CHARACTBRIZATION OF VOLATILE AROMA CONSTITUENTS IN AVOCADOS (PERSEA AMERICANA l.4iILL) GROWN IN SRI LANKA

" ABSTRACT The volatile comporyents of avocados were isolated by Tenex GC and analysed by capillary Gas Chromatography linkedwith Mass Spectrometry. A total of 73 cantponents were rJetected as avocado volqtiles, ofwhich 70 compounds comprising 99% of the isolates tvere positively identffiecl. Sixteen of the identified compounds have not been previously been reported as avocado volatiles including bulqnal, oct-l-ene, dimethyl cyclohexane, nonan-2rone ancl telradecanoic acid. In ripe avocaclos, C-6 alcohols and aldehydes were the major group ofvolatiles ytith trans-hex-3-en-l-ol as the mos.t abundant constituent of 22.04%. The C-6 aldehyde, cis-hex-2-enal accountecl to 6.66% of the crroma isolatesintheripefruits.Terpenoidswerethepredominantclassofconstitttenlsintmripec toccrdos,. )ne monoterpene, limonene (2.97%o) and ten sesquiterpenes'were identified, p-caryophyllene being themaior terpene (14.25%0)followed by B-franescene (10.92%o) and a-cttbebene (10.2%) as the main hydrocqrbons. In an olfactory assessment at odonr port dtrring GC, six aroma compotmds were described possessing lhe characleristics green.flat,our ,tf uvocaclo. The C-6 alcohols ancl atdehydei were signfficant components of aronta together with several compounds deriving fi'ont fotly acidprecursors. The oxidative decotnposition ofcarotenoids leads to theformatio, of terpenoids, in unripe avocados. The carotenoid content of avocados cv. Flass was determined recently as 18pg/g fresh fruit. Onyewn and co-workers (1996) surveyed derivatives of carotenoids (particularly p-carotene) formed upon heat treatment and identified one of them as toluene. Avocado contains phospholipids, glycolipids and triacylglyccrol which are rich in linoleic acid and linolenic acid. On rupture ol'cclls, thcse fatly acids arc t'eleased by the actionof acyl hydrolases andphospholipase D andarc further converted by lipoxygenasc into thc 9- and 13-hydroperoxides ofboth linoleic and linolenic acids. The volatile cleavagc product is hcxanal when 13-hydroperoxy linoleic acid.js thc substrate while crs-hcx-2-enal is procluced on cleavage of l3Jrydroperoxy linoleriicacid.Previousworkinthis(Young etal.,l999)hadcstablishedthathomogenizationofavocado tissue caused the conversion of linoleic and linolenic acid to volatile carbonyl fragmenls. at ripc stage. Terpenes ale thc important zu'olna constituents of unripe avocados and expected to contribute a pcppery after taste. A grcater numbcr of alcohols and carbonyl compounds identified in aroma isolates are classic volatiles produced derivcd from fatty acid oxidati6n and degradation. Olfbctory assessments of odourport during GC six peaks were described as posscssing significant geen note of avocado flavour.


INTRODUCTION
The avocado (Persea americanaMlll) belongs to the family Latraceae and is one ofthe major fruit crops in the world' It is native to ccntral America but is grown in all llopical and subtropical regions of the world.
During ttre past decade, the demand for this fiuit has increased significantly as a resuh of increased consumer awareness offruit's diateryvalue andimprrvedfruitqualiqrresultingfi.omirnplementationofinahrity standards and improved storage and transportation facilities. In Sri Lanka, avocado is grown in central high lands most often as seedlings among crop planls on estatcs and in back gardens. I hc avocado seasons normally exists fromApril to July when the fruits are relalivcly abundant. Avocado is a favorite arlicle of diet in Sri AGRIEAST2OO9(8) Mahendran Lanka and is mainly consumed in the form of fresh fruit. It is also used as an ingredient in salads and tortillas with lemon juice, pepper and salt.
Avocado is an oleaginous fruit and the lipid content in the mesocarp varies froml2.4 -24.2% at maturc ripe stage Q.{agalingam ,1994). Because of the high oil content, the fruits have the highest energy value of any fruit. The high oil content also contributes to the consistency and the special taste of the fruit. Unlike the other fruits, the avocado is high in fat, protein, vitamins and minerals but low in sugars; therefore it can be recommehded as a high energy food for the diabetic (Swisher e/ a\.,2001).Knowledge ofthe identities of the constituents which are responsible for avocado flavour is important for the conhoi of flavow, both in the fresh and processed products. The high oil content ofthe fruit has attracted special attention with respect 1o compositionoffruitbutrelatively little workhas beendone onthe compositionofthe volatile constituents of avocado fiuit and therefore the present study was undertaken to characterize thc volatile aroma components ofavocados.

Avocado Samples
Fresh, locally harvestedAvocado fiuits cv. Hass were obtained from a cortmercial grower at Kandy, Sri Lanka. Fruits were washed with water and dried in the air before use. Fruits were ripened in a humidity control cabinet at20"C and the RH of 90-9 4Yo.To obtain such humidity level, the air plrmp rvas used to bubblethe airthroughwaterin atray. Each day, the stages ofripening offruits were detenlined subjectively by applying gentle pressurc to the fruit held the palm of the hand. Categories were hald, soft and ripe, Thcsc stages were confirmed by texture measurement using Sleven's Compression Rcsponse AnalyzeL using 50 kg load cell. Fruits at different ripening stagcs were halved longitudinally and ilre seeds removed.
Hard fruits were peeled and thc flesh put through a food shredder. For soft and ripe fiuits, the pulp was scrapped and homogenized for 5 min.

Isolation of Volatiles
A weighed quantity of 300 g sample from 3 avocados was transferred to an aroma isolation apparatus with a screw top containing both an inlet and outlet tubes. The flask was placed in a water bath at 40oC. A SGC trap, containing Tenax-GC (polym er of 2,6 diphenyl-P-phenylene) as thc absorbent, was connected to the outlet and a oxygen free nitrogen was connected to the inlet. The nitrogcn was passed over the pulp at a flow rate of 40ml/min for t hour and the avocado volatiles rvcre swept onto the trap. The inlet was also connected to a tube containing an absorbent (charcoal) to prevcnt volatilcs fiom the gas supply reaching AGRIEAST 200e(8) p. 1 9-29 the trap. At the end of collection time, the flask was removed and the trap was connected directly to the nitrogen supply at the same flow rate for 5 min to remove the moisture. A blank isolate was also prepared using an empty isolation apparatus. Alkanes standards (Cu to Crr) were used in the measurement of retention indices of the standard and the compounds separated from avocados. Ethanol, ethyl acetate, hexanal, nonanal. hexan-l-ol, hex-2-en-1-ol, hex-3-en-1-ol and B-pinene were purchased from BDH Chemicals, UK and B-caryophyllene, o-cubebene, p-franescene and s-humulene fromAldrich Chemical Ltd, UK. Gas Chromatography Arrutyri. of Volatiles A Perkin-Elmer Sigma 3B equipped with flame ionization detector (FID) was used for routine gas clnomatographic analysis.Afused silicacapillarycolumnof60mx 0.32mmI.D., coatedwith 1pmfilm thickness of g5ohdimethyl siloxane + 5Yophenyl siloxane (SE 52154)bonded phase was used to separate the volatile components' The trap was placed directly in the injectionport ofthe gas chromatograph and its contents were desorbed with the helium carrier gas at the flow rate of 1.5 ml/min for 5 min onto the front end of the capillary column. 'lhe column was cooled to 0"C with liquid CO, which entered the oven tluough a metal tube, sunounding the column. After desorption the oven was heated rapidly to 3 0"C, followed by heating at3"C/minto 250"C. The columntemperature was maintaine d,at2l}'Cuntil the completionofthe separation' The injector port and the detector temperature were maintained at 250"C and,260oC,respectively. A Hewlett Packard model-3 3 90A reporting integrator was used to determine the peak area during routine analysis' Linear retention indices for the volatile components were calculated by chromatographing nl alkanes (Cu to Crr) mixed with the samples. Linear rctention indices for thc authentic al'olna compounds were similarly determined. Gas Chromatographic-Odour Port Analysis Aromas ofthe separated components wcre assessed at the column outlet during the cluomatographic separation using the technique of GC-Odow port assessment. Chromatographic effluents were split in a ratio of 1:l by means ofthe outlet splitter containing a two fold vespel femrle, such that trvo equal lengtlis of deactivated fused silica (0.3 x 0.32 mm I.D.) led fiom the fenule, one lenglh ofthe GC cletector and the other length of the extemal sniffing port. 'fhe odours ofthe separatecl components were described by two assessors experienced in descriptive analysis of odours. Triplicate assessmcnts were perfomred for each ripening stages of avocados.
Gas chromatographic-Mass spectrometric Analysis of volatiles Volatile components were identified as lar as possible by Gas Chromatography -Mass Spectromelry analysis using a Kratos MS 80RFA Mass Spectrometer linked on line to a Kratos DS 90 data processing system coupled to a Carlo-Erba 5300 Gas Chromatography. The same GC conditions were used in this instrument.Thesignificantoperatingparameterswere:ionizationvoltage -7}eY;Emissioncunent-100pA; Ion source temperature -200"C;Accelerating voltage -4KV; Resolution -1000; Scan speed -1sl decade; Scantime -0.2s.

Component ldentification and Quantitative Ass essment
Interpretation of Mass Spectra was done by computeizeddatamatching, using libraries on the GC-MS data systems (Eight peak Index of the Mass Spectra) and by manual comparisons with published mass spectra. Identifications were confirmed by comparing the Linear Retentive lndices (LRI) or Covats Index of each component. The mass spectra and retention indexes of compounds which were not available, were matched, where possible with values recorded in thc literature. euantitative data were derived from the TIC monitor obtained during GC -MS and for trace components by extrapolation from integator (Hewlett Packard 3370B) data obtained from the GC-FID chromatogram recorded during routine GC analysis. Triplicate analyses were performed'

RESULTS AND DISCUSSION
Components identified in headspace concentrates from avocado samples at three ripeness stages are listed inTable L Overall 73 components were detected as avocado volatiles, ofwhich 70 compounds comprising 99yo of thesamples were positively identihed. The three unidentified compounds were present in the sample in such low amounts that no mass spectrum could be recorded. Clianges in individual volatiles &ring ripening were also assessed. In ripe fruits, the isolates were dominated by C-6 alcohols and aldehydes a but terpenes were the abundant classes of volatiles in unripe fruits.
In avocados, the levels of C-6 alcohols and aldehydes remarkably increascd during ripening' Three C-6 alcohols were present at high concentration in ripe fruits. The predominant alcohol rs lrans-hex-3-en-1-01 which accounts 22.04%in the ripe fruits. The geometric isomer, trans-hex-2-en-1-ol is present at about I7 .43%.In unripe fruits, these two components are present at7 .7lo/o and 4.2lYo respectively. No work appearstohavebeenpublishedonthe changes involatile constituents duringripening ofavocados' However, only tu,o reports are available onthe volatile components of avocadod. Yamaguchi (1989)reported that trans-hex-2-en-1-olandhexan-1-olcomprised58.95% andg.S2Yoinlheripeavocadoisolatebutthc variety of the avocado gxamined was not mentioned. They also reported the presence of low levels of hexanal (0.39%)andtrans-hex-2-enal(I.61%)intheirisolates.Thisisincontrastwithowfindingswhere hexanal was present 5.51% and, trans-hex-2-enalwas not identified as a component of the aroma isolate' Compared to hexanal and czs-hex -2-enal,the concentrationof trans-hex-3-enal was found to be low at 0.37% inripe fruits. According to the work of Kazeniac and Hall (1990), the formation of some ofthe clshex-2-enal is through trans-hex-3-enal by the action of enzyme isomerase.It is well known that this type of double bond shift occurs readily. Because of this double bond shift, it is difficult to get an accurate analysis of the relative amount gf each compound as it occurs in the avocado.
The quantity and quality of C-6 alcohols and aldehydes is dependant on the amount of oxygen in the sample, enrqeactivity, extent of comminution, isolation procedure and the extent of heat. Therefore, some of the aldehydes may be transformed to alcohols by enzymatic action. Since blending effectively disintegrates the avocado tissues, the natwal enrymes and their substrates arc intimately mixcd' [n addition, an cxccss air is whipped into the mixture during blending the pulp. The combination ofthese factors would be expected to greatly favotr the development of flavour compounds by enzymatic oxidation (Whitefield er c/., 1980). The domination of aroma isolates from ripe avocado, by C-6 alcohols and aldehydes appeals to be unique amongtropical fruits. These two classes of compounds accotrnte dover 650/o ofthe total aroma isolates and the characteristics flavour come from the C-6 alcohols and aldehyde:,. In wide range ofplant tissues, the carbonyl compounds are derived from thc reaction of fatty acids with oxygen and lipoxygenase enzyme' The dominant compounds identified in the ripe avocado aroma are reported to be derived from fatty acid degradation. Avocaods are known to be rich in oil. The average oil content ofunripe and ripe fiuits used in this studywas15.4o/oand2L lo% respectively. Scveral authors examinedilre formation of C-6 alcohols andaldehyd'esfromunsaturatedfattyacids. (Stone etal.,1999andGallialdetal.,200l).
Investigating the fatty acid composition of lipids \n Hass avocados, the unsalurated fatly acids such as oleic acid -70.20/o,linoleic acid -l0.l%and linolenic acid -I.}3%were found to prevail in the ripe fruits (lvtalrendranandThireganathan,lgg8). Onmacerationoftissues,enrqaticdccompositionoflipidproceeds veryrapidly. Kazeniacand Hall (1990) showed that in the presence of II* and under the influence of heat' cis-hex-3-enal is transformed into trans-hex-2-enal isomer. These aldehydes may be converted to C-6 alcohols bv the enzym e aJcohol dehydrogenase during storagc and ripening offruits'   Alcohols, a part from C-6 compounds, accounted for about 8% and IIo/o of the isolates obtained from the unripe and ripe fruits respectively. Similarly, total aldehydes proportions of the isolates other than C-6 compounds accountedabout1o/oandl2ohntheunripe andripe fruitsrespectively. The level ofacetaldehyde increased from}.78Yoto 3 .94o/oduring ripening of avocados. The concentration of ethanol was higher than that of acetaldehyde suggesting a rapid conversion of acetaldehyde to ethanolby enzymatic action. Apart from acetaldehyde, nonanal and decanal are reputed to be important volatile constituents in avocado.
Moreover, 2-methyl propanal, butanal, 3-methyl butanal, 2-methyl butanal, pentanal, heptanal, and octanal were also identified. the total amount of aldehydes concentration of avocado increased withprogressive ripening. The amino acid profile of avocado has been reported by (Ahmed and Barmore, 1983) and strecker degradation of alanine, valine, leucine and isoleucine explains the formation of acetaldehyde,2methyl propanal, 2-methyl butanal and 3-methyl butanal, respectively. Many ofthe aldehydes identified in this study can be related to the major groups of the non-volatile constituents found in fiuits. The straight chain saturated and unsaturated aldehydes are fairly tlpical of those formed from oxidative degradation of the fatty acids. These are common to many fruits although each fruit appears to have its own relative proportions of the different volatile constituents.
Aliphatic ketones account less than 2Yo in the ripe and unripe isolates. Acetone, 3-pentanone, 3hydroxybut an-2-one,nonan-2-one and 2-methyl-5-hepten-2-one were identified in botli ripe and unripe fruits. The number ofesters identified inthis study is limited. The lcvel of estcrs isnearly 4o/oand2o/ornthe ripe and iuuipe fruits respectively. Methyl acetate and ethyl acetate are thc only two esters identified from both isolates. These two esters commonly occur in aroma volatiles ofmany tropical fruits including avocados (Kader, 1989). As the endogcnous level of acetaldehyde and ethanol increased, the concentration ofmethyl acetate and ethyl acetatewere found to increase during ripening. The levels of these two esters remained high at edlble ripe stage. This may indicate the biospthesis ofthese two estcrs continues with progessive ripcning, as happened with other esters in apples treated with carboxylic acids. Apples were found to produce carboxylic esters from added aliphatic volatile alcohols and acids apparently by absorbing the alcohols and acids from the surrounding ahnosphere and convelting them iuto esters which were released into the atmosphere again. (De Potter et a\.,2003). As an effect of non-euzymatic blowning reaction between the reducing sugars and amino acids upon heating, volatile carbonyl compounds are formed and these may higtrly affect the character of aroma. In avocado, these comporurds were identified as frfffilrals, 2-methyl fhran and 2-ethylfi.ran.
'fhe most important aroma constituents ofunripe avocadoes are terpencs. These compounds eomprise over 600% of the total volatiles. tn ripe fiuits these compounds are present at atotal level less than l%' One monoterpene hydrocarbon, limonene (2.97%) and ten sesquiterpene hydrocarbons were identified from both ripe and unripe isolates. The major sesquiterpene hydrocarbon identified in this study was p-caryophyllenewhichaccountsl4.2So/ontheunripefruitsandthelevelsdecreasedto 0.L2%vnthprogessive ripening ( Figure: 1). Stevens et al (l999)reported that p-caryophyllene was the major sesquiterpene hydrocarbon component identified from guava. Softening results in a considerable relative decrease in the concentrations of s-cubebene and B-famescene. Other terpene hydrocarbons which have been identifiecl to contribute aroma of unripe avocados were o-copaene (4.86%),B-gurjunene (4.57%),calarene (3.28%), D-cadinene (3.74%)and o-humul ene (2.07o/o). Young et al (1999) found no sesquiterpenes in ripe avocado mesocarp extracted under the reduced pressure, but found 2-methyl-2-bu1anal, dimethyl formaldehydc and methanol which were not found in this study.
The normal flavour of avocado fruit is usually described as bland with a green and peppery afler taste . Thc characteristics green flavour is due to presence bf C-6 alcohols and aldehydes which comprises over 650/o of the total aroma isolates in ripe ftuits, The likely cause of peppery flavour is at prescnt unknown but atleastthree ofthe sesquiterpenes hydrocarbons, p-caryophyllene, o,-cubebene and cr-copaene could impart a distinctive flavour to the rather bland tasting flesh of the avooado. Some of the identifiecl volatilcs arc derived from the nonJipid precursors. The oxidative decomposition of carotenoids leads to the formation ofterpene compounds.
Porlod of ripcning (days) The carotenoid content of avocados cv. Flass was determined recently as 18pg/g fresh fruit. Onyewn and co-workers (1996) surveyed derivatives of carotenoids (particularly p-carotene) formed upon heat treatment and identified one of them as toluene. Avocado contains phospholipids, glycolipids and triacylglyccrol which are rich in linoleic acid and linolenic acid. On rupture ol'cclls, thcse fatly acids arc t'eleased by the actionof acyl hydrolases andphospholipase D andarc further converted by lipoxygenasc into thc 9-and 13-hydroperoxides ofboth linoleic and linolenic acids. The volatile cleavagc product is hcxanal when 13hydroperoxy linoleic acid.js thc substrate while crs-hcx-2-enal is procluced on cleavage of l3Jrydroperoxy linoleriicacid.Previousworkinthis(Young etal.,l999)hadcstablishedthathomogenizationofavocado tissue caused the conversion of linoleic and linolenic acid to volatile carbonyl fragmenls.
In this study, as expected, a large number of volatilc compounds were oblained in addition to terpenes, especially compounds relating from lipid degradationproducl"s which included alcohols and aldehydes.
Partly because ofthe enzl.rnatic procedurcs instantaneously occuning upon thc maceration ofthe fiuit, it is difficult to establish which ofthe volatile components were prescnt originally in the fruit and rvhich developed during the comminution and homogenization. ln addition to the compounds identified, avocado extracts contained a number of related higher molecular wcight compounds which arc probably complexcs of substituted phenols and studies to identifrthese continue.

CONCLUSIONS
The characteristics green flavour of avocado is due to the prescnce of C-6 alcohols and aldehydes which comprises over 65o/oofthe lotal aroma isolates at ripc stage. Terpenes ale thc important zu'olna constituents of unripe avocados and expected to contribute a pcppery after taste. A grcater numbcr of alcohols and carbonyl compounds identified in aroma isolates are classic volatiles produced derivcd from fatty acid oxidati6n and degradation. Olfbctory assessments of odourport during GC six peaks were described as posscssing significant geen note of avocado flavour.