J. Sci. Food Agric. 1983,34, 81-85

Volatile Aroma Constituents of Kiwifruit

Harry Young, Vivienne J. Paterson and Donald J. W. Burns

Division of Horticulture and Processing, Dept of Scientific and Industrial Research, Mt Albert Research Centre, Private Bag, Auckland, New Zealand

(Manuscript received 26 January 1982)

The volatile constituents of kiwifruit (Actinidia chinensis Planch.) have been investi- gated. The volatiles were collected and concentrated using vacuum steam distillation and freeze concentration. The concentrated distillate was analysed by gas chromato- graphy, gas chromatography-mass spectrometry and reaction gas chromatography. Apart from methyl benzoate, all the components identified were alkyl and alkenyl esters, alcohols, aldehydes and ketones. The most abundant component was trans hex-2-enal. Odour evaluation of the components at the exit port of the gas chromato- graph indicated that ethyl butanoate, hexanal and trans hex-2-enal are important contributors to the aroma of kiwifruit. One other component which does not show a peak in the chromatogram and which is not yet completely characterised may have particular significance.

1. Introduction

Kiwifruit (Actinidia chinensis Planch.) is a native of China which over the last 10-15 years has enjoyed spectacular success as a commercial horticultural crop in New Zealand. Present annual production is about 26 000 t per annum with over 80 % destined for the export trade and it has been predicted that production will increase 8-10 fold over the next decade.1 The present market is based primarily on the fresh fruit which stores well, but as production levels rise processing of fruit unsuitable for this trade is assuming increasing importance. However, the flavour of processed kiwifruit, particularly where processing has involved heat treatment, bears little resemblance to that of the fresh fruit. Instead, it is similar to that of the cooked green European gooseberry (Ribes grossuiaria L). Flavour changes also occur during production of kiwifruit juice, even under con- ditions where heat treatment has been avoided (Wilson and Burns, unpublished).

As part of a programme directed at minimising flavour changes during processing we have undertaken a chemical investigation of the volatile compounds in fresh kiwifruit, since taste and aroma are intimately associated with these compounds. In addition to the identification of the volatile constituents, some sensory assessment of those compounds which make a significant contribution to the aroma is reported in this paper.

2. Experimental 2.1. Fruit Kiwifruit of the major commercial cultivar, Hayward, were obtained from the DSIR Research Orchard at Te Puke. The juice of the harvested fruit had a soluble solids content of 7 4 % by refractometer. The fruits were ripened to the eating stage by dipping in Ethrel solution (lo00 mg litre-1) and storing at 20-25°C for 6 days. At this stage the flesh gave a penetrometer (7.9 mm head) reading of about 0.5 kg and the juice had a soluble solids content of 14-15%.

2.2. Collection of the volatiles The ripe fruits were cut in half, the flesh was scooped into a stainless steel bucket, and pulped with a potato masher. It was then passed through a paddle finisher fitted with a perforated stainless steel

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82 H. Young ef al.

screen (0.84 mm holes) which removed seeds and fibrous material (3 kg of fruit gave c. 2.5 kg of finished pulp). The pulp was immediately distilled, under reduced pressure, in a 5 litre wide mouth Quickfit reaction flask fitted with a paddle-stirrer, thermocouple and a 500 ml addition funnel. A Davies condenser (Quickfit, 200 mm) used in the reflux mode and cooled with 0°C coolant, was used as a fractionating column/splash trap. Heat was supplied from a heating mantle to maintain the reflux condenser in a partially flooded state. The distillate was collected in liquid nitrogen-cooled traps connected to the top of the condenser via a 6 mm Rotaflo stopcock (Quickfit) which enabled the trap to be changed without venting the pulp to atmosphere. A second liquid nitrogen-cooled trap was connected in series as a safety trap. The vacuum pump was attached after this safety trap. In operation the apparatus was evacuated to 1.3 kPa before the pulp was added from the addition funnel, at such a rate that the sample did not foam up into the condenser. Distillation was allowed to proceed as soon as the pulp entered the distillation flask. The final pressure was adjusted so that the pot teniperature was maintained between 18-20°C.

When the liquid nitrogen-cooled trap became blocked the Rotaflo stopcock was closed and the trap replaced by a second trap. This trap was evacuated to the operating pressure before the Rotaflo stopcock was reopened. Two fractions (1 and 2) of c. 300 ml each were collected (over c. 5 h). Three batches of fruit (3 kg each) were processed in this way.

Fraction 1 from each batch was combined (1 170 ml) and freeze-concentrated to 140 ml over a period of 12 h in a closed vessel fitted with a mechanical stirrer. The vessel (a 1.8 litre preserving jar) was partially immersed in a bath so that the coolant level was higher than the liquid level inside. The coolant temperature ( - 2°C to - 3°C) was adjusted so that the ice layer formed in the vessel was clear. The concentrated solution was stored in 3 ml aliquots at - 10°C.

Fraction 2 from each batch was combined (1000 ml) and similarly concentrated. However, because it was reduced to a smaller volume, the concentration was carried out in two stages (1000 ml to 200 ml and 200 ml to 15 ml). A 1 litre vessel was used for the second stage.

2.3. Gas chromatography (g.c.) The Varian model 2700 gas chromatograph used was fitted with a flame ionisation detector (FID) and a variable outlet splitter (Scientific Glass Engineering, Australia). The injector and detector were maintained at 175°C and 150°C respectively.

4 r

I I I 0 2 5 50 min

Figure 1. Typical gas chromatogram of con- centrated (fraction 1) kiwifruit distillate (30 PI) 'injected' with a Chromosorb 105 desorption cartridge. Column: 60 m x 0.5 mm i.d. SCOT coated with Carbowax 20M. Carrier gas: He at 18 cm s-l. Temperature programme: isothermal at 50°C for 15 min, then 1°C min-1 for 15 min and finally 4°C min-1 to 150°C. Temperatures: injector 175"C, detector 150°C. a , ethyl acetate; b, ethanol; c, methyl butanoate; d, penten-3-one; e, ethyl butanoate; f, hexanal; g, pentenal; h, penten-3-01; i, hex-2-enal; j, trans hex-2-enal ; k, pentan-1-01; I, pent-2-en-1-01; m, hexan-1-01; n, trans hex-3-en-1-01; 0, cis hex-3-en-1-01; p , trans hex-2-en-1-01; q, unknown; r, methyl benzoate.

Columns used were a 60 m x 0.5 nim i.d. Carbowax 20M support coated open tubular (SCOT) column (Scientific Glass Engineering, Australia) and a 25 m x 0.3 mm i.d. OVlOl wall coated open tubular (WCOT) fused silica column (Hewlett-Packard, USA). Carrier gas (nitrogen) flow rate was 18 cm s-1. The temperature programme for the Carbowax column is given in Figure 1. The tempera- ture of the OVlOl column was varied according to the components being analysed. It was used isothermally between 30°C and 120°C.

Aroma constituents of kiwifruit 83

The aqueous samples were injected using stainless steel cartridges packed with Chromosorb 105.2 For analytical runs 10-20 pl of the fraction 1 concentrate were used. For semi-preparative work up to 200 pl of the concentrate was used in each run.

The collection port of the gas chromatograph consisted of a 3.2 mm to 1.6 mm stainless steel reducing union (Swagelok) connected to the splitter valve with 1.6 mm 0.d. x 0.76 mm i.d. stainless steel capillary tubing. A short portion of this capillary tubing was flattened to create a pressure drop of 20 kPa (at 20 ml min-1) across it. This minimised to a negligible amount the change in split ratio caused by the small back pressure of the packed traps. Traps similar t o the stainless steel cartridges used for injection, were attached to the 3.2 mm end of the union using ferrules machined from teflon rod. When required, the traps were cooled by a 13 mm diameter aluminium alloy rod slotted at one end to fit over a short portion of the trap. The other end of the rod was immersed into the desired coolant (e.g. ice/water or liquid nitrogen). For the Chromosorb 105 packed traps ice/water mixture was sufficient. In general when collection of the individual peaks was required the outlet splitter was set so that 90% of the effluent was diverted to the collection port.

2.4. Gas chromatography-mass spectrometry (g.c.-m.s.) G.c.-m.s. was carried out on a Varian model 2700 gas chromatograph interfaced to a AEI MS30 mass spectometer via a membrane separator maintained at 130°C. The g.c. conditions were similar to those described above except the carrier gas was replaced with helium. Low resolution mass spectra were recorded at 20 eV using an in-house designed and built data system. Accurate mass measurements (using the data system) were carried out at 70ev with perfluorokerosene as an internal reference. For these measurements the mass spectrometer was operated at nominal 3000 and 10000 resolving power (10% valley definition).

2.5. Micro-reaction g.c. Micro-reaction g.c. was carried out using a modification of the method described by Stanley and Kennett.3 Samples were collected from the gas chromatograph using the adaptor shown in Figure 2. At the end of collection, the trap (100 pl Microcap, Drummond Scientific, USA) was withdrawn from the collection adaptor and the section of the capillary containing the sample was immediately placed in a slot at the end of an aluminium alloy rod (1 3 mm diameter) partially immersed in liquid nitrogen. The capillary was then flushed with Ha or 0 3 as appropriate. Adam’s catalyst, introduced according to Stanley & Kennett,3 was used for the hydrogenation. For ozonolysis, the 0 3 stream was allowed to flow over the sample for 30 s after 0 3 was detected (KI paper) at the outlet end of the Microcap. The excess 0 3 was purged with oxygen-free NZ before the capillary was flame-sealed.

The capillary crusher (Figure 2) used to inject the reaction products into the gas chromatograph is a simplified version of that described by Stanley and K e n ~ ~ e t t . ~ It was designed to use the same injector insert as that used by the stainless steel injection cartridges.

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. F gg7gg

Figure 2. Capillary crusher and collection adaptor. ~ 1 1 dimensions are in mm. A, stainless steel plunger A 6 C D for crushing capillary; B, seal retainer (13.6 mm 0.d.); C, silicone rubber seals; D, coupling to injection port (replaces septum holder); E, carrier gas inlet (1 mni diam.); F, capillary crushing chamber (3.3 mm 0.d. cold-drawn stainless steel tubing silver-soldered to B); G, stainless steel tip (3.2 mm 0.d. x 1 mm i.d.); H, teflon ferrule; I, 3.2 mm Swagelok nut (fits g.c. collection port); J, stainless steel capillary trap guide (2.2 mm 0.d. x 1.7 mm Ld.); K, 13 mm diam A1 rod partially immersed in liquid nitrogen; L, teflon tubing; M, capillary trap (100 p1 Microcap).

Capillary crusher

-60-- 9 -I

K Collection adaptor

84 H. Young et al.

2.6. Freon extraction of the concentrate The fraction 1 aqueous concentrate (40 ml) was extracted in a liquid-liquid extractor with redistilled (2 x ) Freon 114 (b.p. 3.5”C) (20 ml). The concentrate was cooled in ice and the condenser was cooled with 0°C coolant. After 4 h the Freon was distilled off through a fractionating column packed with Fenske helices, by allowing the flask to come slowly to ambient temperature. When the volume had been reduced to less than 1 ml the extract was cooled to 0°C and transferred to a 1 ml Reacti-vial (Pierce Chemical Co., USA).

A packed (Fenske helices) fractionating column (100 mm x 9 mm i.d.) was attached to the Reacti- vial and the remainder of the Freon distilled off by allowing the vial to slowly warmtoroom tempera- ture. Traces of the volatile components were detected (by nose) at the top of the fractionating column.

2.7. Identification of the components All the identified peaks (Figure 1) were checked for homogeneity by trapping the individual peaks separated on the Carbowax column in stainless steel traps packed with Chromosorb 105 and analysing the collected material by g.c.-m.s. on the OVlOl WCOT column. Whenever possible the identification was confirmed by direct comparison with authentic standards. Where standard compounds were not available the identifications were verified by chemical transformation using micro-reaction g.c. and high resolution mass spectrometry.

3. Results and discussion

The actual time of sampling kiwifruit for collection of volatile components is critical. The soluble solids content (74%) of the fruit at harvest was within the recommended range (7-10%) for harvest maturity of kiwifruit intended for long term storage (longer than 4-5 month^).^ Once it has reached eating ripeness kiwifruit has a short shelf life. At ambient temperature it changes within 4-5 days from a pleasant tasting fruit with a delicate aroma to one that has a marked ‘estery’ aroma which many consumers find objectionable. The fruit used in this study was considered optimal for eating; it gave soluble solids and penetrometer readings typical of ripe fruit4 and lacked any of the aromatic notes characteristic of overripe fruit.

The low temperature vacuum distillation method for separating the volatile components from the non-volatile material avoided heating the fruit but gave a dilute aqueous solution of the volatile compounds. Attempts to concentrate the dilute solution by further vacuum distillation5 gave a very low concentration ratio. Freeze concentration6 on the other hand readily gave 8-fold concentration of the pooled fraction 1 distillate before liquid occlusion in the ice layer started to occur. Further concentration of this fraction prior to g.c. and g.c.-m.s. analysis was not necessary when the Chromo- sorb 105 cartridges were used for sample introduction. The combined fraction 2 distillate, which was more dilute, was readily freeze concentrated 66 fold. The distillate before concentration had an aroma different from that of the fresh fruit but it was definitely identifiable with kiwifruit. After freeze concentrating, re-dilution with water was necessary to reproduce the characteristic aroma.

A chromatogram of the concentrated fraction 1 distillate obtained on a Carbowax 20M SCOT column is shown in Figure 1. The most prominent peak is that due to trans hex-2-enal.

G.c.-m.s. analysis and ozonolysis (production of n-butanal and COz) of i showed that it isa hex-2- enal. Since j has been identified as trans hex-2-enal by comparison with an authentic sample using mass spectrometry, g.c. retention time and ozonolysis (production of n-butanal and COZ), i must be the cis isomer. Because of the broadness of peak i there was the possibility that it was an artifact caused by overloading of the column by trans hex-2-enal ( j ) . However, when trapped a rechromato- graphed i gave a sharp peak, with a different retention time to that of trans hex-2-enal.

As expected, g.c. analysis of the fraction 2 concentrate which was collected after longer distillation, gave a chromatogram with higher proportions of the less volatile compounds. No additional compounds were detected.

The unidentified low intensity peaks eluting before ethyl acetate on the Carbowax column are

Aroma constituents of kiwifruit 85

probably due to contaminants from the trap or N2 gas since the intensities relative to ethyl acetate, varied from run to run.

The chromatogram of the Freon extract was contaminated with halogenated compounds of low retention time but at the longer retention time it was similar to that obtained using the Chromosorb 105 packed cartridges. Significantly, no new peaks were found at the longer retention time.

When kiwifruit pulp was mashed with sufficient NaC15’ to saturate the final mixture with salt, the distillate obtained gave a chromatogram similar to that of other distillates.

In one series of experiments the column effluent was continuously monitored by nose at the g.c. collection port. The most intense olfactory responses were obtained during the elution of ethyl butanoate (sweet, estery aroma), hexanal (intense grassy aroma) and trans hex-2-enal (grassy to almond-like aroma depending on concentration). There was only one region in the chromatogram in which the eluted compound was not in sufficient quantity to give a FID response but had an aroma attributable to kiwifruit. This compound (or compounds) which was eluted on the tail of the methyl butanoate peak had an intense ‘grassy’ odour characteristic of freshly sliced kiwifruit. On the basis of sensory assessment of freshly cut fruit the level of this compound appeared to vary with the ripeness of the fruit. Further work is in progress to isolate and to identify this compound. Ethyl butanoate and hexanal, two compounds with low olfactory threshold7 contributed more to the kiwifruit aroma than did trans hex-2-ena1, despite its abundance.

The compounds identified in the kiwifruit distillate are all simple compounds frequently found in a wide range of fruit, e.g. passionfruit,5 apple,7 f e i j ~ a ~ , ~ and plumlo. No terpenoid compounds were found.

In conclusion, although ethyl butanoate, hexanal, and trans hex-2-enal have been shown to be important components of kiwifruit aroma, the compound detected on the tail of the methyl buta- noate peak has still to be identified. When this is done a better idea of the importance of each of the volatile constituents in the overall aroma will be obtained.

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Stanley, G. ; Kennett, B. Reaction gas chromatography of microgram and sub-microgram samples using sealed capillaries. J. Chromatogr. 1973, 75, 304-307. Harrnan, J. E. Kiwifruit maturity. Orchardist N.Z. 1981, 54, 126-130. Murray, K. E.; Shipton, J.; Whitfield, F. B. The chemistry of food flavour. 1. Volatile constituents of passion- fruit, Passifloru edulis. Aust. J . Chem. 1972, 25, 1921-1933. Weurman, C. Isolation and concentration of volatiles in food odour research. J. Agric. Food Chem. 1969, 17,

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