J Sci Food Agric 1994,65, 241-247

Volatile Compounds found in Expired Air during Eating of Fresh Tomatoes and in the Headspace above Tomatoes Rob S T Linforth, Isabelle Savary, Becky Pattenden and Andrew J Taylor* Applied Biochemistry and Food Science, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK (Received 3 November 1993; revised version received 14 February 1994; accepted 3 March 1994)

Abstract: Volatile compounds from tomatoes were measured in the headspace above tomatoes and in the air expired from the noses of people eating tomatoes (nosespace). Eleven target compounds, representative of the different metabolic pathways that contribute to tomato aroma were chosen for analysis. The pro- cedure consisted of trapping volumes of headspace or nosespace on Tenax, desorbing and chromatographing the samples on gas chromatographs and quan- tification by integration of characteristic ion chromatograms. Small volumes (8.5 cm3) of headspace were used to develop the method which was then applied to study potential sources of variation in the raw material and in the sampling procedure. The variation in headspace profiles, with time after dicing tomatoes, and with tissue maturity, demonstrated that the amounts of some volatiles changed with time and with maturity. Sampling was therefore undertaken using batches of tomatoes with similar histories and the time of sampling was fixed. The headspace and nosespace profiles from tomatoes produced raw data with substantial variation (percentage coeficient of variation 50-60%) but this appeared to be due to different amounts of volatiles in the replicates. When data were expressed on a relative basis by normalisation, the profiles from groups of replicates were seen to be quite similar for nine of the compounds but values for 3-methylnitrobutane and 2-isobutylthiazole showed considerable variation. Nosespace profiles of tomato volatiles were broadly similar between operators when expressed on a relative basis; the actual total amounts varied considerably. The headspace profiles from diced and stomached tomatoes were comparable but distinctly different from the nosespace profile. There were differences between the headspace and nosespace profiles particularly in the amounts of 3- methylbutanal, dimethyl disulphide and hexanal which were present to a greater extent in the headspace on a relative basis.

Key words: Lycopersicom esculentum, flavour release, trace analytical technique, perceived odour, sensory-analytical technique.

INTRODUCTION system. Mints contain relatively large amounts of vola- tiles ( -2 g kg-I), the aroma derives from about four

A previous paper (Linforth and Taylor 1993), described major components; mints have a regular shape and a technique which was capable of measuring the vola- have a uniform distribution of volatiles. Results showed tiles in expired air collected from the nose during eating. that trapping of the nosespace volatiles on Tenax fol- The term nosespace was coined to describe this method lowed by desorption and gas chromatography-mass of volatile analysis. Experiments were performed on spectroscopy (GC-MS) was sufticiently sensitive to mint-flavoured sweets (mints for short) because they measure the volatiles in nosespace. The method was provide many advantages for studying the analytical reproducible within the normal limits of this type of

analysis (%CV 12-15%; Larsen and Poll 1990) and * To whom correspondence should be addressed. there were substantial differences between the nosespace

J Sci Food Agric 0022-5142/94/%09.00 0 1994 SCI. Printed in Great Britain 24 1

242

and headspace volatile profiles. This difference was expected as the mints were solid when headspace was analysed; in the mouth they were mixed with saliva and chewed to form an aqueous suspension which parti- tioned volatiles between the aqueous and air phases thus changing the volatile profile expressed above the solid mints.

The current paper describes the application of this technique to tomatoes. The aroma volatiles from tomato have been the subject of extensive research and the major compounds responsible for aroma in toma- toes have been identified (Buttery et a1 1987). However, most of the data have been derived from experiments where tomatoes have been mechanically homogenised or damaged to simulate eating and then sampled using solvent extraction of headspace techniques. Some workers have optimised their extractions for quantitat- ive recovery of volatiles which has given data on the total amount of volatiles in the tomato (the base profile), others have inhibited enzyme activity to sim- plify the range of volatiles formed. These experiments have generated important data on the volatile composi- tion of tomatoes but they do not measure directly the profile that is sensed by humans when they eat toma- toes. If the nosespace from tomatoes is to be measured, it is important to realise that the levels of volatiles in tomatoes are much lower than in mints and the profile is also more complex (for a review see Petro-Turza (1986/87)). Tomatoes, like some other plant foods (Galliard et a1 1977), generate some of the flavour vola- tiles when the food is eaten and this adds to the com- plexity of the analysis and makes the use of direct measurement of volatiles in the nosespace of consumers even more relevant. It seems unlikely that new volatile compounds will be discovered in tomato nosespace but a comparison of the profiles obtained by this method and those obtained by other techniques is of interest. Besides sampling the volatiles at a location that seems more relevant for comparison with sensory analysis, the technique used for analysis may also overcome some of the problems encountered by other workers. Typically, the methods for volatile analysis involve trapping, then solvent extraction followed by GC to give the volatile profile (Buttery et al 1987). It is acknowledged that the use of a solvent to desorb samples concentrates the volatiles and the solvent peak also obscures some vola- tiles when they are subjected to GC (Buttery et a1 1987).

It was recognised that tomatoes provided consider- able challenges for the technique of nosespace analysis in terms of sensitivity and complexity of the volatile profile when compared with the mint system used pre- viously. In addition, tomatoes are natural products and the variation between fruits is likely to be considerably greater than the variation in a fabricated food like mints. There is also variation in the way people eat tomatoes in terms of how frequently they chew, swallow or breathe. The route of breathing also has implications

R S T Linforth, I Savary, B Pattenden, A J Taylor

for the analysis as it is not clear how air flows between the mouth, nose and lungs during eating. The experi- ments were designed to test the nosespace methodology with one of the most difficult systems possible and to determine whether it was applicable to the analysis of fruits like tomatoes.

MATERIALS AND METHODS

Tomatoes

The tomatoes used during these experiments were judged ripe by their overall red colour and purchased from a local store. They were kept at room temperature prior to analysis. Tomatoes were used within 1-2 days of purchase but, for the experiments on over-ripe toma- toes, the storage period was 4-5 days after purchase.

Volatile sampling

Headspace samples (diced tomatoes) Three tomatoes were cut into quarters, and cut again to give 12 segments per tomato. Three portions of approx- imately 50 g were transferred to 100 cm3 flasks which were then sealed with lids that had two openings. One was fitted with a Tenax trap (Unijector, SGE, Milton Keynes, UK), the other was fitted with a septum through which 10 cm3 of laboratory air was injected after a 5 min equilibration period. The injected air dis- placed 8.5 cm3 of headspace through the Tenax trap over a further 5 min period. The trap was then removed and the volatiles chromatographed.

For the experiments on the effect of time on the vola- tile composition of headspace, tomatoes (250 g) were diced and a representative subsample (50 g) was placed in a sealed glass jar. At 1, 3, 6 and 10 min, 8.5 cm3 of headspace was trapped on Tenax and analysed by GC-MS. The same method was used for analysis of over-ripe tomatoes from the same batch. All headspace analyses were repeated three times.

Headspace samples (stomached tomatoes) Four tomatoes (approximately 200 g) were placed in a stomacher bag and stomached for 1.5 min (Seward M50-110, London, UK). Portions (40 cm3) of the homogenate were transferred to 10-cm3 flasks, which were then sealed and sampled as described above.

Nosespace samples (quantitative analysis) Tomatoes were cut into quarters and eaten by the oper- ators at the rate of one quarter every 20 s for 3 min. Operators were requested to eat normally and no attempt was made to standardise their chewing or swal- lowing. Each operator had a soft plastic tube fitted to their nostril and sampling was effected with the trap

Volatile compounds of fresh tomatoes 243

inserted through the plastic tube at right angles so that the tip of the trap was in the stream of expired air from the nostril. Air was drawn through the Tenax trap by a vacuum pump at a rate of 75 cm3 min-’. This arrange- ment allowed the operators to breathe normally through the soft plastic tube while air was sampled through the trap. The samples were then desorbed and chromatographed as described below.

Nosespace samples The collection of nosespace samples for odour port analysis OPA required the collection of a larger sample of expired air. Air (about 2.5 litres) from the nose of an operator was drawn through Tenax traps (CHISA, SGE) over a period of 10 min whilst the operator ate three tomatoes, which had each been cut into eight pieces. The CHISA traps allowed greater rates of gas flow due to their larger diameter compared to the Unijector traps (gas flow rate 250 cm3 min-‘). The samples were then desorbed, chromatographed and the compounds ‘sniffed as they left the column. For each odour noted by the operators, the time and an odour description were recorded.

Chromatographic conditions

Quantitative analysis The Tenax traps were thermally desorbed at 240°C (Unijector; SGE) for 2 min and the volatiles re-focused on a 400 mm region of the column (25 m x 0.22 mm ID, BP-1, 1 pm film thickness; SGE), which was cooled with liquid N,. The compounds were then chromato- graphed (Hewlett Packard 5890, Manchester, UK) using a linear temperature gradient from 30°C to 110°C at 4°C min-’ (head pressure 18 psi, helium), after a 2 min delay: followed by a 10°C min-’ linear gradient to 220°C. Mass spectra were recorded from m/z 30 to 150 using an MD 800 mass spectrometer (VG Mass Labs, Manchester, UK). Compounds were identified based on their mass spectra and Kovats linear retention indices (LRI). Peak area data were obtained by the inte- gration of single ion chromatograms of ions character- istic for the compounds of interest (Table 1).

OPA The chromatographic conditions were similar to those above except the column used had an internal diameter of 0.33 mm (head pressure 12 psi: SGE CHISA injector) and the eluent was split at a ratio of about 1 : 1 as it left the column by way of a simple union (SGE). Part of the carrier gas entered a Hewlett Packard MSD 5970 via a restriction capillary to limit the flow to around 1 ml min-’, the remainder was conducted to a home made odour port for ‘sniffing’. The path lengths to the odour port and to the MSD interface were approximately equal so that compounds would arrive

TABLE 1 Volatile constituents and the ions used for their detection and

peak area determination

Compound Identification’ Ionsb LRI

Dimethyl sulphide MS 62,47 <600 3-Methylbutanal MS/LRI 58, 71 639 Penten-3-one MS/LRI 55, 84 670 2-Methylbut-2-enal MS 55, 84 125 Dimethyl disulphide MS/LRI 94,79 725 3-Methylbutanol MS/LRI 55, 70 730 (E)-Pent-2-enal MS/LRI 83, 55 732 (Z)-Hex-3-enal MS 98, 69 778 Hexanal MS/LRI 82, 57 780 3-Methylnitrobutane MS 55, 71 867 2-Isobutylthiazole MS/LRI 99, 58 1019

’ MS, electron impact mass spectrometry; LRI, linear reten- tion index (BP-1). ‘The first ion was used for peak area determination; the second ion was used for peak confirmation.

at about the same time at both the MS and odour detectors. Humidified make-up gas (30 cm’ min- ’) was added to the column emuent just before it reached the nose cone on the odour port.

RESULTS

Preliminary experiments

There are many qualitative and quantitative reports of volatile compounds from tomatoes (see, for example, Buttery et a l ( l987) and Petro-Turza (1986/87). It is rec- ognised that volatile analysis of tomatoes varies con- siderably depending on the methodology utilised and it was necessary to define the volatiles found in this method. From a preliminary experiment using head- space from a diced tomato, 11 compounds were chosen which represented some of the different metabolic path- ways that contribute to tomato aroma. Due to low signal to noise ratios and incomplete resolution of some compounds in the chromatogram, the eleven com- pounds were identified and quantified using the LRI values and characteristic ions listed in Table 1. It was not possible to estimate the amount of each particular compound due to the unavailability of all authentic standards. Those that were available suggested that the amounts of volatiles on the column were in the region of 1-50 ng.

Variation in tomato material

Following the preliminary experiments which showed that the 11 volatiles could be quantified, at least in a headspace sample, the variation in headspace volatile

244 R S T Linjorth, I Savary, B Pattenden, A J Taylor

profile was measured as a function of time after tissue damage and with increasing tissue maturity. The peak areas of the 11 target compounds were determined and are presented in Table 2. The data show substantial variations. However, there are trends in the behaviour of the volatiles between the different times of collection and between the ripe and over-ripe tomatoes. For instance, the amount of dimethyl sulphide seems to remain fairly constant over the sampling period and the amounts are also very similar in the ripe and over-ripe samples. In contrast, the amounts of hexanal in the ripe tomatoes change substantially with time and, overall, the amounts of hexanal are greater in the over-ripe than the ripe fruits. These two examples illustrate the main differences that can be seen in Table 2. There may be other differences that are hidden in the variation between samples.

Comparison of headspace and nosespace volatile profiles

The experiment above demonstrated that volatile pro- files could be obtained from fairly small volumes of headspace (8.5 cm’) and the next stage was to deter- mine whether the technique was sufficiently sensitive for the nosespace system. A batch of tomatoes was analysed using headspace and nosespace sampling. Prior to the experiment, subjects were sampled over a 3 min period with no food in the mouth to determine the background volatiles present. No significant peaks were observed under these conditions and there was no interference with detection of the target compounds.

The value in Table 3 shows the raw data from nose- space and headspace experiments. The data show the

variation between the replicates and the variation between operator. The percentage coefficient of varia- tion (%CV) of the headspace samples was 29% while the variation amongst the nosespace samples was higher at between 50 and 60% CV). However, analysis of the raw data suggested that, although the quantities of volatiles varied between replicates and between oper- ators, the actual volatile profiles were similar. It was thought that, if the data could be normalised in some way, comparisons between the headspace and nosespace profiles (and between operator profiles) could be made. Inspection of the raw data indicated that the relative amount of 2-methylbut-2-enal was fairly constant in all samples and the raw data were normalised by taking the peak area of this compound as 100. The data were replotted in graphical form in Fig 1 which shows more clearly the similarities and differences of the various samples. Because the data in Fig 1 are normalised, error bars are inappropriate.

For the normalised noespace profiles, there were similarities between the profiles for all three operators with nine of the components present in similar propor- tions while two components (3-methylnitrobutane and 2-isobutylthiazole) showed five-fold and seven-fold variations respectively. This confirms that some of the variation in the raw data was due to different amounts of compounds being present in the samples and showed that normalisation could assist in removing this source of variation. The technique also showed that three dif- ferent operators consuming three samples each at differ- ent times (but from the same batch of tomatoes) had very similar profiles in their noses. Perhaps this should be expected as there must be a degree of similarity between the profiles so that they are all recognised as

TABLE 2 Time course of aroma development in the headspace above ripe and over-ripe diced tomatoes’

Compound Equilibrium time (rnin)

Ripe tomatoes Over-ripe tomatoes

1 3 6 10 1 3 6 10

Dimethyl sulphide 22 21 76 28 34 46 20 63 3-Methylbutanal 149 790 2548 993 754 1588 505 664 Penten-3-one 59 246 430 178 848 738 261 204 2-Methylbut-2-enal 200 200 580 333 437 437 233 476 Dimethyl disulphide 6 216 1034 409 1431 1875 276 1428 3-Methylbutanol 144 224 471 286 258 405 314 393 (E)-Pent-2-enal 8 17 43 7 32 20 13 28 (Z)-Hex-3-enal 108 192 142 288 1102 1032 496 476 Hexanal 91 880 2635 1443 1701 3867 2647 3350 3-Methylnitrobutane 435 515 1019 606 1311 1523 953 1211 2-Isobutylt hiazole 492 285 59 321 623 860 382 622

Headspace samples were collected from above the same 50 g of tomato in a 100 ml flask after 1, 3, 6 and 10 min of equilibration. Amounts of volatile compounds are expressed in arbitrary peak area units.

Volatile compounds of fresh tomatoes 245

Pent-2-enal

TABLE 3 Mean ion peak areas for volatiles present in the headspace above diced tomatoes, or in expired breath (nosespace) of three

operators eating tomatoes'

Compound Operators

I

Headspace Rob Becky Imad

M E A N S D %CV M E A N S D %CV M E A N S D %CV M E A N S D %CV

Dimethyl sulphide 3-Methylbutanal Pen ten-3-one 2-Methylbut-2-enal Dimethyl disulphide 3-Methylbutanol (Q-Pent-2-enal (Z)-Hex-3-enal Hexanal 3-Methylnitrobutane 2-Isobut ylthiazole

118 29 25 3 2 67 2142 273 13 105 78 74 284 51 18 61 59 97 576 74 13 135 73 54

6370 3454 54 17 2 12 772 314 41 151 98 65

17 6 35 26 36 138 546 201 37 111 57 51

2342 820 35 67 48 72 2163 372 17 1004 417 42

507 134 26 450 396 88

2 1 50 7 8 114 106 64 60 116 71 61

38 151 78 52 147 83 56

5 1 20 11 4 36 110 83 75 149 108 72

5 5 loo 5 2 40 115 53 46 147 80 54 77 31 40 118 53 45

361 159 44 1407 887 63 474 247 52 1029 314 36

64 24 50 20 40

Mean %CV 29 69 53 56

a Each value is the mean of three replicates.

tomato aroma. It was not clear whether the variations in the proportions of 2-isobutylthiazole and 3- methylnitrobutane were significant and whether they were due to the analytical procedure (instability of the compounds on GC for instance) or whether they rep- resented real differences at the sensory level. Further work is needed to confirm these differences.

Comparison of the profiles of headspace and nose- space showed that some compounds were present in the same ratios in both profiles but others showed varia-

tion. 3-Methylbutanal was present in the headspace profile at a four-fold relatively greater concentration than in the nosespace samples. For dimethyl disulphide and hexanal, headspace contained ten times and four times more, on a relative basis, than nosespace samples.

These experiments suggested that the experimental procedures described above were capable of measuring the profile of volatiles in both nosespace and headspace of tomatoes. In addition, the studies showed that there were differences in the nosespace and headspace profiles

0 200 400 600 800 1000 1200 Relative amounts

m I" NS

hi NS

ii NS Headspace

Fig 1. Relative amounts of the target volatiles in the headspace and in the nosespace of three operators after normalisation of the mean data in Table 3. The amounts are expressed relative to 2-methylbut-2-enal(lOO).

246 R S T Linforth, I Savary, B Pattenden, A J Taylor

which may be significant in correlating sensory and instrumental analyses of tomatoes.

Comparison of headspace of diced and stomached tomatoes with nosespace analysis

The headspace experiments described above had used diced tomatoes as they represented an intermediate state between whole and homogenised tomatoes. Dicing tomatoes however, caused variation between the samples due to different surface areas and different degrees of tissue damage. Since part of tomato flavour is generated as tissues are macerated and enzymes and substrates come into contact, the degree of tissue homogenisation may play a role in the amount of some volatiles. Indeed, Kazeniac and Hall (1970) reported that aldehyde production from tomatoes increased with the severity of maceration. To verify this, a large batch of tomatoes was obtained and portions treated as follows. One portion of tomatoes was mechanically damaged using a stomacher machine that is commonly used to prepare samples for microbiological analysis. The stomacher has two flat pistons that act alternately on a stout plastic bag in which the sample is contained. The amount of mechanical damage can be standardised by stomaching for a set time and the bag allows fairly large samples (200 g) to be prepared from which repre- sentative samples can be taken. Since the stomacher reduces the tomatoes to the consistency of a pulp, the technique also lends itself to the incorporation of an internal standard if required. Three portions of the pulp were taken and the headspace profile determined.

Another portion of tomatoes was diced in the usual way and headspace profiles obtained from three sub samples. A third portion of tomatoes was quartered and three samples were eaten by one operator to give tripli- cate nosespace profiles. The raw ion peak areas were then normalised by expressing the amount of the target compounds as a percentage of the total area for all eleven target compounds. The results are shown in Fig 2.

A degree of similarity between the two headspace samples can be seen although after normalisation it is not possible to determine whether it is statistically sig- nificant. Lipid oxidation products like pent-Zenal, (Z)- hex-3-ena1, hexanal and penten-3-one might be expected to show changes between the diced and stomached samples (Kazeniac and Hall 1970) but this is not the case. From these data, the method of tissue damage appears to have little effect on the volatile profiles over this time period, and stomaching tomatoes appears preferable to dicing as it allows better standardisation of mechanical damage and allows the incorporation of an internal standard which could be used to normalise the profiles. Homogenisation using high shear condi- tions is effective in damaging tissue but may have unde- sirable effects like localised heating, seed disintegration, incorporation of air and potential denaturation of enzymes.

Comparison of the nosespace and headspace profiles in Fig 2 showed that some compounds were present at the same relative content. Among these, 2-methylbut-2- enal was important as it was the basis for comparison in the previous experiment and these result validate the choice of 2-methylbut-2-enal for normalisation of the

3-Methylnitrobutane

Hexanal

Hex-3-enal

Pent-2-enal

3-Methylbutanol

Dirnethyl sulphide

2-Methylbut-2-enal

Penten-3-one

3-Methylbutanal - Dirnethyl dtsulphide k 7 . _____r_. y-1-

0 10 20 30 40 Relative amounts

~~ ~~ -~

~IHSD IHSSTINS

Fig 2. Relative amounts of the target volatiles in the headspace from diced tomatoes (HS-D), the headspace from stomached tomatoes (HS-ST) and in the nosespace of a single operator (NS). The mean values from the raw data were normalised as a

percentage of the total target volatile peak area.

Volatile compounds of fresh tomatoes 247

data in Fig 1. Comparison of Figs 1 and 2 shows that the compounds that are present in different amounts between the headspace and nosespace profiles are largely the same which is reassuring and suggests that the analysis has a certain amount of consistency, at least on the two batches of tomatoes analysed here.

Odour port analysis of nosespace sample

Initially, the 11 target volatiles had been chosen on the basis of literature reports on tomato volatiles but it was not certain whether these eleven were actually impor- tant in the aroma profile sensed by the nose. To study this idea, a volume of nosespace was collected and analysed by GC-OPA to determine where odorous compounds eluted. A blank sample was collected from the operator, chromatographed and, although it con- tained a number of peaks on GC-MS, the only odour noted was a lemony odour at 23 min. The sample col- lected during eating tomatoes however, contained 17 odorous components, nine of which coincided with the target compounds. This demonstrated that the target compounds selected were relevant to nosespace aroma although there were other odorous compounds in the nosespace which should be identified and included in future analyses.

CONCLUSIONS

The experiments above demonstrated that the technique of trapping expired air on Tenax, followed by GC-MS could be used for studying the volatile profile during eating of tomatoes. The technique needs further devel- opment to improve reproducibility and sensitivity, and to reduce the time required for the experimental work and the subsequent data handling. The results above suggested that there are differences in the headspace and nosespace profiles of tomatoes and these have implications when related to sensory analyses of toma-

toes. Although the variation in the instrumental analyses is higher than usual, normalisation improves the situation and, according to Acree and Barnard (1994), it is necessary for the concentration of an odour to change by a factor of two before a human can detect a change in odour. It appears that humans have excel- lent sensitivity to aromas but their ability to quantify them is not so good and, as long as the instrumental analysis measures within the sensory variance, the tech- nique is valid. More work is needed to study the differ- ences between volatile profiles in tomatoes to ensure that the changes noted here are representative.

ACKNOWLEDGEMENTS

The authors acknowledge the advice and support from Dr P Dunphy and Mr G Channel1 of Unilever Research, Colworth House.

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