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Application of headspace solid-phase microextraction to volatile flavour profile development during storage and ripening of kiwifruit
Food Research International 32 (1999) 175±183
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Application of headspace solid-phase microextraction to volatile ¯avour pro®le development during storage and ripening of kiwifruit X.M. Wan a,1, R.J. Stevenson a,b,1, X.D. Chen b, L.D. Melton a,*,1 a Department of Chemistry, The University of Auckland, New Zealand Food Science and Process Engineering Group, Department of Chemical and Materials Engineering, The University of Auckland, Private Bag 92019, Auckland, New Zealand
b
Received 7 October 1998; accepted 30 April 1999
Abstract Kiwifruit volatile ¯avour compounds were evaluated with headspace solid-phase microextraction (SPME) as a sample concentration technique. Gas chromatography±mass spectrometry (GC±MS) was used for qualitative and semi-quantitative analysis after SPME. Components such as heptanal, ethyl hex-3-enoate, 6-methylhept-5-en-2-one, acetic acid, c/t-2-nonenal (mixture) and c/ t-2-decenal (mixture), which were previously found only in kiwifruit juice, were detected by headspace SPME±GC±MS. Other compounds (pent-4-enal, t,t-nona-2,4-dienal, 2-nonanone, ethyl octanoate, butyrolactone and 2-propenyl butanoate), which had not been found previously, were identi®ed. Flavour volatiles of kiwifruit were very sensitive to storage time and state of ripeness. # 1999 Published by Elsevier Science Ltd on behalf of the Canadian Institute of Food Science and Technology. All rights reserved. Keywords: Kiwifruit; Storage; Ripening; Solid-phase microextraction; Volatile ¯avour compounds
1. Introduction The kiwifruit, a native of China which was ®rst developed commercially in New Zealand, is now a commercial crop in many countries. Numerous eorts have been made to characterise the ¯avour pro®les of fresh kiwifruit grown in dierent countries (Bartley & Schwede, 1989; Cossa, Trova & Gandolfo, 1988; Shiota, 1982; Takeoka, Guntert, Flath, Wurtz & Jennings, 1986; Young & Paterson, 1985; Young, Stec, Paterson, McMath & Ball, 1995). Frozen kiwifruit puree (Pfannhauser, 1987) and kiwifruit juice (Young et al., 1992) have also been investigated. Signi®cant components found by these groups were methyl and ethyl butanoate, hexanal, t-hex2-enal, 1-hexanol, t-hex-2-enol, c/t-hex-3-enol, all of which are typical degradation products of unsaturated fatty acids (Winterhalter, 1991). * Corresponding authors Tel.: +64-9-373-7599 ext. 6658 or ext. 2875; fax: +64-9-373-7463. E-mail addresses: [email protected] (R.J. Stevenson), [email protected] (L.D. Melton) 1 Member of the Food Science Postgraduate Program, Department of Chemistry, The University of Auckland.
Three analytical approaches have been applied for isolating and concentrating volatile components of kiwifruit ¯avours: simultaneous distillation and extraction (Shiota, 1982), vacuum-distillation (Takeoka et al., 1986; Young, Paterson & Burn, 1983) and dynamic headspace sampling (Bartley & Schwede, 1989; Young & Paterson, 1985). Solid-phase microextraction (SPME) was ®rst developed mainly for analysis of pollutants in environmental water samples (Arthur & Pawliszyn, 1990; Belardi & Pawliszyn, 1989). A SPME unit consists of a length (1±2 cm) fused-silica ®bre, coated with a stationary phase such as polydimethylsiloxane, polyimide, polyacrylate, Carbowax-divinylbenzene, polydimethylsiloxane-divinylbenzene, Carboxen-polydimethylsiloxane or pencil lead (Wan, Chi, Wang & Mok, 1994), and bonded to a stainless steel plunger and a holder. The ®bre is sheathed in a syringe needle tube, which is used to pierce the septum of a sample container. When sampling, the ®bre is extended from its sheath and exposed to the headspace or directly to the sample matrix to extract analytes onto the coating. Then the ®bre, with the concentrated analytes, is transferred to a gas chromatograph (GC) injection port for desorption, followed by separation, identi®cation and quantitation.
0963-9969/99/$20.00 # 1999 Published by Elsevier Science Ltd on behalf of the Canadian Institute of Food Science and Technology. All rights reserved. PII: S0963-9969(99)00074-5
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The principle of SPME is based on the adsorption of analytes onto the phase-coated fused silica ®bre and the partitioning of analytes between the stationary phase of the ®bre and the extraction medium (sample matrix). During SPME sampling, exhaustive extraction does not occur but equilibrium is established as analytes partition between the sample matrix and the stationary phase. For SPME±GC analysis, cryogenic focusing is often used to improve peak resolution. Yang and Peppard (1994) found that the internal diameter of the GC injection liner can in¯uence the peak width, especially for eluting compounds. SPME has been recently applied to the analysis of volatile ¯avour compounds in foods (Page & Lacroix, 1993; Yang & Pepard, 1994) and beverages (Constant & Collier, 1997; Gandini & Riguzzi, 1997; Hawthorne, Miller, Pawliszyn & Arthur, 1992; Yang M.J., Opton & Pawliszyn, 1997) such as dairy products (Mariaca & Bosset, 1997; Stevenson & Chen, 1996; Stevenson, Chen & Mills, 1996), apples, strawberry, sour cherry and tomato (Matich, Rowan & Banks, 1996; Paliyath, Whiting, Stasuak, Murr & Clegg, 1997; Song, Gardner, Holland & Beaudry, 1997; Ulrich, Krumbein & Rapp, 1997). Quantitative information on the ¯avour volatile development during kiwifruit ripening is limited. The objective of the present study was to apply SPME combined with GC±MS to identify the headspace volatile ¯avour compounds of kiwifruit, and to evaluate the evolving pro®le of kiwifruit volatile ¯avour compounds after storage followed by ripening. 2. Materials and methods 2.1. Materials Kiwifruit (Actinidia deliciosa (A. Chevalier) C. F. Liang et A. R. Ferguson Var deliciosa cv Hayward) were harvested from the South Auckland region (New Zealand) on 9 May, 1997. Soluble solids concentration (SS) was approximately 8% and ®rmness was approximately 8 kgf at harvest. 2.2. Design of experiments The ¯avour pro®les of the freshly picked fruit (storage time=0) and those after storage of 1, 2, 3 and 5 months were analysed. Firmness and SS were measured before and after ripening, after each period of storage. 2.3. Experimental methods 2.3.1. Storage Kiwifruit (500) were loosely packed inside a conventional modular bulk container with a polyliner and stored at 0 C and 88±90% relative humidity. The humidity inside the polyliner was approximately 95%.
The air was exchanged with fans four times daily to remove ethylene. 2.3.2. Evaluation of ripening On removal from storage, 60±80 kiwifruits were repacked in standard kiwifruit trays, which consisted of an outer case of cardboard, a preformed plastic pocket tray-pack and a polyliner. The repacked kiwifruit were held at 20 C. Paterson, MacRae and Young (1991) reported that the ``eating-ripe'' range of kiwifruit ®rmness was 0.4±1.0 kgf, whereas McDonald (1990) used 0.50.8 kgf. Young and Paterson (1985) used the terms ripe, ``under-ripe'', and ``over-ripe'' to describe fruit softened to 0.5±0.6 kgf, 0.8±1.1 kgf, and 0.3±0.4 kgf, respectively. Since it was dicult in practice to control the ®rmness between 0.5±0.6 kgf and as the number of kiwifruit were limited, a ripeness standard of 0.6±0.8 kgf was chosen. 2.3.3. Measurements of ®rmness Penetrometers Models FT 327 and FT 011 (Eegi, Italy) were used to measure ®rmness. After each storage period of 0, 1, 2, 3 and 5 months, 10 fruits were randomly selected from 60 to 80 fruits. The ®rmness of all fruits were measured twice, once on each side (Fig. 1). The skin was removed using a slicer, of which the blade in the centre was set to a height of 1 mm, from the area to be tested (about 20±25 mm diameter) midway between the stem and calyx ends of the fruits. The fruits were placed on a hard surface and held ®rmly during testing (Watkins & Harman, 1981). 2.3.4. Measurements of soluble solids (SS) A refractometer Model ATC-1 E (Brix 0±32%, ATAGO, Japan) was used for SS measurements. The stem and calyx ends of the kiwifruit were cut o approximately 1.5 cm away from each end at right angles to the length axis. In order to obtain an average reading for each fruit, both ends were used for SS measurements. The same 10 fruits used for ®rmness measurements, which were randomly selected from 60 to 80 fruits after each storage period of 0, 1, 2, 3 and 5 months, were used for SS measurements before ripening (Fig. 2). The remaining fruits (about 50±70) were ripened (method described above). Eighteen fruit ripened to ®rmness of 0.6±0.8 kgf were selected from these 50±70 fruits and used for SS measurements after ripening. 2.3.5. Sampling method for headspace SPME±GC±MS After storage of 1, 2 and 3 months, 12 fruits ripened to 0.6±0.8 kgf ®rmness on the same day (ripening times were 8, 6 and 5 days, respectively) were selected from the 50±70 fruits of each group and used for SPME±GC± MS analysis. For fruits stored for 5 months, 18 fruits (0.6± 0.8 kgf ®rmness) ripened over 3 days were selected and used for SPME±GC±MS analysis. Results of SPME±GC±MS analysis from these four groups were used to compare
R.J. Stevenson, / Food Research International 32 (1999) 175±183
177
Fig. 1. Change of kiwifruit ®rmness during storage.
Fig. 2. Comparison of the SS changes before and after ripening during storage (error bars indicate RSD's): BR, before ripening; AR, after ripening.
the trends in development of kiwifruit volatile ¯avour compounds during storage. SPME±GC±MS analysis was also carried out on another 12 ripe fruit (0.6±0.8 kgf ®rmness) from those stored for 5 months with ripening time of 2 days. These results were compared with those of which the ripening time was 3 days to study the in¯uence of the ripening time on the volatile ¯avour compounds.
The fruits were sliced into four parts and the skin was discarded. Pieces (20±22 in number; 250 g) which had been chosen randomly were placed in a round-bottom ¯ask (500 ml) immersed in a water bath (25 C). A glass beaker (5 ml) was placed on the surface of the chopped kiwifruit. A glass tube (1 ml) containing 2-octanone (800 ml of a 50 mg/ml solution in water±methanol) as an internal standard was placed in the beaker. The ¯ask
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was then tightly closed with a screw-top quick®t adaptor and Te¯on-coated silicone septum. For the fruits stored for 5 months, and ripened for 3 days, triplicate sampling runs (each with six fruits) were carried out. Duplicate sampling runs (each with six fruits) were performed for the remaining three storage periods. A number of the kiwifruit stored for 5 months were allowed to become ``over-ripe'' [®rmness 0.3±0.4 kgf, as de®ned by Young and Paterson (1985)]. Sampling was performed separately on ``over-ripe'' kiwifruit (5±6 days ripening) sliced into four parts with the skin discarded (250 g), and ``over-ripe'' macerated kiwifruit (250 g). A portion of six fruit were used for each sampling. Macerated kiwifruit were prepared by blending in a blending device for 4 s, four times. The sampling method, GC±MS analysis and estimation of the relative amounts of compounds were as for other groups of fruit. The sample-containing ¯ask was ®rst held in the water bath (25 C) for 30 min to allow equilibrium between the fruit sample and the headspace, prior to SPME sampling. The SPME manual device (Supelco Co., Bellefonte, PA, USA) was equipped with a fused-silica ®bre coated with Carbowax-divinylbenzene (65 mm). The ®bre was inserted into the sample container through the septum, and exposed to the headspace for 30 min. 2.3.6. GC±MS analysis Volatile compounds adsorbed on the SPME ®bre were thermally desorbed in the injector port of the GC and then separated on the GC column. The desorption time was 5 min. GC±MS analysis was performed in a Shimadzu GC-17A GC/Shimadzu QP-5000 quadruple MS system (Shimadzu, Kyoto, Japan). Separation was achieved on a 30 m0.25 mm id. fused-silica capillary column coated with cross-linked polyethene glycol 20 M, ®lm thickness 0.25 (m (DB-Wax; J and W Scienti®c, Folsom, CA, USA). The column temperature was held at 27 C for 6 min, then increased to 135 C (at 3 C/min), then to 230 C (at 6 C /min), ®nally held at 230 C for 5 min. The injection port (splitless mode) and detector temperatures were 220 and 230 C, respectively. Helium (column ¯ow rate 0.8 ml/min) was the carrier gas. Cryogenic focussing was performed by immersing about 4 cm of the front of the column in dry ice (ÿ80 C) for the ®rst 6 min of the temperature program. Compounds were identi®ed by matching mass spectra with NIST62 library of standard compounds. When available, MS identi®cations were con®rmed by comparing GC retention times with authentic compounds. 2.3.7. Calculation method of the relative amounts of compounds For each sampling, the same amount of kiwifruit (250 g), the same ¯ask, the same amount of internal standard (800 ml of a 50 mg/ml solution of 2-octanone), equilibrium time, and SPME headspace sampling time were
applied (the SPME ®bre was conditioned at 220 C for 20 min before each sampling). The relative amount of a speci®c compound was de®ned as: the height of the compound GC peak divided by the height of the internal standard GC peak, then times 100%, or the height of the compound selected ion monitoring (SIM) GC peak divided by the intensity of the internal standard SIM peak, then times 100%. Under such conditions, it was possible to compare the relative changes in concentration of certain compounds during storage. Relative standard deviations (RSD) were included with average peak heights for samplings carried out in triplicate but were not calculated for average peak heights for samplings carried out in duplicate. 3. Results and discussion 3.1. Firmness Fig. 1 demonstrates that the kiwifruit ®rmness decreased with increasing storage time. Firmness declined considerably during the ®rst two months of storage; it then decreased very slowly. Previous work (McDonald, 1990) described a similar pattern of kiwifruit ®rmness change. 3.2. Soluble solids measurements The changes of SS before (immediately after storage) and after ripening have been compared (Fig. 2). During storage, the SS before ripening increased with time, indicating that the fruit ripened slowly during storage. Large changes occurred during the ®rst two months and then the SS levels remained nearly constant. Interestingly, the SS after ripening (ripened to ®rmness of 0.6±0.8 kgf) decreased with increasing storage time. In the ®rst two months the levels decreased rapidly, then slightly or not at all. The dierence between the SS before ripening and after ripening was at ®rst easily apparent, then became smaller, and eventually negligible (Fig. 2). This is possibly because the freshly harvested fruit (storage time=0 month) took approximately 8 weeks to ripen to ®rmness 0.6±0.8 kgf, and the fruit stored for 1, 2, 3 and 5 months took 8±9, 5±6, 2±5 and 2±3 days, respectively, to ripen to ®rmness 0.6±0.8 kgf. The longer the ripening time, the greater the dierence of SS between before ripening and after ripening. 3.3. Headspace SPME±GC±MS analysis 3.3.1. Flavour volatile compounds in kiwifruit Typical headspace SPME±GC±MS pro®les of the kiwifruit volatiles at ripe and ``over-ripe'' stages are shown in Fig. 3. The peak numbering refers to the compounds in Table 1., which lists 42 ¯avour volatile
R.J. Stevenson, / Food Research International 32 (1999) 175±183
179
Fig. 3. Typical headspace SPME-GC-MS pro®les of (a) ripe (®rmness of 0.6±0.8 kgf) kiwfruit (stored for 5 months prior to ripening); (b) macerated ``over-ripe'' kiwfruit (stored for 5 months prior to ripening) (i, internal standard; h, hydrocarbon).
compounds (compound 26 was identi®ed as a mixture of c/t-hex-3-en-1-ol). The results can be compared with those found by dynamic headspace sampling methods of Young and Paterson (1985) and Bartley and Schwede (1989), who identi®ed 26 and 27 compounds, respectively. Except for pent-4-enal, t,t-nona-2,4-dienal, 2nonanone, ethyl octanoate, butyrolactone and 2-propenyl butanoate, the other 33 ¯avour volatiles have been reported previously. Of these 33 ¯avour volatiles, 23 compounds were identi®ed in the headspace of macerated kiwifruit using dynamic headspace sampling techniques (Bartley & Schwede, 1989; Young & Paterson, 1985). Twenty-two compounds were identi®ed by Takeoka et al. (1986), using a vacuum-distillation method. Other compounds (heptanal, ethyl hex-3-enoate, 6methylhept-5-en-2-one, acetic acid, c/t-2-nonenal and c/t2-decenal) have been detected only in kiwifruit juice (Young & Perara, 1992). There were 12 compounds (ethyl ethanoate, methyl butanoate, pent-1-en-3-one, ethyl butanoate, hexanal, c-hex-2-enal, t-hex-2-enal, 1-hexanol, c/t-hex-3-en-1-ol, t-hex-2-en-1-ol and methyl benzoate) commonly found by Young et al. (1983), Young and Paterson (1983), Bartley and Schwede (1989) and Takeoka et al. (1986). All of them, except c-hex-2-enal, were detected by headspace SPME±GC±MS. Of the 19 compounds (ethyl ethanoate, methyl propanoate, ethyl propanoate, methyl butanoate, pent-1-en-3-one, ethyl butanoate, hexanal, propyl butanoate, ethyl pentanoate,
methyl hexanoate, c-hex-2-enal, t-hex-2-enal, ethyl hexanoate, 1-hexanol, c/t-hex-3-en-1-ol, t-hex-2-en-1-ol, methyl benzoate and ethyl benzoate) commonly found by Young and Paterson (1985) and Bartley and Schwede (1989) in the headspace of macerated kiwifruit, 18 (except c-hex-2-enal which was present in very low amount) were detected by headspace SPME±GC±MS. The compounds labelled with * in Table 1 (pent-4-enal, t,t-nona-2,4-dienal, 2-nonanone, ethyl octanoate, butyrolactone and 2-propenyl butanoate) have not been previously reported in kiwifruit. 3.3.2. The development of volatile ¯avour pro®les during storage Table 2 lists the ¯avour volatiles found in ripe kiwifruit during storage and their relative amounts. It is thought that the main contributors to kiwifruit aroma (Paterson et al., 1991; Young et al., 1992; Young et al., 1995) were methyl and ethyl butanoate, hexanal, t-hex-2-enal, 1hexanol, t-hex-2-en-1-ol and c/t-hex-3-en-1-ol. Fig. 4 shows the evolution of the main ¯avour components of ripe kiwifruit after storage for various time periods. It demonstrates an increasing trend with all the above important aroma components (although methyl butanoate increased initially, then decreased from 3 to 5 months) during storage. During the ®rst 2 months, the changes were slight or negligible. From 2 to 3 months, the enrichments were more substantial. Other volatiles listed
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R.J. Stevenson, / Food Research International 32 (1999) 175±183
Table 1 Identi®cation of volatile ¯avour compounds in a typical headspace SPME±GC±MS pro®le of ``over-ripe'' kiwifruit
Table 2 Volatile ¯avour compounds developed during storage in the headspace SPME±GC±MS pro®les of kiwifruit
Peaka
No. of Compounda
1 month (2)/b
2 months (2)
3 months (2)
5 months (3)
2 4 5 8 10 11 20 25 26 28 32 35 38
11.75c 43.66 7.39 12.49 34.21 2.17 26.17 21.40 0.52 9.10 0.64 ud 4.04
udd 48.60 ud 19.62 12.17 2.90 22.72 20.10 1.40 9.51 1.01 2.73 20.96
ud 46.42 ud 74.86 35.33 5.50 54.77 53.08 1.85 28.04 1.93 3.31 20.93
72.1012.78e 98.8516.71 27.5412.39 39.6615.39 178.8487.27 7.121.49 93.7230.82 66.5716.52 3.940.32 41.4714.89 2.470.38 5.002.52 11.043.31
b
1 c d e f 2y1 ;y2 ;b ;t y2,b,t 3 4y1,y2 5y2,b,t 6y2,t 7t 8y1,y2b,t 9y1,y2,b,t 10y1,y2,b,t 11y1,y3,b,t 12y2,t 13g 14y2,b 15y1,y2,t 16y2,b 17 18y2,b 19 20y1,y2,b,t 21y2,b,t 22 23t 24 25y1,y2,b,t 26y1,y2,b,t 27 28y1,y2,b,t 29 30 31T 32t 33 34y1,y2,b,t 35 36 37y2,b,t 38
Compounds 2-Propanone Ethyl acetate Methyl propanoate Ethanol Ethyl propanoate Ethyl 2-methylpropanoate Pentanal Methyl butanoate Pent-1-en-3-one Ethyl butanoate Hexanal Methyl pentanoate Pent-4-enal Propyl butanoate t-pent-2-enalh Ethyl pentanoate Heptanal Methyl hexanoate t; t-Nona-2,4-dienal t-Hex-2-enal Ethyl hexanoate Ethyl hex-3-ennoate c=t-Hept-3-enali 6-Methylhept-5-en-2-one 1-Hexanol c=t-Hex-3-en-1-ol 2-Nonanone t-Hex-2-en-1-ol Ethyl octanoate Acetic acid t; t-Heota-2,4-dienal Benzaldehyde c=t-2-nonenal Methyl benzoate Butyrolactone c=t-2-Decenal Ethyl benzoate 2-Propenyl butanoate
RT (min) 9.02 10.22 10.53 11.21 11.52 11.38 12.07 12.29 13.22 13.85 15.22 15.42 16.15 16.68 16.95 17.11 18.86 18.89 20.19 20.55 20.89 24.07 25.14 25.85 25.51 26.73 28.06 29.24 29.99 31.03 31.38 33.56 34.31 37.71 36.51 38.83 39.55 47.08
Identi®cation b
MS,RT MS,RT MS,RT MS,RT MS,RT MS MS MS,RT MS MS,RT MS,RT MS,RT MS MS,RT MS MS MS MS,RT MS MS,RT MS,RT MS MS MS MS,RT MS,RT MS,RT MS,RT MS MS MS MS,RT MS MS,RT MS MS MS,RT MS
a
Peak number in Fig. 4. MS, mass spectrometry; RT, retention time. c y1, detected by Young et al. (1983) using vacuum-distillation sampling. d y2, detected by Young and Paterson (1985) using dynamic headspace sampling. e b, detected by Bartley and Schwede (1989) using dynamic headspace sampling. f t, detected by Takeoka et al. (1986) using vacuum-distillation sampling. g compounds not previously reported in kiwifruit. i c, cis. h t, trans. b
in Table 2 such as ethanol, benzaldehyde and butyrolactone showed a similar trend. Young and Paterson (1985) showed that fruit harvested at maturity 8.0 Brix (SS 8%, same as the fruit at harvest used in this study) had slightly less total volatile ¯avours than those after 9 weeks (about 2 months) storage, although the level of total volatile ¯avours
a
Compound's number in Table 1. Figures in parentheses indicate the number of sampling replications. c Relative peak heights expressed as means. d ud, undetected. e Relative peak heights expressed as ``meanRSD''. b
Fig. 4. Evolution of important kiwifruit volatile ¯avour compounds during storage. A: methyl butanoate; B, ethyl butanoate; C, hexanal; D, t-hex-2-enal; E, 1-hexanol; F, cis- and trans-hex-3-en-1-ol; G, t-hex2-en-1-ol.
decreased after 21 weeks (about 5 months) storage. The changes observed between 1 and 2 months in the present work were comparable with their results. However, in contrast to Young and Paterson, it was found that after 5 months, the total volatiles were still increasing, which may be due to the use of a dierent ripening method. In this work, the fruit were covered with polyliner packed in a standard tray and ripened in an incubator set at 20 C, while the above workers triggered the ripening with Ethrel at 22 C. A ®rmness of 0.6±0.8 kgf was used as the ripe standard, while they used ®rmness of 0.5±0.6 kgf. In their research, the ripening time for the fruit stored 9 and 21 weeks prior to ripening was 4 and 2 days, respectively. In our study, the ripening time for the fruit stored for 2 and 5 months were 6 and 3 days, respectively. As discussed by Young and Paterson (1985), the level of kiwifruit volatile ¯avour compounds
R.J. Stevenson, / Food Research International 32 (1999) 175±183
was related to the storage time prior to ripening as well as ripeness. Unlike apples and oranges, the levels of kiwifruit ¯avour volatiles are tremendously sensitive to ripeness, ®rmness and storage (Young et al., 1995). Large variations in levels of compounds are found between individual fruit in any one batch. Informal sensory evaluation showed that individual fruit, even from the same batch, tasted quite dierent. Generally, the ``under-ripe'' fruit had a green grassy note which was possibly due to thex-2-enal, while the more ripe fruit had a sweet and fruity ¯avour which was attributed to butanoate esters (Young et al., 1983). In the current research, each sampling was carried out with a portion of at least six fruit in an attempt obtain statistically meaningful results. 3.3.3. The in¯uence of ripening time on the kiwifruit volatile ¯avour compounds As discussed above, the quantity of ¯avour volatiles was in¯uenced by both storage time and stage of ripeness. Bartley and Schwede (1989) also showed that the ripening time aected the levels of kiwifruit volatile ¯avour compounds. During our study, it was found that dierent ripening times resulted in dierent levels of ¯avour volatiles among the fruit stored for the same time prior to ripening (®rmness 0.6±0.8 kgf). Twelve fruit (5 months storage) with a ripening time of 2 days were compared with 18 fruit (5 months storage) with a ripening time of 3 days. Semi-quantitative headspace SPME±GC±MS results of fruit with dierent ripening times are listed in Table 3 and comparisons of main component levels are illustrated in Fig. 5. There were dramatic increases in the amounts of esters, especially
Table 3 Volatile ¯avour compounds in the headspace SPME±GC±MS pro®le of kiwifruit (stored for 5 months) with dierent ripening times No. of compounda
Ripening time 2 days (2)b
Ripening time 3 days (3)
2 4 5 8 10 11 20 25 26 28 32 35 38
15.69c 102.03 10.17 2.83 11.36 7.88 100.83 65.52 4.61 48.78 3.81 8.47 10.99
72.1012.78d 98.8516.71 27.5412.39 39.6615.39 178.8487.27 7.121.49 93.7230.82 66.5716.52 3.940.32 41.4714.89 2.470.38 5.002.52 11.043.31
a b c d
Compound's number in Table 1. Figures in parentheses indicate the number of sampling replications. Relative peak heights expressed as means. Relative peak heights expresed as ``meanRSD''.
181
Fig. 5. Comparison of important kiwifruit volatile ¯avour compounds with ripening times of 2 days and 3 days. A, methyl butanoate; B, ethyl butanoate; C, hexanal; D, t-hex-2-enal; E, 1-hexanol; F, cis- and trans-hex-3-en-1-ol; G, t-hex-2-en-1-ol.
ethyl butanoate, between the kiwifruit samples with a ripening time of 2 days and those of 3 days. 3.3.4. The pro®les of ``over-ripe'' kiwifruit volatile ¯avour compounds Fig. 3(b) shows a typical headspace SPME±GC±MS pro®le of volatiles from macerated ``over-ripe'' kiwifruit. Table 4 lists the compounds identi®ed by headspace SPME-GC-MS and their relative amounts of chopped ``over-ripe'' kiwifruit and macerated ``overripe'' kiwifruit. Tables 3 and 4 demonstrate that in the kiwifruit stored for 5 months prior to ripening, more ¯avour compounds were found in the ``over-ripe'' fruit than in ripe fruit. The largest increases were esters such as methyl propanoate, propyl butanoate, methyl pentanoate, ethyl pentanoate, methyl hexanoate, ethyl hexanaote, methyl benzoate and ethyl benzoate. These results agree with those of Young and Paterson (1985). Table 4 indicates more compounds (pent-4-enal, t-pent2-enal, c/t-hept-2-enal, 6-methylhept-5-en-2-one, 2-nonanone, ethyl octanoate, t,t-hepta-2,4-dienal, c/t-2-nonenal and c/t-2-decenal) were found in the macerated kiwifruit than in the chopped kiwifruit pieces. The amounts of aldehydes such as hexanal, t-hex-2-enal, pent-4-enal, tpent-2-enal, c/t-hept-2-enal, t,t-hepta-2,4-dienal, c/t-2nonenal and c/t-2-decenal also increased. This was probably due to more ¯avour volatiles being released from the cells after the fruit had been macerated. However, the amounts of alcohols changed only slightly or not at all and the amounts of some esters decreased. It is possible that the additional experimental step (blending the fruit and transferring them from the vegetable blender to the glass ¯ask for sampling) resulted in liberation of some of the ¯avour volatiles. Reproducibility appeared to be better in the macerated fruit than in the fruit pieces. Headspace SPME sampling is sensitive to sample volume/headspace volume ratio and the distance from the ®bre to the surface of the sample (Matich et al., 1996). The surface and the headspace volume of the macerated fruit were more controllable.
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Table 4 Volatile ¯avour compounds in headspace SPME±GC±MS pro®les of ``over-ripe'' chopped kiwifruit and macerated kiwifruit No. of compounda
c
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 a b c d e f
Chopped kiwifruit (2)b tr 778.32d tr 331.19 25.49 7.36 udf 621.69 ud 2665.73 3.39 tr tr tr ud 22.37 ud 21.11 ud 24.76 124.91 7.83 ud ud 30.32 1.30 ud 8.94 ud 3.53 ud 0.27 ud 9.10 3.01 ud 27.55 9.43
Macerated kiwifruit (3) tr 520.4015.93e 2.5825.05 210.9313.28 39.0514.28 2.6510.86 7.5521.76 434.1026.02 21.3420.84 1414.387.30 67.4628.04 1.4225.64 4.6913.22 2.7028.34 13.3915.39 11.8016.29 6.9226.46 26.7730.60 7.2219.98 298.8423.34 66.3317.12 3.3929.90 7.1915.56 tr 23.8529.33 0.2123.16 4.6738.49 25.2718.42 3.4418.27 5.0340.44 3.3732.54 0.7245.97 4.7514.22 9.911.86 1.0918.01 1.758.14 24.8243.65 ud
Compound's number in Table 1. Figures in parentheses indicate the number of sampling replications. tr, trace amounts. Relative peak heights expressed as means. Relative peak heights expressed as ``meanRSD''. ud, undetected.
4. Conclusions Headspace SPME is a complementary sampling technique for kiwifruit volatile ¯avour compound analysis and can be operated in parallel with and is more convenient than vacuum-distillation and dynamic headspace sampling. A number of compounds (pent-4-enal, t,t-nona-2,4dienal, 2-nonanone, ethyl octanoate, butyrolactone and 2-propenyl butanoate) were reported for the ®rst time in kiwifruit. Other components detected by headspace SPME±GC±MS, i.e. heptanal, ethyl hex-3-enoate, 6methylhept-5-en-2-one, acetic acid, c/t-2-nonenal and c/
t-2-decenal, have been found before only in the juice of kiwifruit (Young et al., 1992). It has been demonstrated that the volatile ¯avour compounds of kiwifruit are very sensitive to storage time and stage of ripeness. In general, total volatiles increase with with increasing storage time and advancing ripeness. It appears that the headspace SPME± GC±MS pro®les of macerated kiwifruit are more reproducible than those of sliced kiwifruit. Acknowledgements The authors would like to thank Dr. J. Burdon and Dr. H. Young from Horticultural and Food Research Institute, Auckland, for their help in providing kiwifruit and a number of chemicals as volatile standards. References Arthur, C. L., & Pawliszyn, J. (1990). Solid phase microextraction with thermal desorption using fused silca optical ®bers. Analytical Chemistry, 62, 2145±2148. Bartley, J. P., & Schwede, A. M. (1989). Production of volatile compounds in ripening kiwifruit (Actinidia chinensis). Journal of Agriculture and Food chemistry, 37, 1023±1025. Belardi, R. P., & Pawliszyn, J. (1989). The application of chemically modi®ed fused silica ®bres in the extraction of organics from water matrix samples and their rapid transfer to capillary columns. Water Pollution Research in Canada, 24, 179±191. Constant, M., & Collier, J. (1997). Headspace gas chromatography pro®les of fruit-¯avored malt beverages using solid-phase microextraction. Journal of the American Society of Brewing Chemists, 55, 112±118. Cossa, G., Trova, C., & Gandolfo, G. (1988). Estrazione ed identi®cazione dei componenti volatili del kiwi. Industrie Alimentaria, 17, 531±534. Gandini, N., & Riguzzi, R. (1997). Headspace solid-phase microextraction analysis of methyl isothiocyanate in wine. Journal of Agriculture and Food Chemistry, 45, 3092±3094. Hawthorne, S. B., Miller, D. L., Pawliszyn, J., & Arthur, C. L. (1992). Solventless determination of caeine in beverages using solid-phase microextraction with fused-silica ®bres. Journal of Chromatography, 603, 185±191. McDonald, B. (1990). Precooling, storage and transport of kiwifruit. In I. J. Warrington, & G. C. Weston, Kiwifruit: Science and Management (pp. 429±459). Auckland: Ray Richard Publisher. Mariaca, R., & Bosset, J. O. (1997). Instrumental analysis of volatile ¯avour compounds in milk and dairy products. Lait, 77, 13±40. Matich, A. J., Rowan, D. D., & Banks, N. H. (1996). Solid-phase microextraction for quantitative headspace sampling of apple volatiles. Analytical Chemistry, 68, 4114±4118. Page, B. D., & Lacroix, G. (1993). Application of solid-phase microextraction to the headspace gas chromatogaphic analysis of halogenated volatiles in selected foods. Journal of Chromatography, 648, 199±211. Paliyath, G., Whiting, M. D., Stasiak, M. R., Murr, D. P., & Clegg, B. S. (1997). Volatile production and fruit quality during development of super®cial scald in red delicious apples. Food Research International, 30, 95±103. Paterson, V. J., MacRae, E. A., & Young, H. (1991). Relationships between sensory properties and chemical composition of kiwifruit
R.J. Stevenson, / Food Research International 32 (1999) 175±183 (Actinidia deliciosa). Journal of the Science of Food and Agriculture, 57, 235±251. Pfannhauser, W. (1987). Identi®cation of kiwi aroma compounds by GC/MS and sning analysis. In Proceedings of European food chemistry. IV: Rapid analysis in food chemistry (Vol. 1, pp. 86±90). Norwegian Food Research Institute. Shiota, H. (1982). Kiwifruit. Koryo, 137, 59±64. Song, J., Gardner, B. D., Holland, J. F., & Beaudry, R. M. (1997). Rapid analysis of volatile ¯avour compounds in apple fruit using SPME and GC/time of ¯ight mass spectrometry. Journal of Agriculture and Food Chemistry, 45, 1801±1807. Stevenson, R. J., Chen, X. D., & Mills, O. E. (1996). Modern analysis and binding studies of ¯avour volatiles with particular reference to dairy protein products. Food Research International, 29, 265± 290. Stevenson, R. J., & Chen, X. D. (1996). A study of volatile ``trapping'' in spray dried whey protein concentrate by ``crushing'' and/or vacuuming, and detection by solid phase microextraction/gas chromatography/mass spectrometry. Food Research International, 29, 495±504. Takeoka, G. R., Guntert, M., Flath, R. A., Wurtz, R. E., & Jennings, W. (1986). Volatile constituents of kiwifruit (Actinidia chinensis Planch). Journal of Agriculture and Food Chemistry, 34, 576±578. Ulrich, D., Krumbein, A., & Rapp, A. (1997). Analysis of aroma compounds in strawberry, sour cherry and tomato by gas chromatography after solid phase microextraction. Deutsche LebensmittelRundschau, 93, 311±316.
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Wan, H. B., Chi, H., Wong, M. K., & Mok, C. Y. (1994). Solid phase microextraction using pencil lead as sorbent for analysis of organic pollutants. Analytica Chimica Acta, 298, 219±223. Watkins, C., & Harman, J. (1981). Use of penetrometer to measure ®rmness of fruit. The Orchardist of New Zealand, 54, 14±16. Winterhalter, P. (1991). Fruit IV. In H. Maarse, Volatile compounds in foods and beverages, (pp. 389±410). The Netherlands: TNO-CIVO Food Analysis Institute Zeist. Yang, M. J., Orton, M. L., & Pawliszyn, J. (1997). Quantitative determination of caeine in beverages using a combined SPME± GC/MS method. Journal of Chemical Education, 74, 1130±1132. Yang, X., & Peppard, T. (1994). Solid phase microextraction for ¯avour analysis. Journal of Agriculture and Food Chemistry, 42, 1925±1930. Young, H., & Paterson, V. J. (1985). The eects of harvest maturity, ripeness and storage on kiwifruit aroma. Journal of the Science of Food and Agriculture, 36, 352±358. Young, H., Paterson, V.J. & Burn, D.J.W. (1983) Volatile aroma constituents of kiwifruit. Journal of the Science of Food and Agriculture 34, 81±85. Young, H., Perera, C. O., & Paterson, V. J. (1992). Identi®cation of E-hex-3-enal as an important contributor to the o-¯avour aroma in the kiwifruit juice. Journal of the Science of Food and Agriculture, 58, 519±522. Young, H., Stec, M., Paterson, V. J., McMath, K., & Ball, R. (1995). Volatile compounds aecting kiwifruit ¯avour. In R. L. Rousell, & M. Leahy, Fruit ¯avour (pp. 59±67). Washington: American Chemical Society.
www.elsevier.com/locate/foodres
Application of headspace solid-phase microextraction to volatile ¯avour pro®le development during storage and ripening of kiwifruit X.M. Wan a,1, R.J. Stevenson a,b,1, X.D. Chen b, L.D. Melton a,*,1 a Department of Chemistry, The University of Auckland, New Zealand Food Science and Process Engineering Group, Department of Chemical and Materials Engineering, The University of Auckland, Private Bag 92019, Auckland, New Zealand
b
Received 7 October 1998; accepted 30 April 1999
Abstract Kiwifruit volatile ¯avour compounds were evaluated with headspace solid-phase microextraction (SPME) as a sample concentration technique. Gas chromatography±mass spectrometry (GC±MS) was used for qualitative and semi-quantitative analysis after SPME. Components such as heptanal, ethyl hex-3-enoate, 6-methylhept-5-en-2-one, acetic acid, c/t-2-nonenal (mixture) and c/ t-2-decenal (mixture), which were previously found only in kiwifruit juice, were detected by headspace SPME±GC±MS. Other compounds (pent-4-enal, t,t-nona-2,4-dienal, 2-nonanone, ethyl octanoate, butyrolactone and 2-propenyl butanoate), which had not been found previously, were identi®ed. Flavour volatiles of kiwifruit were very sensitive to storage time and state of ripeness. # 1999 Published by Elsevier Science Ltd on behalf of the Canadian Institute of Food Science and Technology. All rights reserved. Keywords: Kiwifruit; Storage; Ripening; Solid-phase microextraction; Volatile ¯avour compounds
1. Introduction The kiwifruit, a native of China which was ®rst developed commercially in New Zealand, is now a commercial crop in many countries. Numerous eorts have been made to characterise the ¯avour pro®les of fresh kiwifruit grown in dierent countries (Bartley & Schwede, 1989; Cossa, Trova & Gandolfo, 1988; Shiota, 1982; Takeoka, Guntert, Flath, Wurtz & Jennings, 1986; Young & Paterson, 1985; Young, Stec, Paterson, McMath & Ball, 1995). Frozen kiwifruit puree (Pfannhauser, 1987) and kiwifruit juice (Young et al., 1992) have also been investigated. Signi®cant components found by these groups were methyl and ethyl butanoate, hexanal, t-hex2-enal, 1-hexanol, t-hex-2-enol, c/t-hex-3-enol, all of which are typical degradation products of unsaturated fatty acids (Winterhalter, 1991). * Corresponding authors Tel.: +64-9-373-7599 ext. 6658 or ext. 2875; fax: +64-9-373-7463. E-mail addresses: [email protected] (R.J. Stevenson), [email protected] (L.D. Melton) 1 Member of the Food Science Postgraduate Program, Department of Chemistry, The University of Auckland.
Three analytical approaches have been applied for isolating and concentrating volatile components of kiwifruit ¯avours: simultaneous distillation and extraction (Shiota, 1982), vacuum-distillation (Takeoka et al., 1986; Young, Paterson & Burn, 1983) and dynamic headspace sampling (Bartley & Schwede, 1989; Young & Paterson, 1985). Solid-phase microextraction (SPME) was ®rst developed mainly for analysis of pollutants in environmental water samples (Arthur & Pawliszyn, 1990; Belardi & Pawliszyn, 1989). A SPME unit consists of a length (1±2 cm) fused-silica ®bre, coated with a stationary phase such as polydimethylsiloxane, polyimide, polyacrylate, Carbowax-divinylbenzene, polydimethylsiloxane-divinylbenzene, Carboxen-polydimethylsiloxane or pencil lead (Wan, Chi, Wang & Mok, 1994), and bonded to a stainless steel plunger and a holder. The ®bre is sheathed in a syringe needle tube, which is used to pierce the septum of a sample container. When sampling, the ®bre is extended from its sheath and exposed to the headspace or directly to the sample matrix to extract analytes onto the coating. Then the ®bre, with the concentrated analytes, is transferred to a gas chromatograph (GC) injection port for desorption, followed by separation, identi®cation and quantitation.
0963-9969/99/$20.00 # 1999 Published by Elsevier Science Ltd on behalf of the Canadian Institute of Food Science and Technology. All rights reserved. PII: S0963-9969(99)00074-5
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The principle of SPME is based on the adsorption of analytes onto the phase-coated fused silica ®bre and the partitioning of analytes between the stationary phase of the ®bre and the extraction medium (sample matrix). During SPME sampling, exhaustive extraction does not occur but equilibrium is established as analytes partition between the sample matrix and the stationary phase. For SPME±GC analysis, cryogenic focusing is often used to improve peak resolution. Yang and Peppard (1994) found that the internal diameter of the GC injection liner can in¯uence the peak width, especially for eluting compounds. SPME has been recently applied to the analysis of volatile ¯avour compounds in foods (Page & Lacroix, 1993; Yang & Pepard, 1994) and beverages (Constant & Collier, 1997; Gandini & Riguzzi, 1997; Hawthorne, Miller, Pawliszyn & Arthur, 1992; Yang M.J., Opton & Pawliszyn, 1997) such as dairy products (Mariaca & Bosset, 1997; Stevenson & Chen, 1996; Stevenson, Chen & Mills, 1996), apples, strawberry, sour cherry and tomato (Matich, Rowan & Banks, 1996; Paliyath, Whiting, Stasuak, Murr & Clegg, 1997; Song, Gardner, Holland & Beaudry, 1997; Ulrich, Krumbein & Rapp, 1997). Quantitative information on the ¯avour volatile development during kiwifruit ripening is limited. The objective of the present study was to apply SPME combined with GC±MS to identify the headspace volatile ¯avour compounds of kiwifruit, and to evaluate the evolving pro®le of kiwifruit volatile ¯avour compounds after storage followed by ripening. 2. Materials and methods 2.1. Materials Kiwifruit (Actinidia deliciosa (A. Chevalier) C. F. Liang et A. R. Ferguson Var deliciosa cv Hayward) were harvested from the South Auckland region (New Zealand) on 9 May, 1997. Soluble solids concentration (SS) was approximately 8% and ®rmness was approximately 8 kgf at harvest. 2.2. Design of experiments The ¯avour pro®les of the freshly picked fruit (storage time=0) and those after storage of 1, 2, 3 and 5 months were analysed. Firmness and SS were measured before and after ripening, after each period of storage. 2.3. Experimental methods 2.3.1. Storage Kiwifruit (500) were loosely packed inside a conventional modular bulk container with a polyliner and stored at 0 C and 88±90% relative humidity. The humidity inside the polyliner was approximately 95%.
The air was exchanged with fans four times daily to remove ethylene. 2.3.2. Evaluation of ripening On removal from storage, 60±80 kiwifruits were repacked in standard kiwifruit trays, which consisted of an outer case of cardboard, a preformed plastic pocket tray-pack and a polyliner. The repacked kiwifruit were held at 20 C. Paterson, MacRae and Young (1991) reported that the ``eating-ripe'' range of kiwifruit ®rmness was 0.4±1.0 kgf, whereas McDonald (1990) used 0.50.8 kgf. Young and Paterson (1985) used the terms ripe, ``under-ripe'', and ``over-ripe'' to describe fruit softened to 0.5±0.6 kgf, 0.8±1.1 kgf, and 0.3±0.4 kgf, respectively. Since it was dicult in practice to control the ®rmness between 0.5±0.6 kgf and as the number of kiwifruit were limited, a ripeness standard of 0.6±0.8 kgf was chosen. 2.3.3. Measurements of ®rmness Penetrometers Models FT 327 and FT 011 (Eegi, Italy) were used to measure ®rmness. After each storage period of 0, 1, 2, 3 and 5 months, 10 fruits were randomly selected from 60 to 80 fruits. The ®rmness of all fruits were measured twice, once on each side (Fig. 1). The skin was removed using a slicer, of which the blade in the centre was set to a height of 1 mm, from the area to be tested (about 20±25 mm diameter) midway between the stem and calyx ends of the fruits. The fruits were placed on a hard surface and held ®rmly during testing (Watkins & Harman, 1981). 2.3.4. Measurements of soluble solids (SS) A refractometer Model ATC-1 E (Brix 0±32%, ATAGO, Japan) was used for SS measurements. The stem and calyx ends of the kiwifruit were cut o approximately 1.5 cm away from each end at right angles to the length axis. In order to obtain an average reading for each fruit, both ends were used for SS measurements. The same 10 fruits used for ®rmness measurements, which were randomly selected from 60 to 80 fruits after each storage period of 0, 1, 2, 3 and 5 months, were used for SS measurements before ripening (Fig. 2). The remaining fruits (about 50±70) were ripened (method described above). Eighteen fruit ripened to ®rmness of 0.6±0.8 kgf were selected from these 50±70 fruits and used for SS measurements after ripening. 2.3.5. Sampling method for headspace SPME±GC±MS After storage of 1, 2 and 3 months, 12 fruits ripened to 0.6±0.8 kgf ®rmness on the same day (ripening times were 8, 6 and 5 days, respectively) were selected from the 50±70 fruits of each group and used for SPME±GC± MS analysis. For fruits stored for 5 months, 18 fruits (0.6± 0.8 kgf ®rmness) ripened over 3 days were selected and used for SPME±GC±MS analysis. Results of SPME±GC±MS analysis from these four groups were used to compare
R.J. Stevenson, / Food Research International 32 (1999) 175±183
177
Fig. 1. Change of kiwifruit ®rmness during storage.
Fig. 2. Comparison of the SS changes before and after ripening during storage (error bars indicate RSD's): BR, before ripening; AR, after ripening.
the trends in development of kiwifruit volatile ¯avour compounds during storage. SPME±GC±MS analysis was also carried out on another 12 ripe fruit (0.6±0.8 kgf ®rmness) from those stored for 5 months with ripening time of 2 days. These results were compared with those of which the ripening time was 3 days to study the in¯uence of the ripening time on the volatile ¯avour compounds.
The fruits were sliced into four parts and the skin was discarded. Pieces (20±22 in number; 250 g) which had been chosen randomly were placed in a round-bottom ¯ask (500 ml) immersed in a water bath (25 C). A glass beaker (5 ml) was placed on the surface of the chopped kiwifruit. A glass tube (1 ml) containing 2-octanone (800 ml of a 50 mg/ml solution in water±methanol) as an internal standard was placed in the beaker. The ¯ask
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was then tightly closed with a screw-top quick®t adaptor and Te¯on-coated silicone septum. For the fruits stored for 5 months, and ripened for 3 days, triplicate sampling runs (each with six fruits) were carried out. Duplicate sampling runs (each with six fruits) were performed for the remaining three storage periods. A number of the kiwifruit stored for 5 months were allowed to become ``over-ripe'' [®rmness 0.3±0.4 kgf, as de®ned by Young and Paterson (1985)]. Sampling was performed separately on ``over-ripe'' kiwifruit (5±6 days ripening) sliced into four parts with the skin discarded (250 g), and ``over-ripe'' macerated kiwifruit (250 g). A portion of six fruit were used for each sampling. Macerated kiwifruit were prepared by blending in a blending device for 4 s, four times. The sampling method, GC±MS analysis and estimation of the relative amounts of compounds were as for other groups of fruit. The sample-containing ¯ask was ®rst held in the water bath (25 C) for 30 min to allow equilibrium between the fruit sample and the headspace, prior to SPME sampling. The SPME manual device (Supelco Co., Bellefonte, PA, USA) was equipped with a fused-silica ®bre coated with Carbowax-divinylbenzene (65 mm). The ®bre was inserted into the sample container through the septum, and exposed to the headspace for 30 min. 2.3.6. GC±MS analysis Volatile compounds adsorbed on the SPME ®bre were thermally desorbed in the injector port of the GC and then separated on the GC column. The desorption time was 5 min. GC±MS analysis was performed in a Shimadzu GC-17A GC/Shimadzu QP-5000 quadruple MS system (Shimadzu, Kyoto, Japan). Separation was achieved on a 30 m0.25 mm id. fused-silica capillary column coated with cross-linked polyethene glycol 20 M, ®lm thickness 0.25 (m (DB-Wax; J and W Scienti®c, Folsom, CA, USA). The column temperature was held at 27 C for 6 min, then increased to 135 C (at 3 C/min), then to 230 C (at 6 C /min), ®nally held at 230 C for 5 min. The injection port (splitless mode) and detector temperatures were 220 and 230 C, respectively. Helium (column ¯ow rate 0.8 ml/min) was the carrier gas. Cryogenic focussing was performed by immersing about 4 cm of the front of the column in dry ice (ÿ80 C) for the ®rst 6 min of the temperature program. Compounds were identi®ed by matching mass spectra with NIST62 library of standard compounds. When available, MS identi®cations were con®rmed by comparing GC retention times with authentic compounds. 2.3.7. Calculation method of the relative amounts of compounds For each sampling, the same amount of kiwifruit (250 g), the same ¯ask, the same amount of internal standard (800 ml of a 50 mg/ml solution of 2-octanone), equilibrium time, and SPME headspace sampling time were
applied (the SPME ®bre was conditioned at 220 C for 20 min before each sampling). The relative amount of a speci®c compound was de®ned as: the height of the compound GC peak divided by the height of the internal standard GC peak, then times 100%, or the height of the compound selected ion monitoring (SIM) GC peak divided by the intensity of the internal standard SIM peak, then times 100%. Under such conditions, it was possible to compare the relative changes in concentration of certain compounds during storage. Relative standard deviations (RSD) were included with average peak heights for samplings carried out in triplicate but were not calculated for average peak heights for samplings carried out in duplicate. 3. Results and discussion 3.1. Firmness Fig. 1 demonstrates that the kiwifruit ®rmness decreased with increasing storage time. Firmness declined considerably during the ®rst two months of storage; it then decreased very slowly. Previous work (McDonald, 1990) described a similar pattern of kiwifruit ®rmness change. 3.2. Soluble solids measurements The changes of SS before (immediately after storage) and after ripening have been compared (Fig. 2). During storage, the SS before ripening increased with time, indicating that the fruit ripened slowly during storage. Large changes occurred during the ®rst two months and then the SS levels remained nearly constant. Interestingly, the SS after ripening (ripened to ®rmness of 0.6±0.8 kgf) decreased with increasing storage time. In the ®rst two months the levels decreased rapidly, then slightly or not at all. The dierence between the SS before ripening and after ripening was at ®rst easily apparent, then became smaller, and eventually negligible (Fig. 2). This is possibly because the freshly harvested fruit (storage time=0 month) took approximately 8 weeks to ripen to ®rmness 0.6±0.8 kgf, and the fruit stored for 1, 2, 3 and 5 months took 8±9, 5±6, 2±5 and 2±3 days, respectively, to ripen to ®rmness 0.6±0.8 kgf. The longer the ripening time, the greater the dierence of SS between before ripening and after ripening. 3.3. Headspace SPME±GC±MS analysis 3.3.1. Flavour volatile compounds in kiwifruit Typical headspace SPME±GC±MS pro®les of the kiwifruit volatiles at ripe and ``over-ripe'' stages are shown in Fig. 3. The peak numbering refers to the compounds in Table 1., which lists 42 ¯avour volatile
R.J. Stevenson, / Food Research International 32 (1999) 175±183
179
Fig. 3. Typical headspace SPME-GC-MS pro®les of (a) ripe (®rmness of 0.6±0.8 kgf) kiwfruit (stored for 5 months prior to ripening); (b) macerated ``over-ripe'' kiwfruit (stored for 5 months prior to ripening) (i, internal standard; h, hydrocarbon).
compounds (compound 26 was identi®ed as a mixture of c/t-hex-3-en-1-ol). The results can be compared with those found by dynamic headspace sampling methods of Young and Paterson (1985) and Bartley and Schwede (1989), who identi®ed 26 and 27 compounds, respectively. Except for pent-4-enal, t,t-nona-2,4-dienal, 2nonanone, ethyl octanoate, butyrolactone and 2-propenyl butanoate, the other 33 ¯avour volatiles have been reported previously. Of these 33 ¯avour volatiles, 23 compounds were identi®ed in the headspace of macerated kiwifruit using dynamic headspace sampling techniques (Bartley & Schwede, 1989; Young & Paterson, 1985). Twenty-two compounds were identi®ed by Takeoka et al. (1986), using a vacuum-distillation method. Other compounds (heptanal, ethyl hex-3-enoate, 6methylhept-5-en-2-one, acetic acid, c/t-2-nonenal and c/t2-decenal) have been detected only in kiwifruit juice (Young & Perara, 1992). There were 12 compounds (ethyl ethanoate, methyl butanoate, pent-1-en-3-one, ethyl butanoate, hexanal, c-hex-2-enal, t-hex-2-enal, 1-hexanol, c/t-hex-3-en-1-ol, t-hex-2-en-1-ol and methyl benzoate) commonly found by Young et al. (1983), Young and Paterson (1983), Bartley and Schwede (1989) and Takeoka et al. (1986). All of them, except c-hex-2-enal, were detected by headspace SPME±GC±MS. Of the 19 compounds (ethyl ethanoate, methyl propanoate, ethyl propanoate, methyl butanoate, pent-1-en-3-one, ethyl butanoate, hexanal, propyl butanoate, ethyl pentanoate,
methyl hexanoate, c-hex-2-enal, t-hex-2-enal, ethyl hexanoate, 1-hexanol, c/t-hex-3-en-1-ol, t-hex-2-en-1-ol, methyl benzoate and ethyl benzoate) commonly found by Young and Paterson (1985) and Bartley and Schwede (1989) in the headspace of macerated kiwifruit, 18 (except c-hex-2-enal which was present in very low amount) were detected by headspace SPME±GC±MS. The compounds labelled with * in Table 1 (pent-4-enal, t,t-nona-2,4-dienal, 2-nonanone, ethyl octanoate, butyrolactone and 2-propenyl butanoate) have not been previously reported in kiwifruit. 3.3.2. The development of volatile ¯avour pro®les during storage Table 2 lists the ¯avour volatiles found in ripe kiwifruit during storage and their relative amounts. It is thought that the main contributors to kiwifruit aroma (Paterson et al., 1991; Young et al., 1992; Young et al., 1995) were methyl and ethyl butanoate, hexanal, t-hex-2-enal, 1hexanol, t-hex-2-en-1-ol and c/t-hex-3-en-1-ol. Fig. 4 shows the evolution of the main ¯avour components of ripe kiwifruit after storage for various time periods. It demonstrates an increasing trend with all the above important aroma components (although methyl butanoate increased initially, then decreased from 3 to 5 months) during storage. During the ®rst 2 months, the changes were slight or negligible. From 2 to 3 months, the enrichments were more substantial. Other volatiles listed
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R.J. Stevenson, / Food Research International 32 (1999) 175±183
Table 1 Identi®cation of volatile ¯avour compounds in a typical headspace SPME±GC±MS pro®le of ``over-ripe'' kiwifruit
Table 2 Volatile ¯avour compounds developed during storage in the headspace SPME±GC±MS pro®les of kiwifruit
Peaka
No. of Compounda
1 month (2)/b
2 months (2)
3 months (2)
5 months (3)
2 4 5 8 10 11 20 25 26 28 32 35 38
11.75c 43.66 7.39 12.49 34.21 2.17 26.17 21.40 0.52 9.10 0.64 ud 4.04
udd 48.60 ud 19.62 12.17 2.90 22.72 20.10 1.40 9.51 1.01 2.73 20.96
ud 46.42 ud 74.86 35.33 5.50 54.77 53.08 1.85 28.04 1.93 3.31 20.93
72.1012.78e 98.8516.71 27.5412.39 39.6615.39 178.8487.27 7.121.49 93.7230.82 66.5716.52 3.940.32 41.4714.89 2.470.38 5.002.52 11.043.31
b
1 c d e f 2y1 ;y2 ;b ;t y2,b,t 3 4y1,y2 5y2,b,t 6y2,t 7t 8y1,y2b,t 9y1,y2,b,t 10y1,y2,b,t 11y1,y3,b,t 12y2,t 13g 14y2,b 15y1,y2,t 16y2,b 17 18y2,b 19 20y1,y2,b,t 21y2,b,t 22 23t 24 25y1,y2,b,t 26y1,y2,b,t 27 28y1,y2,b,t 29 30 31T 32t 33 34y1,y2,b,t 35 36 37y2,b,t 38
Compounds 2-Propanone Ethyl acetate Methyl propanoate Ethanol Ethyl propanoate Ethyl 2-methylpropanoate Pentanal Methyl butanoate Pent-1-en-3-one Ethyl butanoate Hexanal Methyl pentanoate Pent-4-enal Propyl butanoate t-pent-2-enalh Ethyl pentanoate Heptanal Methyl hexanoate t; t-Nona-2,4-dienal t-Hex-2-enal Ethyl hexanoate Ethyl hex-3-ennoate c=t-Hept-3-enali 6-Methylhept-5-en-2-one 1-Hexanol c=t-Hex-3-en-1-ol 2-Nonanone t-Hex-2-en-1-ol Ethyl octanoate Acetic acid t; t-Heota-2,4-dienal Benzaldehyde c=t-2-nonenal Methyl benzoate Butyrolactone c=t-2-Decenal Ethyl benzoate 2-Propenyl butanoate
RT (min) 9.02 10.22 10.53 11.21 11.52 11.38 12.07 12.29 13.22 13.85 15.22 15.42 16.15 16.68 16.95 17.11 18.86 18.89 20.19 20.55 20.89 24.07 25.14 25.85 25.51 26.73 28.06 29.24 29.99 31.03 31.38 33.56 34.31 37.71 36.51 38.83 39.55 47.08
Identi®cation b
MS,RT MS,RT MS,RT MS,RT MS,RT MS MS MS,RT MS MS,RT MS,RT MS,RT MS MS,RT MS MS MS MS,RT MS MS,RT MS,RT MS MS MS MS,RT MS,RT MS,RT MS,RT MS MS MS MS,RT MS MS,RT MS MS MS,RT MS
a
Peak number in Fig. 4. MS, mass spectrometry; RT, retention time. c y1, detected by Young et al. (1983) using vacuum-distillation sampling. d y2, detected by Young and Paterson (1985) using dynamic headspace sampling. e b, detected by Bartley and Schwede (1989) using dynamic headspace sampling. f t, detected by Takeoka et al. (1986) using vacuum-distillation sampling. g compounds not previously reported in kiwifruit. i c, cis. h t, trans. b
in Table 2 such as ethanol, benzaldehyde and butyrolactone showed a similar trend. Young and Paterson (1985) showed that fruit harvested at maturity 8.0 Brix (SS 8%, same as the fruit at harvest used in this study) had slightly less total volatile ¯avours than those after 9 weeks (about 2 months) storage, although the level of total volatile ¯avours
a
Compound's number in Table 1. Figures in parentheses indicate the number of sampling replications. c Relative peak heights expressed as means. d ud, undetected. e Relative peak heights expressed as ``meanRSD''. b
Fig. 4. Evolution of important kiwifruit volatile ¯avour compounds during storage. A: methyl butanoate; B, ethyl butanoate; C, hexanal; D, t-hex-2-enal; E, 1-hexanol; F, cis- and trans-hex-3-en-1-ol; G, t-hex2-en-1-ol.
decreased after 21 weeks (about 5 months) storage. The changes observed between 1 and 2 months in the present work were comparable with their results. However, in contrast to Young and Paterson, it was found that after 5 months, the total volatiles were still increasing, which may be due to the use of a dierent ripening method. In this work, the fruit were covered with polyliner packed in a standard tray and ripened in an incubator set at 20 C, while the above workers triggered the ripening with Ethrel at 22 C. A ®rmness of 0.6±0.8 kgf was used as the ripe standard, while they used ®rmness of 0.5±0.6 kgf. In their research, the ripening time for the fruit stored 9 and 21 weeks prior to ripening was 4 and 2 days, respectively. In our study, the ripening time for the fruit stored for 2 and 5 months were 6 and 3 days, respectively. As discussed by Young and Paterson (1985), the level of kiwifruit volatile ¯avour compounds
R.J. Stevenson, / Food Research International 32 (1999) 175±183
was related to the storage time prior to ripening as well as ripeness. Unlike apples and oranges, the levels of kiwifruit ¯avour volatiles are tremendously sensitive to ripeness, ®rmness and storage (Young et al., 1995). Large variations in levels of compounds are found between individual fruit in any one batch. Informal sensory evaluation showed that individual fruit, even from the same batch, tasted quite dierent. Generally, the ``under-ripe'' fruit had a green grassy note which was possibly due to thex-2-enal, while the more ripe fruit had a sweet and fruity ¯avour which was attributed to butanoate esters (Young et al., 1983). In the current research, each sampling was carried out with a portion of at least six fruit in an attempt obtain statistically meaningful results. 3.3.3. The in¯uence of ripening time on the kiwifruit volatile ¯avour compounds As discussed above, the quantity of ¯avour volatiles was in¯uenced by both storage time and stage of ripeness. Bartley and Schwede (1989) also showed that the ripening time aected the levels of kiwifruit volatile ¯avour compounds. During our study, it was found that dierent ripening times resulted in dierent levels of ¯avour volatiles among the fruit stored for the same time prior to ripening (®rmness 0.6±0.8 kgf). Twelve fruit (5 months storage) with a ripening time of 2 days were compared with 18 fruit (5 months storage) with a ripening time of 3 days. Semi-quantitative headspace SPME±GC±MS results of fruit with dierent ripening times are listed in Table 3 and comparisons of main component levels are illustrated in Fig. 5. There were dramatic increases in the amounts of esters, especially
Table 3 Volatile ¯avour compounds in the headspace SPME±GC±MS pro®le of kiwifruit (stored for 5 months) with dierent ripening times No. of compounda
Ripening time 2 days (2)b
Ripening time 3 days (3)
2 4 5 8 10 11 20 25 26 28 32 35 38
15.69c 102.03 10.17 2.83 11.36 7.88 100.83 65.52 4.61 48.78 3.81 8.47 10.99
72.1012.78d 98.8516.71 27.5412.39 39.6615.39 178.8487.27 7.121.49 93.7230.82 66.5716.52 3.940.32 41.4714.89 2.470.38 5.002.52 11.043.31
a b c d
Compound's number in Table 1. Figures in parentheses indicate the number of sampling replications. Relative peak heights expressed as means. Relative peak heights expresed as ``meanRSD''.
181
Fig. 5. Comparison of important kiwifruit volatile ¯avour compounds with ripening times of 2 days and 3 days. A, methyl butanoate; B, ethyl butanoate; C, hexanal; D, t-hex-2-enal; E, 1-hexanol; F, cis- and trans-hex-3-en-1-ol; G, t-hex-2-en-1-ol.
ethyl butanoate, between the kiwifruit samples with a ripening time of 2 days and those of 3 days. 3.3.4. The pro®les of ``over-ripe'' kiwifruit volatile ¯avour compounds Fig. 3(b) shows a typical headspace SPME±GC±MS pro®le of volatiles from macerated ``over-ripe'' kiwifruit. Table 4 lists the compounds identi®ed by headspace SPME-GC-MS and their relative amounts of chopped ``over-ripe'' kiwifruit and macerated ``overripe'' kiwifruit. Tables 3 and 4 demonstrate that in the kiwifruit stored for 5 months prior to ripening, more ¯avour compounds were found in the ``over-ripe'' fruit than in ripe fruit. The largest increases were esters such as methyl propanoate, propyl butanoate, methyl pentanoate, ethyl pentanoate, methyl hexanoate, ethyl hexanaote, methyl benzoate and ethyl benzoate. These results agree with those of Young and Paterson (1985). Table 4 indicates more compounds (pent-4-enal, t-pent2-enal, c/t-hept-2-enal, 6-methylhept-5-en-2-one, 2-nonanone, ethyl octanoate, t,t-hepta-2,4-dienal, c/t-2-nonenal and c/t-2-decenal) were found in the macerated kiwifruit than in the chopped kiwifruit pieces. The amounts of aldehydes such as hexanal, t-hex-2-enal, pent-4-enal, tpent-2-enal, c/t-hept-2-enal, t,t-hepta-2,4-dienal, c/t-2nonenal and c/t-2-decenal also increased. This was probably due to more ¯avour volatiles being released from the cells after the fruit had been macerated. However, the amounts of alcohols changed only slightly or not at all and the amounts of some esters decreased. It is possible that the additional experimental step (blending the fruit and transferring them from the vegetable blender to the glass ¯ask for sampling) resulted in liberation of some of the ¯avour volatiles. Reproducibility appeared to be better in the macerated fruit than in the fruit pieces. Headspace SPME sampling is sensitive to sample volume/headspace volume ratio and the distance from the ®bre to the surface of the sample (Matich et al., 1996). The surface and the headspace volume of the macerated fruit were more controllable.
182
R.J. Stevenson, / Food Research International 32 (1999) 175±183
Table 4 Volatile ¯avour compounds in headspace SPME±GC±MS pro®les of ``over-ripe'' chopped kiwifruit and macerated kiwifruit No. of compounda
c
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 a b c d e f
Chopped kiwifruit (2)b tr 778.32d tr 331.19 25.49 7.36 udf 621.69 ud 2665.73 3.39 tr tr tr ud 22.37 ud 21.11 ud 24.76 124.91 7.83 ud ud 30.32 1.30 ud 8.94 ud 3.53 ud 0.27 ud 9.10 3.01 ud 27.55 9.43
Macerated kiwifruit (3) tr 520.4015.93e 2.5825.05 210.9313.28 39.0514.28 2.6510.86 7.5521.76 434.1026.02 21.3420.84 1414.387.30 67.4628.04 1.4225.64 4.6913.22 2.7028.34 13.3915.39 11.8016.29 6.9226.46 26.7730.60 7.2219.98 298.8423.34 66.3317.12 3.3929.90 7.1915.56 tr 23.8529.33 0.2123.16 4.6738.49 25.2718.42 3.4418.27 5.0340.44 3.3732.54 0.7245.97 4.7514.22 9.911.86 1.0918.01 1.758.14 24.8243.65 ud
Compound's number in Table 1. Figures in parentheses indicate the number of sampling replications. tr, trace amounts. Relative peak heights expressed as means. Relative peak heights expressed as ``meanRSD''. ud, undetected.
4. Conclusions Headspace SPME is a complementary sampling technique for kiwifruit volatile ¯avour compound analysis and can be operated in parallel with and is more convenient than vacuum-distillation and dynamic headspace sampling. A number of compounds (pent-4-enal, t,t-nona-2,4dienal, 2-nonanone, ethyl octanoate, butyrolactone and 2-propenyl butanoate) were reported for the ®rst time in kiwifruit. Other components detected by headspace SPME±GC±MS, i.e. heptanal, ethyl hex-3-enoate, 6methylhept-5-en-2-one, acetic acid, c/t-2-nonenal and c/
t-2-decenal, have been found before only in the juice of kiwifruit (Young et al., 1992). It has been demonstrated that the volatile ¯avour compounds of kiwifruit are very sensitive to storage time and stage of ripeness. In general, total volatiles increase with with increasing storage time and advancing ripeness. It appears that the headspace SPME± GC±MS pro®les of macerated kiwifruit are more reproducible than those of sliced kiwifruit. Acknowledgements The authors would like to thank Dr. J. Burdon and Dr. H. Young from Horticultural and Food Research Institute, Auckland, for their help in providing kiwifruit and a number of chemicals as volatile standards. References Arthur, C. L., & Pawliszyn, J. (1990). Solid phase microextraction with thermal desorption using fused silca optical ®bers. Analytical Chemistry, 62, 2145±2148. Bartley, J. P., & Schwede, A. M. (1989). Production of volatile compounds in ripening kiwifruit (Actinidia chinensis). Journal of Agriculture and Food chemistry, 37, 1023±1025. Belardi, R. P., & Pawliszyn, J. (1989). The application of chemically modi®ed fused silica ®bres in the extraction of organics from water matrix samples and their rapid transfer to capillary columns. Water Pollution Research in Canada, 24, 179±191. Constant, M., & Collier, J. (1997). Headspace gas chromatography pro®les of fruit-¯avored malt beverages using solid-phase microextraction. Journal of the American Society of Brewing Chemists, 55, 112±118. Cossa, G., Trova, C., & Gandolfo, G. (1988). Estrazione ed identi®cazione dei componenti volatili del kiwi. Industrie Alimentaria, 17, 531±534. Gandini, N., & Riguzzi, R. (1997). Headspace solid-phase microextraction analysis of methyl isothiocyanate in wine. Journal of Agriculture and Food Chemistry, 45, 3092±3094. Hawthorne, S. B., Miller, D. L., Pawliszyn, J., & Arthur, C. L. (1992). Solventless determination of caeine in beverages using solid-phase microextraction with fused-silica ®bres. Journal of Chromatography, 603, 185±191. McDonald, B. (1990). Precooling, storage and transport of kiwifruit. In I. J. Warrington, & G. C. Weston, Kiwifruit: Science and Management (pp. 429±459). Auckland: Ray Richard Publisher. Mariaca, R., & Bosset, J. O. (1997). Instrumental analysis of volatile ¯avour compounds in milk and dairy products. Lait, 77, 13±40. Matich, A. J., Rowan, D. D., & Banks, N. H. (1996). Solid-phase microextraction for quantitative headspace sampling of apple volatiles. Analytical Chemistry, 68, 4114±4118. Page, B. D., & Lacroix, G. (1993). Application of solid-phase microextraction to the headspace gas chromatogaphic analysis of halogenated volatiles in selected foods. Journal of Chromatography, 648, 199±211. Paliyath, G., Whiting, M. D., Stasiak, M. R., Murr, D. P., & Clegg, B. S. (1997). Volatile production and fruit quality during development of super®cial scald in red delicious apples. Food Research International, 30, 95±103. Paterson, V. J., MacRae, E. A., & Young, H. (1991). Relationships between sensory properties and chemical composition of kiwifruit
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