L-leucine and L-valine increased 3-fold while otheramino acids remained constant (Tressl & Drawert

Table 3 Relative activity of alcohol dehydrogenase to acetaldehyde for aldehydes and to ethanol for alcohols ofCox's Orange Pippin apples (Malus domestica) and Carignane grapes (Vitus vinifera) (adapted from Bartley &Hindley (1980); Molina et al. (1987)).

Aldehyde

acetaldehydepropanalbutanal2-methylpropanalpentanalhexanal/ra/w-2-hexenal

% activity of ADHApple

100.0*30.137.24.3

31.15.77.2

Grape

100.0

63.06.3

Alcohol

methanolethanolpropan-1-olpropan-2-olbutan-1-ol2-methylpropan-1 -olpentan-1-olhexan-1 -oltrans-2-hexen-1 -ol

% activityApple

0.0100.0+

44.78.5

45.00.0

19.118.3

121.3

of ADHGrape

100.031.0

64.0

117.0

*100% activity equivalent to 0.21 mmol NADH oxidised min ' £activity equivalent to 0.03 mmol NAD reduced min"1 g~'

~' tissue,tissue.


1973). Amino acids are converted to branched chainalcohols and esters in post-climacteric banana slicesand may involve enzymes from three biosyntheticpathways: aminotransferase, decarboxylase, and ADH(Sanz et al. 1997). Iso-leucine is considered to be thebiosynthetic precursor of 2-methyl butanoic acid andits esters in apples (Paillard 1990). Deuterated iso-leucine was metabolised by 'Red Delicious' apples to2-methyl butan-1-ol and to 2-methyl butyl and 2-methyl-2-butenyl esters, whereas 'Granny Smith'apples produced ethyl-2-methyl butanoate almostexclusively (Rowan et al. 1996). Different ratios ofamino acid conversion to volatiles, in particular thedifferential rates of metabolism of leucine and iso-leucine, occur in 'Braeburn', 'Granny Smith', 'Fuji','Red Delicious', and 'Royal Gala' apples (Rowan etal. 1997). This suggests that different enzyme activityand selectivity, rather than substrate availability of theamino acid degradation pathway, determines theconcentration of branched chain esters for eachcultivar. Presence of labelled butyl and hexyl acetateindicates that amino acids provide substrates for acetateesters via pVoxidation (Rowan et al. 1997). As little isknown about the concentration and availability ofdifferent amino acids during ripening and senescenceof apples further research is required to identify ifamino acid concentrations determine the type ofvolatile compounds produced by apples duringripening.

Aldehydes

Conversion of aldehydes to alcohols involves ADHthat catalyses oxidation of alcohols and reduction ofaldehydes with NAD and NADH as cofactors(Bartley & Hindley 1980). Alcohol dehydrogenasefrom 'Cox's Orange Pippin' apples has optimalactivity at pH 5.5-6.0 when reducing acetaldehydeand pH 7.0-10.0 when oxidising ethanol. It has a 13-fold higher affinity for acetaldehyde than ethanol inapples (Bartley & Hindley 1980) and is the preferredreaction in grapes (Molina et al. 1987). AlthoughADH in apples is active on a wide range of aldehydecompounds, substrate affinity for compounds longerthan C2 carbon chain aldehydes and alcohols isgreater for straight chain than for branched chaincompounds (Table 3).

Esters

Esters form the largest group of volatile compoundsproduced by fruit, but the ester biosynthetic pathwayis not fully understood. Few studies have investi-gated detailed biochemical aspects of ester forma-tion in contrast to P-oxidation or LOX breakdown

of fatty acids. Ester production in fruit tissue is theresult of esterification of alcohols, carboxylic acids,and acyl CoAs in an oxygen dependent reaction(Drawert & Berger 1983) and is considered to be themost active in epidermis (Berger et al. 1992). Thereare some similarities between substrate specificityof AAT enzymes from different fruits. Optimal tem-perature for maximum activity is c. 30°C, pH rangeis 7-8.5, sulfydryl groups are essential for activity.Activity is linked to lipid metabolism in micro-organisms (Sanz et al. 1997). However, ester form-ing enzymes in yeasts are localised in the cell mem-brane (Yoshioka & Hashimoto 1981) whereas inbanana AAT may be localised in the cytoplasm(Harada et al. 1985). The mixture of esters producedin different fruits depends on the activity andsubstrate specificity of AAT. Strawberry AAT hasgreater activity with straight-chain alcohols than withbranched chain alcohols of the same carbon number,with acetyl CoA and hexan- l-ol being the preferredsubstrates (Perez et al. 1993). Apples exposed toatmospheres containing low molecular weightalcohols ("Precursor Atmosphere Technology")have enhanced concentration of esters with the cor-responding alcohol moiety (Berger 1995).

Substrate specificity of AAT differs from fruit tofruit and esterification of straight-chain alcohols ispreferred over branched-chain alcohols (Olias et al.1995; Rowan et al. 1996). Such differences in pref-erence for acyl CoAs and alcohols may determineconcentration of different esters in fruit aroma pro-files. ' Jonagold' apples exposed to hexanal vapourssynthesised hexan-1 -ol and related volatiles such ashexyl acetate, butyl hexanoate, and hexyl hexanoate,whereas 'Golden Delicious' apples had a greatercapacity to convert hexanal to hexan-l-ol than'Jonagold' apples (Song et al. 1996). In yeast AATactivity is competitively inhibited in vitro by synthe-sis of unsaturated fatty acids (Mauricio et al. 1993).The relationship between lipid synthesis and estersynthesis is not known for apples. Apples kept in lowoxygen storage conditions known to have reducedfatty acid concentration, have a reduced pool of al-cohol precursors (Brackmann et al. 1993). Activityof AAT increases with advancing maturity and issuppressed by atmospheres containing 0.5 and 1%O2 (Fellman et af 1993b).

In addition to AAT, the enzyme esterase, whichconvert esters to alcohols and carboxylic acids, mayhave some synthetic capacity as well as its ability tohydrolyse esters (Bartley & Stevens 1981; Sanz etal. 1997). Therefore, ester synthesis in apple tissuemay be the sum of ester formation by AAT, reverse


reaction of ester hydrolysis and ester hydrolysis(Knee & Hatfield 1981). Apple juice has highconcentrations of alcohols and esters; for example,'Cox's Orange Pippin' apple juice contains largeamounts of hexan-1-ol, butan-1-ol, and pentan-1-oland hexyl acetate, butyl acetate, and pentyl acetate(Goodenough 1983). The high concentration ofalcohols may result from esterase activity whichincreases during the climacteric (Goodenough 1983).

Ester biosynthesis is considered to be limited byalcohol concentration (Gilliver & Nursten 1976;Berger et al. 1992). Alcohol concentrations canchange the composition of volatiles emanating from'Red Delicious' apple disks. High ethanolconcentrations promote formation of C8 compoundsor longer acyl moieties, whereas low ethanolconcentrations increase short chain acyl moiety(Berger & Drawert 1984). Maximum production ofesters was achieved using butan-1-ol and pentan-1-ol, the least using methanol and ethanol (Berger &Drawert 1984). Addition of butan-1-ol increasedbutyl acetate concentrations and butanoate esters ofall alcohol moieties indicating that alcohols werebeing converted to butyl CoA. Ethyl and hexyl estersynthesis was stimulated by ethanol and hexan-1-olat the expense of butyl esters, indicating that esterformation in apple fruit is a competitive reaction(Kollmannsberger & Berger 1992). 'Jonagold'apples converted hexanal to hexan-1-ol immediatelyafter application of hexanal vapours to intact fruitwhile increases in hexyl acetate took c. 5 h andincreases in esters with a hexyl acyl moiety took upto 24 h (Song et al. 1996). This suggests that hexanalis incorporated into fruit, first as an alcohol, then asacetate ester, and is further metabolised into acylCoA compounds. This agrees with the propositionthat substrate availability rather than enzyme activitylimits volatile production in apples (Knee & Hatfield1981; Songetal. 1996).

Effect of hypoxia on volatile concentrationHypoxic treatments before storageExposure of fruit to hypoxic atmospheres for severaldays has been investigated as an alternative non-chemical insect disinfestation treatment to thefumigant methyl bromide (Lay-Yee & Whiting1996; Whiting et al 1996). Methyl bromide is agreenhouse gas and its use is to be phased out by theyear 2001 in the United States and by 2010 in therest of the world (Anon. 1995, 1997). Warmfumigation temperatures generally allow decreasedmethyl bromide concentrations and/or decreasedfumigant exposure time to achieve insect kill, as the

target insect has increased metabolic activity and rateof fumigant uptake (Paull & Armstrong 1994). Adisinfestation treatment using hypoxic atmospheresat warm temperatures (>20°C) will kill insects fasterthan hypoxic atmospheres at low (<5°C)temperatures (Ke & Kader 1992).

The effect of exposure to hypoxia on apple qual-ity at warm temperatures has been investigated byAmpun (1997) who determined that exposure tohypoxia induces substantial qualitative and quanti-tative changes in concentration of volatiles thoughtto be important in apple aroma. The type of com-pounds enhanced included low odour thresholdvolatiles such as ethyl-2-methyl butanoate and ethylbutanoate which are of commercial significance tothe apple juice processing industry. The potentialexists to manipulate juice quality to meet particularmarket demands by adding specific volatiles to juiceconcentrates from cultivars that lack these com-pounds. For example, ethyl-2-methyl butanoatecould be added to juice from 'Golden Delicious'apples that contain relatively low concentrations ofthis compound. It may also be possible to isolatespecific compounds for use as fragrances for per-fumes and food additives (Ampun 1997).

Fruit held in air (c. 20% O2) are considered to bein normoxic conditions where aerobic respirationoccurs. Anoxic conditions are those where totalabsence of O2 prevents mitochondrial activity(Ricard et al. 1994) whereas, hypoxic conditions arethose where O2 partial pressure limits mitochondrialactivity and fruit are predominantly respiring anaero-bically (Pradet & Bomsel 1978). True anoxic con-ditions are difficult to achieve in practice thereforemost experimental conditions reported as being an-oxic are redefined as "deep hypoxia" (Roberts et al.1992). Many hypoxic treatments applied to apples(Table 4) would be considered as deep hypoxia.

Effect on general fruit quality

Apples maintained at low temperatures or in CAhave reduced softening as well as reduced CO2 andethylene production (Table 4) for longer than fruitstored in air (Kader 1986) because of inhibitoryeffects of high concentrations of CO2, acetaldehyde,ethanol or low O2 concentrations on enzyme systemswithin fruit. Fruit exposed to hypoxic atmospheresranging from 10 to 100% CO2, 50 to 100% N2, and0 to 17% O2 in temperatures from -1.1 to 32°C anddurations of a few hours to 42 days will, dependingon the fruit: (1) have enhanced maintenance ofquality during storage (Eaves et al. 1968; Pesis et al.1988; Pesis & Avissar 1989; Pesis et al. 1994); (2)

ON

Table 4 Effects of hypoxic and high carbon dioxide (CO2) treatments before storage on quality attributes of apples (Malus domestica). (Deer. = decrease; Incr.increase.)

Cultivar

BraeburnCox's Orange Pippir

Golden Delicious

Granny Smith

Mclntosh

Pacific Rose™Red Delicious

Royal GalaYellow NewtonWagener

Treatment

100%CO21 100%N2

15-30%CO2, 6%O2*100% CO2

10-30% CO2, 10% O2*14orl8%CO2 , 6%O2*15%CO2, 18%O2*20%CO2, 17%O2

99.75% N2

95% CO299.75-100% N2

99.5% N2, 0.5% O2*100%CO2

100% N2*12%CO2, 5%O2*100% N2*100% CO2

100% N2*100%CO2

100% CO2

99.75-100% N2

100% N2*

'Treatment before controlled atmosphere storage.+Compared to air-stored controls.+CO2 production.^Usually an alcoholic off-flavour.

Duration(days)

13-10

15110102044

1-23-353-9

1714

1.5-4.51711

3-357

Temp.(°Q

203.53.520_100

202020

0-10-0.5203.50202002020

0-100

Firmness

Deer.Incr.Incr.Incr.Incr.Incr.Incr.

Incr.Incr.Incr.Deer.Incr.Incr.

Incr.Incr.

Incr.Incr.

Effect of treatment*Flavour

Deer.

Incr.

Incr.Deer. 5Deer. 5

Incr.

Incr.

Decr.§Incr.

Resp.*

Incr.Incr.

Deer.

Incr.Deer.

Incr.

Deer.

Incr.

Incr,

Deer.

Ethylene

Deer.

Deer.Incr.Deer.

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Deer.

Incr.Incr.Deer.

Reference

Ampun(1997)Fidler&North(1971)Stow (1988)Ampun(1997)Couey&Olsen(1975)Lau&Looney(1978)Hribaretal. (1994)Gorny &Kader(1996)Gorny & Kader(1996)Pesisetal. (1994)Keetal. (1991b)Little etal. (1982)Ampun(1997)Eaves etal. (1968)Bramlage et al. (1977)Dilley etal. (1963)Ampun(1997)Eaves etal. (1968)Ampun(1997)Ampun(1997)Keetal. (1991b)Eaves etal. (1968)

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0

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X03.0 '

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ft2000, Vol. 28


Fig. 1 Anaerobic biosyntheticpathway for the formation ofacetaldehyde, ethanol, and esters(adapted from Mathews & vanHold 1996). Highlighted text rep-resents compounds that accumu-late under hypoxic conditions.(PDH = pyruvate dehydrogenase,PDC = pyruvate decarboxylase,ADH = alcohol dehydrogenase,AAT = alcohol acyl CoA trans-ferase, TCA = tri-carboxylic acid.)

( TCA cycle )

Acyl CoAs

| Esters |

(Ethyl acetate)

be conditioned to low oxygen storage conditions(Little et al. 1982); (3) have tolerance to non-chemical disinfestation treatments (Ke et al. 1991a,b;Yahia & Vazquez-Moreno 1993); (4) have enhancedaroma/flavour volatile concentrations in fruit (Shawetal. 1991,1992;Dourtoglouetal. 1994;Pesis 1994;Ampun 1997). Following a brief exposure tohypoxia, 86-95% CO2 or 97% N2 for 1 day, 'GoldenDelicious' apples, peaches, and nectarines were ratedby panellists as better flavoured than control fruitafter 2 weeks or 5 days at 20°C (Lurie & Pesis 1992;Pesis et al. 1994).

In addition fruit may incorporate CO2 into malatethrough dark CO2 fixation as in lemon (Bogin &Wallace 1966) and persimmon fruit (Pesis & Ben-Arie 1986). Persimmon fruit produced moreacetaldehyde when exposed to a hypoxic atmosphereof 99% CO2 compared to 99% N2 (Pesis & Ben-Arie1986). Apple fruit may also fix CO2 into malatealthough Ampun (1997) found no difference inconcentration of acetaldehyde produced by apples in100% CO2 or 100% N2 atmospheres.

Respiration and hypoxia

Refrigeration is the most common storage tech-nology used to preserve quality of horticultural pro-duce; controlled or modified atmosphere storage(CA or MA) further supplements refrigeration toextend storage life and maintain quality (Kader1986). Once O2 concentration in air around fruit isreduced to less than 10%, respiration rate is reducedin proportion to O2 concentration. A concentrationof c. 1-3% O2, depending on the fruit, is required toinduce anaerobic respiration (Kader 1986). Underhypoxic conditions the glycolytic pathway replacesthe tricarboxylic acid cycle (TCA) as the main sourceof energy provided by plant tissue, where oxidationof pyruvate in mitochondria is greatly reduced andaccumulation of pyruvate and acetyl CoA activatethe fermentation biosynthetic pathway (Fig. 1). Pyru-vate is converted to acetaldehyde and CO2 by the en-zyme pyruvate decarboxylase (PDC) and acetaldehydeis reduced to ethanol by NADH by ADH (Mathews& van Holde 1996). Most fruit produce ethanol whenexposed to anaerobic or hypoxic conditions.


Fermentation may be regulated by two mecha-nisms: molecular control of PDC and ADH by in-creased concentration or production of newisozymes; and metabolic control by feedback mecha-nisms of products and co-factors inhibiting enzymefunction (Perata & Alpi 1993; Ke et al. 1995). Al-though induction of PDC, ADH, and their isozymes(Pradet et al. 1985; Sachs et al. 1985; Longhurst etal. 1990; Chen & Chase 1993) occurs in hypoxicconditions, enzyme concentration is not well corre-lated with enzyme activity except at very low con-centrations (Roberts et al. 1989; Ke at al. 1995). Thisimplies that metabolic regulation of anaerobic en-zymes takes place as a result of other factors suchas changes in pH, substrate concentration, cofactorsand/or inhibitors.

Induction of fermentation may involve reductionin cellular pH that selectively activates PDC andADH. A decrease in cytosolic pH, associated withtransient lactate fermentation, has been reported inavocado (Hess et al. 1993), 'Bartlett' pears (Nanos& Kader 1993), and in tomato root cultures (Rivoal& Hanson 1994).

However, not all plants produce lactic acid beforean ethanol increase occurs (Andreev & Vartapetian1992). A decrease in pH could come about followinginhibition of proton pumping at low ATP concen-tration and proton release by ATP hydrolysis (Saint-Ges et al. 1991; Chervin et al. 1996), by release ofmalic acid into the cytoplasm from the vacuole(Bufler & Bangerth 1982), or as a result of high CO2

concentrations decreasing cytoplasm pH and thusinducing PDC activity (Blanke 1991).

Hypoxic conditions consistently enhance acetal-dehyde and ethanol concentrations in a wide rangeof fruits. Under hypoxic conditions acetaldehydeand ethanol can greatly exceed concentrations ofseveral hundred (0.1 litre1 (Knee 1991) with ethanolaccumulation as high as 47 ((j.1 litre~') kg"1 day ' at0°C (Knee 1991). When returned to air acetaldehydeand ethanol concentrations decrease to initial val-ues over 1-2 weeks (Saltviet & Ballinger 1983a,b;Ampun 1997). Fruit metabolism may be affected byacetaldehyde and ethanol that stimulate or inhibitvarious biochemical pathways involved in ripening.Application of acetaldehyde vapours to apples(Fidler 1968), blueberries (Paz et al. 1981), feijoa(Pesis 1994), oranges (Pesis & Avissar 1989;Shawetal. 1991), peaches (Pesis 1994), and pears(Janes & Frenkel 1978) induced an ethylene-likestimulation of ripening by enhancing ethyleneproduction and directly stimulating CO2 produc-tion.

Ester formation, (3-oxidation activity, and LOXare suppressed by low O2 concentration and this isprobably the reason why apples maintained in CAconditions have reduced volatile production (Tough1999). A decrease in acetaldehyde and ethanol afterremoval from hypoxia is associated with a several-fold enhancement in ethyl esters and some alcohols,as well as decreases in non-ethyl esters and alde-hydes (Mattheis et al. 1991a; Ampun 1997). In-creased ethyl ester concentration after removal fromhypoxia may be because of enhanced ethanol con-centrations (Mattheis et al. 1991a) as exogenousapplication of ethanol vapour increased ethyl estersand reduced non-ethyl esters (Berger et al. 1992).Ethyl ester production of apples was enhanced moreafter exposure to hypoxia for 18 and 24 h than after6, 12, or 48 h. The magnitude of enhancement wasthe same regardless of whether CO2 or N2 was usedto generate hypoxia (Ampun 1997). Oxygen concen-trations of <5% O2 were required to induce enhance-ment of ethyl esters; the lower the O2 concentrationthe greater the response (Ampun 1997). When O2

concentrations were maintained at 20% and CO2

concentrations ranged from 10 to 80%, only fruitexposed to CO2 concentrations >20% had enhancedethyl esters after treatment; the higher the CO2 con-centrations the greater the change in volatile concen-tration (Ampun 1997). It is not known whether thisincrease in ethyl esters because of the increase inethanol during hypoxia or to new isozymes of AATand ADH with substrate specificity for ethanol be-ing induced in response to hypoxia.

Pre-treatment factorsRipeness and maturity

Ripening is a process of physical, metabolic, andbiochemical changes initiated and/or co-ordinated byethylene, either on or off the tree, and includes lossof background green colour, softening of fruit tis-sue, and development of characteristic aroma andflavour (Wills et al. 1997). Typical flavour com-pounds of apples are only produced after ripeninghas been initiated by ethylene (Tressl et al. 1975).Apples are classified as having a climacteric ripen-ing pattern which is a rapid increase in productionof ethylene and/or respiration rate to a maximumafter which the rate declines (Wills et al. 1997).Different stages of ripeness and maturity can bedefined by their production of ethylene and/or carbondioxide. Physiologically immature pre-climactericapples have low aminocyclopropane-1-carboxylicacid (ACC) concentration and ACC synthase (ACS)activity, low ethylene production and fail to ripen


normally. Physiologically mature pre-climactericapples on the other hand have increased ACS activ-ity, are accumulating ACC and producing endog-enous ethylene and can be induced to ripen byexposure to exogenous ethylene (Lelievre et al.1997). Fruit harvested at this stage of maturity havethe greatest postharvest storage life potential. Peakclimacteric apples are those which have reached theirmaximum respiration rate and ethylene productionwhereas post-climacteric apples would be consid-ered ripe having moderate ethylene production, lowfirmness, and a short storage life.

There is a consistent correlation between the cli-macteric and volatile production. Typical aroma/fla-vour volatiles increase in concentration duringclimacteric ripening reaching maxima at the climac-teric (Sapers et al. 1977; Yahia et al. 1990b; Mattheiset al. 1991b; Song & Bangerth 1994, 1996). Applesharvested physiologically immature produce verylow concentrations of volatiles while overripe post-climacteric fruit produce low and declining concen-trations of volatiles (Brown et al. 1966; Hansen etal. 1992a; Vanoli et al. 1995; Song & Bangerth1996). Volatile production follows ethylene produc-tion during ripening in some cultivars (Brown et al.1966) but not in others (Hansen et al. 1992a). In-creased ethylene production and respiration may beneeded to provide precursors for increased volatilesynthesis (Song & Bangerth 1996). There is littleevidence that a direct relationship exists betweenethylene production and volatile production, but sucha relationship has not been investigated fully.

Specific volatiles in intact fruit appear to have fivepatterns of production as the fruit ripen that mayrelate to patterns of substrate production/availabil-ity (Brown et al. 1966; Sapers et al. 1977; Yahia etal. 1990b; Mattheis et al. 1991b). The patterns are:a continuous decline; steady stable levels; a transientrise in production followed by a return to previouslevels; a steady rise in production followed by a rapidrise as the fruit become fully ripe; and productiononce the fruit are fully ripe. Volatile production inapples ripened at warm temperatures after harvest orduring coolstorage is, in general, sigmoidal withvolatile production declining as the fruit becomeoverripe (Dirinck et al. 1989; Hansen et al. 1992a;Vanoli et al. 1995). Propyl acetate production of' Jonagold' apples increased for 3 weeks at 20°C afterremoval from 2°C, then decreased (Hansen et al.1992b). In contrast, production of butyl and hexylacetate declined steadily over time at 20°C. Aspropan-1 -ol is thought to be produced by a-oxida-tion and butan-1 -ol and hexan-1 -ol from (3-oxidation,

any differences in acetate ester concentration amongapple cultivars indicate that the metabolic origin ofalcohols affects the pattern of ester production(Hansen et al. 1992b). It would be necessary tomeasure rates of volatile production, rates of (3-oxi-dation, transamination, and AAT activity to estab-lish if they were affected by substrate production/availability.

Temperature

Volatile concentrations increase as temperature in-creases, although production rate is reduced above32°C. Ester and alcohol concentrations and rates ofproduction of 'Jonathan' apples increased as tem-perature increased from -1 to 10°C during 12 weeksin store (Wills & McGlasson 1971). 'Red Delicious'apples had maximum ester production at 22°C; itdecreased at 32°C and was inhibited at 46°C(Guadagni et al. 1971) indicating that heat treatmentmay temporarily inhibit or inactivate enzymes re-sponsible for producing volatiles. A heat treatmentof 38°C for 4 days reduced volatile production in'Golden Delicious' apples compared to fruit at 22°C(Fallik et al. 1997). However, characterisation of therelationship between volatile production and tem-perature over the range o f - 1^ 5° C has not beendone. Therefore, it is not known how apples mayrespond to hypoxic conditions at temperatures otherthan 20°C.

Apples transferred to 20°C after low temperaturestorage produce greater concentrations of volatiles,and reach maximum production earlier, than freshlyharvested apples, this being a cultivar specific effect.Maximum concentration of butyl acetate and hexylacetate in 'Cox's Orange Pippin' apples was reached18 days after harvest and 27 days after harvest forbutan-l-ol and hexan-l-ol (Hatfield & Patterson1975). After 3.5 months at 3.3°C, maximum butylacetate concentration was reached 4 days after returnto 20°C and concentration was about twice that offreshly harvested fruit. Butan-l-ol took 15 days toreach a maximum concentration c. 5 times that offreshly harvested fruit (Hatfield & Patterson 1975).Such increases in volatile concentration may resultfrom accumulation of volatile precursors in fruit atlow temperatures.

Temperature affects volatile concentrations andpatterns of concentration change during storage in acultivar specific manner (Yahia et al. 1990b; Yahiaet al. 1991). For example, maximum total volatileconcentration of 'Cortland' apples at 3.3°C was c.60% that of fruit at 20°C, whereas 'Mclntosh' ap-ples at 3.3°C had only half the total volatile


concentration of fruit at 20°C (Yahia et al. 1990b;Yahia et al. 1991). Hexanal concentrations of'Cortland' and 'Mclntosh' apples had the same pat-tern of change, irrespective of temperature (Yahiaet al. 1990b; Yahia et al. 1991). Ethyl butanoateconcentrations for 'Cortland' and 'Mclntosh' applesat 20°C increased to a peak before decreasing tooriginal levels, whereas in fruit at 0 and 3.3°C, ethylbutanoate concentrations increased steadily withtime (Yahia et al. 1990b; Yahia et al. 1991).

Duration of storageLow temperature storage in air for longer than 3months reduced production and concentration ofvolatiles in apples (Ampun 1997). Such decreasesin volatile production are detectable by sensory pan-ellists after 6 and 8 months of storage (Plotto et al.1997). Maximum concentration of'Red Delicious'volatiles occurred after 2-4 months at 1 °C and de-clined after longer storage periods (Guadagni et al.1971). After 5 months at 1°C 'Golden Delicious'apples had less volatiles than fruit stored for 3months (Streif & Bangerth 1988). Total volatileconcentration of 'Golden Delicious' apples after 3months at 1°C was c. 50% greater than after 8months (Brackmann et al. 1993). Butyl acetate andhexyl acetate concentrations in 'Golden Delicious'apples increased to a maximum after 2.5 months at4°C then decreased after 3.5 months (Bachmann1983). Ester concentrations in 'Law Rome' and '262Rome' apples were lower after 6 months at 0.5°Cthan in freshly harvested fruit and in fruit stored for3 months at 0.5°C (Fellman et al. 1993b). The re-duction in ester concentration was associated withreduced ester biosynthesis where AAT activity wasdecreased after 6 months at 0.5°C compared tofreshly harvested fruit and fruit stored for 3 monthsat 0.5°C (Fellman et al. 1993b). Reduced ester con-centration may also be because of reduced availabil-ity of substrates for esterification although thisrequires measurement.

When hypoxia is applied before CA or low tem-perature storage or at warm temperatures (>20°C),high concentrations of ethanol and ethyl acetate canpersist throughout storage and shelf life of a fruit(Mattheis et al. 1991a). This residual effect of hy-poxic treatment may cause off-flavours (Ke at al.1991b). Exposing apples to hypoxia for 24 h, afterremoval from different periods in CA storage, en-hanced acetaldehyde, ethanol, and ethyl esters whiledecreasing non-ethyl esters (Ampun 1997). Thelonger the duration of CA storage the less the en-hancement of ethyl esters induced by exposure to

hypoxia (Ampun 1997). This suggests that applesstored in CA have a reduced capacity to produceesters; this has been shown to occur in 'Pacific Rose'apples after as little as 4 weeks in CA (Tough 1999).It is possible this is because of decreased AAT ac-tivity (Fellman et al. 1993a) or to lack of substrateavailability (Knee & Hatfield 1981):

Post hypoxia effects

Fruit exposed to brief periods of hypoxia have reducedpostharvest decay (Pesis & Avissar 1989; Ke et al.1991a), improved fruit quality (Pesis 1994), and in-creased aroma volatile concentration in citrus andfeijoa fruit (Pesis et al. 1991; Shaw et al. 1991) com-pared to untro ited fruit. Application of hypoxia hasbeen evaluated as a potential disinfestation treatmentusing <2°/o O2 and up to 100% CO2 or N2 atmospheresfor 1-14 days at c. 20°C (Gaunce et al. 1982; Hallman1994). Sensory panel analysis indicated that fruittreated with hypoxia had increased aroma/flavour.Apples treated with 10-15% CO2 before CA storagewere rated by taste panellists as having better flavourand texture than untreated fruit stored in CA only(Tiejen & Hudson 1984). Peaches and nectarines ex-posed to 86% CO2 or 97% N2 for 1 day at 20°C werepreferred over untreated fruit after 7 days at 20°C bya panel of 15 tasters (Lurie & Pesis 1992). Feijoa fruittreated with 98% N2 + 2% O2 for 24 h at 20°C wererated by a sensory panel as sweeter than control fruitafter 7 days at 20°C (Pesis 1994). 'Golden Delicious'apples exposed to >95% CO2 for 24 or 48 h at 20°Chad better flavour than untreated control fruit after 2weeks at 20°C, with 24 h treatment being the mostpreferred (Ampunpong 1991; Shusiri 1992; Pesis etal.1994). Such improvement in flavour was undoubtablybecause of an increase in concentration of aroma/fla-vour volatiles induced by exposure to hypoxia (Pesis1994). Apart from some preliminary results from Pesiset al. (1994) the effect of short-term exposure to hy-poxic conditions on apple cultivars has not been char-acterised. It is possible that exposure of apples to a briefperiod of hypoxia will increase the concentration ofaroma and flavour volatiles thereby improving flavouracceptability to consumers.

CONCLUSIONS

Flavour typical to apples develops during ripening(Tressl et al. 1975) and may be associated withethylene production and metabolic activity (Song &Bangerth 1996). The greatest concentrations of


volatiles are produced at the climacteric peak inethylene production (Sapers et al. 1977; Yahia et al.1990a; Mattheis et al. 1991b; Song & Bangerth1994). However, it is unknown whether the enzymesinvolved in volatile biosynthesis are induced duringthe climacteric or are constitutive.

Exposing apples to hypoxic conditions induceschanges in volatile concentrations; acetaldehyde andethanol accumulate to high concentrations and afterreturn to aerobic conditions ethyl esters are enhancedand non-ethyl esters are decreased (Mattheis et al.1991a; Ampun 1997). Differences in proportions ofvolatile compounds exist between cultivars (Dirinck& Schamp 1989; Paillard 1990) as does degree ofethyl ester enhancement following hypoxictreatment (Ampun 1997). Possible reasons for thesedifferences might include separate iso-forms of AATand ADH in cultivars each with their own substratespecificity for substrates, or alcohol precursors beingavailable in varying concentrations; hypoxia mayinduce increased activity or synthesis of AAT andADH depending on cultivar.

If some or all enzyme systems involved in volatilebiosynthesis are induced during ripening, thenvolatile changes after exposure to hypoxia coulddiffer according to stage of ripeness. If enzymes areconstitutive then it is likely that volatile changeswould be similar at all stages of ripeness assumingprecursors were not limiting. Changes in volatileconcentration after exposure to hypoxia of apples atdifferent stages of ripeness has not been determined.

Storage at low temperatures is a commonly usedmethod for slowing ripening to allow transport overlong distances to markets or for delaying marketingof fruit to achieve higher returns (Wills et al. 1997).Volatile production is considered to be proportionalto temperature, the higher the temperature, thegreater the production of volatiles (Guadagni et al.1971; Wills & McGlasson 1971; Fallik et al. 1997).Exposure of apples to low temperatures for morethan 3 months decreases volatile concentrations by30-60% (Bachman 1983; Brackmann 1993; Fellmanet al. 1993a,b). Temperature may also affect produc-tion of specific volatiles with some compounds onlybeing produced at certain temperatures by affectingrates of substrate supply and volatile biosynthesis.If this is so then the different biosynthetic pathwaysproducing volatiles may be active at different ratesaccording to temperature. It is currently not known ifthe activity of enzymes active in volatile synthesis arethermally labile or if storage at low temperatures re-duces or enhances the capacity of apples to producevolatile compounds after exposure to hypoxia. If

exposure to hypoxia changes the volatile biosyntheticpathway then hypoxic conditions imposed at differenttemperatures could change the type of volatile com-pounds that increase after such treatment.

Treatment of apples using hypoxic conditions hasthe potential to enhance volatile concentrationsameliorating the decrease in volatiles induced by lowtemperature and CA storage conditions. At presentit is unknown if likely increases in volatile concen-trations induced by hypoxia are detectable orga-noleptically, whether they affect apple aromapositively or negatively, and would require testingby sensory analysis. Hypoxia induced increases involatile concentrations may be a serendipitous andbeneficial side effect of some potential disinfestationtreatments. This could mean that given equivalentefficacy, hypoxia may be preferred to heat treatmentas a disinfestation treatment given the latter maydepress fruit volatile production. When used onfreshly harvested fruit, hypoxia can induce signifi-cant increases in aroma volatile concentrations thatmay be of value in producing high quality appleessences from apple juice.

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factors affecting apple aroma flavour volatile concentration

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unpublished phd thesis

induce significant increases

unpublished postgraduate diploma

freshly harvested fruit

carbon dioxide atmospheres

malus domestica borkh

fresh horticultural products

horticultural science thesis