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Measurement of lipid oxidation-derived volatiles in fresh tomatoes
Postharvest Biology and Technology 23 (2001) 117– 131 www.elsevier.com/locate/postharvbio
Measurement of lipid oxidation-derived volatiles in fresh tomatoes Fabienne Boukobza a, Patrick J. Dunphy b, Andrew J. Taylor a,* a
Di6ision of Food Sciences, School of Biosciences, Uni6ersity of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK b Unile6er Research Colworth Laboratory, Colworth House, Sharnbrook, Bedford MK44 1LQ, UK Received 21 February 2001; accepted 2 May 2001
Abstract A rapid, low-shear, reproducible maceration device was developed for tomato fruit and coupled to an Atmospheric Pressure Chemical Ionisation-Mass Spectrometer (API-MS) so that real time, simultaneous measurement of nine key volatile compounds released upon disruption of the tomato tissue was achieved. The minimum detection level of the volatiles was around the odour threshold concentrations of the compounds and variation between release from macerated tomatoes was between 10 and 25%. Based on their temporal differences in release, the compounds were classified into two groups: the preformed compounds already present in the ripe fruit (isobutylthiazole, 6-methyl-5hepten-2-one, methylbutanal, methylbutanol, ethanol and acetaldehyde) and the enzyme generated compounds formed only upon maceration (hexanal, hexenal and hexenol). The addition of exogenous linoleic and linolenic acids (2 mg/g FW) to tomatoes prior to maceration influenced the amount of C6 aldehydes produced. Hexanal was increased 12-fold in the presence of linoleic acid, while hexenal was increased 14-fold in the presence of linolenic acid. Addition of both fatty acids gave intermediate behaviour. Addition of enzymes from the lipid oxidation pathway (lipase, phospholipase, lipoxygenase and alcohol dehydrogenase) at various concentrations, in buffer or emulsion and with or without fatty acids caused no significant changes in the release profile of tomatoes. Results highlighted the inhibitory effect of Tween 20 used as surfactant in emulsions. Alternative surfactants (lecithin and glyceryl monostearate) were tested and did not influence volatile generation. The paper demonstrates that the maceration device coupled to the API-MS can be used for rapid screening of tomato fruit as well as for studying the factors that influence the lipoxygenase pathway. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Lipoxygenase; Flavour; APCI; Lipid oxidation; Fatty acid substrate
1. Introduction
* Corresponding author. Tel.: + 44-115-9516144; fax: + 44115-9516154. E-mail address: [email protected] (A.J. Taylor).
Fresh tomato flavour is determined by a relatively small number of volatile aroma compounds and non-volatile taste compounds (Petro-Turza, 1986 –1987). Among the volatile components, sixcarbon (C6) compounds play a major role giving
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tomato its fresh ‘top-note’. These C6 volatile compounds are formed upon breakdown of the fruit (Galliard et al., 1977). Tissue disruption results in the degradation of endogenous lipids to fatty acids, then oxidation to yield a hydroperoxide followed by selective lyase cleavage to form hexanal and hexenal from the 13-hydroperoxides of linoleic and linolenic acids, respectively (Galliard et al., 1977). Conversion of the C6 aldehydes to the corresponding alcohols may also occur through alcohol dehydrogenase (ADH). The series of reactions is often termed the lipoxygenase pathway and has been extensively studied (Galliard et al., 1977; Eriksson, 1979; Buttery, 1993; Riley and Thompson, 1998; Gray et al., 1999; Griffiths et al., 1999). Its general importance in plants (Gardner, 1995) as well as its specific role during the ripening of tomatoes has been described (Baldwin et al., 1991; Buttery and Ling, 1993; Riley and Thompson, 1998). The lipoxygenase pathway is involved in the production of flavour compounds as well as in plant defence, plant development and plant communication. Recent studies of the pathway have shown that it is more complex than originally postulated by Galliard, as different isoforms of the lipoxygenase enzyme exist and appear to have different selectivity and activity towards fatty acid substrates (Gray et al., 1999). A transgenic approach was used to determine the relative importance of lipoxygenase (Griffiths et al., 1999) and of alcohol dehydrogenase enzyme (Prestage et al., 1999) in the formation of the C6 aldehydes and alcohols in tomato fruits. These studies demonstrated both the complexity of this system and highlighted the necessity for rapid assays so that representative samples of modified fruits could be assayed for their ability to generate the different C6 compounds. Previous analyses of tomato aroma volatiles have used solvent extraction of tomato fruit macerates (Buttery et al., 1987, 1989; Buttery, 1993), followed by Gas Chromatography-Mass Spectrometry (GC-MS) to identify and quantify the key compounds responsible for tomato aroma. However, extraction, followed by GC-MS is timeconsuming and limits the number of samples that can be analysed in a day (typically 5– 8 in our lab)
as well as making it difficult to follow the dynamics of the reaction. It should also be remembered that tomato fruit is a living organism whose composition varies rapidly with time. It is therefore desirable to analyse tomato fruits at the same developmental stage, but, due to the unpredictable ripening pattern of tomato fruit, this results in an uneven supply of samples for analysis, and can sometimes overwhelm analyses based on extraction/GC-MS. An alternative method of volatile analysis is to analyse the headspace above tomato macerate (Baldwin et al., 1991; Linforth et al., 1994) and concentrate volatiles on some form of trapping material prior to GC-MS analysis. This simplifies the extraction step but provides an indirect measure of the volatiles present in the tomato itself. Depending on the method of maceration and headspace sampling, it is possible to estimate total amounts by assuming the volatile is in equilibrium between the air and liquid phases and then applying a correction for the air–liquid partition coefficient and the trapping efficiencies of each compound. Alternatively the results can be used to compare the volatile compositions of tomatoes providing the assay conditions (and the tomato composition) do not vary significantly. However, GC-MS is still necessary to identify and quantify the volatile components in the headspace, which limits the number of samples that can be analysed in a day. With the advent of direct headspace analyses like Atmospheric Pressure Ionisation Mass Spectrometry (API-MS) (Linforth et al., 1996; Taylor, 1996; Taylor et al., 2000) it is now possible to measure mixtures of volatiles in headspace directly with a millisecond time delay. Using carefully controlled conditions in the ion source (Taylor et al., 2000), quantitative, real-time analysis of volatile mixtures can be obtained at concentrations around 10 ppbv (nl of vapour in a litre of air). The technique has been used for a preliminary study of volatile release from tomatoes by sampling air directly from the nose of a subject eating the fruits to demonstrate the effect of variety and fruit-to-fruit variation (Brauss et al., 1998). Measurement in vivo is desirable in that it reflects the volatile profile reaching the olfactory receptors and therefore relates well with sensory
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perception. Mastication in vivo is variable, however, and, for a rapid, reproducible biochemical assay for volatile products of lipid oxidation, mechanical maceration of tomato is preferred. Released volatiles are then removed from the headspace by a controlled gas flow and direct analysis of the headspace achieved by API-MS. This paper describes real-time monitoring of the C6 compounds (and other volatile compounds) in the headspace of tomatoes using controlled maceration to mimic tomato breakdown in mouth. A further aim of this work is to study the lipoxygenase pathway in tomato macerate by the addition of enzymes and substrates.
2. Material and methods
2.1. Tomato fruits Batches of tomatoes (Lycopersicon esculentum L., cv. Flavia) were purchased from the local
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supermarket throughout the season. Each batch was stored at room temperature (21 °C) for a maximum of 3 days and used for experiments when fruits were all fully red. The average weight was 89 9 4 g. To avoid excessive variation in raw material composition, each batch of tomatoes was used to generate data for a single factor, e.g. maceration optimisation (see Section 2.2 and Section 2.3), effect of enzymes (see Section 2.4) or effect of substrate (see Section 2.5).
2.2. Tomato maceration Tomatoes were blended in the maceration device (volume 355 ml) made from a commercial food blender (Phillips, type HR2810/A; Fig. 1). Three Swagelok bulkhead fittings were bolted into the blender lid. The left and right fittings were connected to PTFE lines (4 mm OD) for flushing air through the blender. The centre aperture was fitted with a septum so that additional substrates and enzymes could be added via a syringe (Fig.
Fig. 1. Schematic of the maceration device and connections to the APCI-MS.
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1). The intact fruit was placed inside the blender, which was then sealed. The headspace within the device was continually flushed with air (170 ml/ min) so that volatiles formed were removed rapidly (total volume of device 355 ml, so one change of air every 2 min). There was dilution of the headspace by the incoming air, but it was constant. On the outlet side, a portion of the airflow was continuously sampled into the API-MS at 11.5 ml/min through a heated transfer line (0.53 mm id fused silica tube held at 100 °C). The excess of air flow was vented to atmosphere (Fig. 1). The headspace was initially monitored for about 30 s to obtain a baseline measure of volatiles above the intact fruits, then the fruit was blended (2– 5 s) and monitoring of the headspace above the macerate carried out for a further 3– 15 min to follow the release of the selected volatile compounds as described below.
2.3. Sensiti6ity and reproducibility of maceration de6ice Sensitivity for the nine compounds was estimated by first calibrating the API-MS interface with a solution of each compound in hexane (Taylor et al., 2000). The gas phase concentration for a macerated tomato sample was then calculated (Cg) and the signal to noise ratio (SNc) for that concentration determined using the Masslynx software on the API-MS (Micromass, Altrincham, UK). The minimum detectable gas phase concentration of volatile (Cgmin) was estimated (assuming a signal to noise ratio of 5:1 is necessary for detection) using the following calculation: Cgmin = (Cg × 5)/SNc. Reproducibility of the device was tested using three large beef-type tomatoes (about 230 g), which were divided into three portions (77 g each) and macerated. It was assumed that each portion of the tomato was identical in terms of composition and release behaviour. Portions of tomato were macerated as described in Section 2.2 and volatile release monitored as described in Section 2.7. The headspace concentrations of the nine selected compounds were determined at 2 min after maceration and variation expressed as the coefficient of variation (standard deviation× 100/mean).
2.4. Addition of enzymes Enzymes from the lipid oxidation pathway were injected individually through the septum onto the top of the intact tomato (about 90 g) before maceration. Enzyme type and amounts introduced per tomato were as follows: phospholipase A2 from porcine pancreas (120, 240, and 480 units/ tomato), phospholipase D from Streptomyces chromofuscus (800 or 3500 units/tomato), lipase from Mucor meihei (20000 or 26000 units/tomato), lipoxygenase from soybean (1500, 5000 or 10000 units/tomato) and alcohol dehydrogenase from bakers yeast (3300 or 6000 units/tomato), all purchased from Sigma. Enzymes were added either in a sodium phosphate buffer alone (pH 7), in buffer with emulsifier (Tween 20) or in an emulsion prepared with an equal amount of Tween 20 and linoleic acid. Solutions and emulsions were freshly prepared (10 ml) and homogenised with an Ultraturrax blender (10 min). For each experiment, an aliquot (1 ml) was added to the tomato fruit.
2.5. Addition of free fatty acids Emulsions were prepared by mixing linoleic or linolenic acid (1 g) with Tween 20 (1 g) in sodium phosphate buffer (60 ml) with an Ultraturrax blender (10 min) at pH 7. When both fatty acids were added together (1 g each), they were mixed with an equal amount of Tween 20 (2 g). Emulsion (10 ml) was injected onto the intact tomato, then the fruit was blended (2–5 s), resulting in efficient mixing of the macerated tissue and the emulsion. This macerated mixture was incubated for 15 min and volatile compounds sampled and analysed as in Section 2.7. The final amount of fatty acid added was around 2 mg/g FW. This corresponds to an excess of free fatty acid. The estimated concentrations of 18:2 and 18:3 in tomato calculated from previous experiments (Gray et al., 1999) are 0.223 and 0.05 mg/g FW, respectively. Therefore, 10–40 times more fatty acids were added compared to the endogenous situation. Blank runs verified that no volatiles were released from the emulsion on its own. As a further control, tomatoes were macerated with the
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same volume of buffer (10 ml) mixed with Tween 20 but containing no enzymes.
2.6. Effect of emulsifier on lipid oxidation pathway The following surfactants/emulsifiers were compared: Tween 20, lecithin and glyceryl monostearate (GMS). Each compound was added individually (1 g) in sodium phosphate buffer (pH 7; 60 ml) and homogenised using an Ultraturrax blender (10 min). Tomatoes (89 g) were macerated with aliquots (2 ml; equivalent to 33 mg of surfactant) of each type of surfactant/emulsifier in buffer.
2.7. API-MS A Platform Quadrupole mass spectrometer (Micromass, Altrincham, UK) operating in the API positive ion mode was fitted with a custombuilt air-sampling interface (Linforth and Taylor, 1999). The following m/z values and cone voltages (CV) were used: isobutylthiazole (m/z 142; CV 37), 6-methyl-5-hepten-2-one (m/z 127; CV 20), hexanal (m/z 101; CV 12), hexenal (m/z 99; CV 15), methylbutanal (m/z 87; CV 18), hexenol (m/z 83; CV 18), methylbutanol (m/z 71; CV 15), ethanol (m/z 47; CV 15) and acetaldehyde (m/z 45; CV 15). Calibration was achieved using authentic standards as described previously (Taylor et al., 2000). Concentrations of the samples were expressed as mg volatile compound/m3 air. Although data collection occurred on a millisecond scale, data points were extracted every 0.2 min so that statistical variation could be more easily calculated at regular time points and to reduce the data file size for easier handling.
3. Results and discussion
3.1. Maceration de6ice The maceration device was designed to macerate individual tomato fruits (up to 100 g) rapidly and in a consistent manner, but without breaking the tomato fruit seeds, which contain lipid and
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can contribute to the products of lipid oxidation. Since this does not usually occur in vivo, it is possible to create artefacts by high shear homogenisation of tomato fruits. The short maceration time proved effective for the ripe fruits used in this study but not for hard, unripe fruit, which were poorly macerated. The rapidity of the technique (and the use of several maceration devices) enabled a typical sample to be run in 3 min with less than 1 min delay for changeover. Total time per sample was less than 5 min. It was therefore possible to analyse around 100 tomato samples per day, allowing measurement of fruit-to-fruit variation as well as minimising the time to analyse a batch of fruit, as it is known that the rapid metabolic changes in ripe fruit can result in a change in flavour quality (Stern et al., 1994). For some experiments, longer incubation times (15 min after maceration) were used to study the appearance of certain products (e.g. C6 alcohols). Analysis by API-MS in the Selected Ion Mode was chosen so as to monitor only the compounds of interest and to increase the sensitivity of detection. Because resolution was by mass alone, the system was unable to differentiate between positional isomers or stereoisomers. Therefore, the signal for ‘methylbutanals’ represented the sum of the two isomers 2-methylbutanal and 3-methylbutanal; similarly, the signal for ‘hexenals’ represented the sum of the isomers (E)-2-hexenal and (Z)-3-hexenal. The sensitivity depends on the type of volatile molecule analysed, with ionisation efficiency and the amount of chemical noise at that mass determining overall sensitivity (Taylor et al., 2000). Table 1 shows the sensitivity of the APIMS for compounds above the tomato macerate and the odour thresholds of those compounds as this is the analytical limit of interest; i.e. if the compounds are present below the odour threshold, they are unlikely to contribute to tomato aroma unless there is synergism between flavour compounds (Fisher and Scott, 1997). The minimum detectable limits are estimates, based on a signal to noise calculation. The odour threshold data in the literature are very variable due to differences in methods of testing and differences between isomeric forms. This is particularly true for the hexenals where the value of 0.09 mg/m3 is
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Table 1 Comparison of the minimum detectable gas phase concentration (described in Section 2.3) for each volatile compound to the corresponding odour threshold Compound
Minimum detectable concentration (Cgmin) (mg/m3)
Isobutylthiazole 2.7 6-Methyl-5-hepten 1.9 -2-one Hexanal 420.5 12.3 Hexenal Methylbutanal 161.2 Hexenol 16.7 Methylbutanol 1.0 Ethanol 1.7 Acetaldehyde 2.0 a b
Odour threshold (mg/m3)
No data 300–500b 40a 0.09–480a 3–6a 4–16a 100–200b 20–76 000b 41a
Data are from (Rychlik et al., 1998). Data are from (Van Gemert and Nettenbreijer, 1977).
Table 2 Variation in release from the nine compounds from three replicates from each of three tomatoes Compound
Tomato 1
Isobutylthia-z Not detected ole 6-Methyl-5-he 3.39 pten-2-one Hexanal 7.81 Hexenal 4.62 Methylbu-tana 7.74 l Hexenol 7.55 Methylbu-tano 1.69 l Ethanol 9.69 Acetaldehyde 3.10
Tomato 2
Tomato 3
Not detected
Not detected
13.30
6.61
17.95 19.27 13.01
27.93 26.24 9.11
16.21 15.13
25.70 12.23
25.18 10.40
12.38 9.81
Values are the percentage coefficients of variation (CV = SD× 100/mean) for the release at 2 min after maceration. Isobutylthiazole was not present in these fruits.
the odour threshold for (Z)-3-hexenal and 480 mg/m3 is the value for (E)-2-hexenal. The isomers of hexenol and methylbutanol do not show such large differences (Table 1). For most compounds, the API-MS is detecting the volatile compounds below, or around, their odour thresholds despite the dilution effect from the maceration vessel and
the data are therefore comparable to the aroma detection of humans when they consume tomatoes. The reproducibility of the maceration device was tested using three replicates cut from a large beef tomato. The amounts of the nine compounds liberated after 2 min were measured and variation expressed as the percentage coefficient of variation (Table 2). For the three tomatoes tested, no isobutylthiazole could be detected and variation for the other compounds ranged from under 10% (tomato 1) to around 20% (tomatoes 2 and 3).
3.2. Fruit-to-fruit 6ariation Individual tomato fruits from one batch were macerated and release of the nine compounds followed. The time course of release of hexanal and methylbutanal is given in Fig. 2 and these traces are typical of the other seven compounds. Data represent the average concentration for five replicates and error bars correspond to the standard deviation for each data point. Error bars were relatively small for hexanal for the first minute and then increased with time as the concentration built up. The corresponding coefficients of variation (SD×100/mean) calculated for these data were all below 25% (data not shown). The variation was less than that noted by Brauss et al. (1998) who measured the concentration in the nasal expired air of people during eating of tomatoes. The in vivo experiments had variation in the range 27 to 61%, which suggests that the eating process was variable or it may reflect the previously reported wide range of fruit-to-fruit variation in tomatoes (Kazeniac and Hall, 1970). For experiments to study the biochemistry of the lipid oxidation pathway in macerated tomato fruits, 25% variation was considered acceptable and further experiments utilised five replicates.
3.3. Temporal differences in release Typical traces showing the release patterns obtained for the nine selected compounds are presented in Fig. 3. Fruits were blended at t= 0 min and monitoring carried out for 3 min after macer-
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ation. The maceration device was disconnected from the API-MS around 3.5 min, which accounted for the rapid drop in volatile concentrations for all compounds around this time (Fig. 3). Care must be taken in interpreting these traces as they represent the net concentration in the headspace which is a function of: (1) partition coefficient; (2) dilution and removal by the air flow; and (3) possible synthesis or degradation of the compounds. However, the temporal differences in release suggest different origins for these compounds. Two clear types of release behaviour can be differentiated in Fig. 3: some compounds showed rapid release immediately after maceration, reaching maximum concentration within the first 30 s while the concentration of other compounds increased at a steady rate, reaching a maximum concentration after 2 min. Isobutylthiazole, 6-methyl-5-hepten-2-one, methylbutanal, methylbutanol and acetaldehyde belonged to the former group while the C6 compounds hexenal, hexanal and hexenol comprised the latter group. The rapid release behaviour suggested these compounds were preformed in fruit prior to maceration. The release pattern observed was indicative of release into the headspace, followed by
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dilution and depletion of the compounds from the tomato matrix. Methylbutanal and, to a lesser extent, acetaldehyde and methylbutanol intensity decreased much faster than the other compounds, which may have been due to their relative volatility or indicative of further degradative processes. The initial concentration of ethanol released during the first 30 s after disruption increased steadily for a further 2 min. This suggests the presence of some ethanol in the intact fruit followed by a slow partition into the gas phase. The second type of release behaviour involved compounds, which were formed only after tissue disruption and were derived from the lipid oxidation pathway (Galliard et al., 1977). Closer examination revealed differences in the release patterns for the C6 aldehydes. Hexanal was produced slowly and reached its maximum intensity after 3 min (typically around 5–6 min, then slowly decreased, data not shown), whereas hexenal exhibited a rapid increase during the first 2 min and reached its maximum intensity around 2–3 min before starting to decrease. Given the various mechanisms that determine headspace concentration of these compounds, precise interpretation of the curves was not possible. It is interesting to note that the decrease in hexenal may be linked to
Fig. 2. Time course release of hexanal and methylbutanal after maceration of tomatoes. Data represent the mean of five replicates; error bars are 9 SD.
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Fig. 3. Time course release of nine different volatile compounds from tomato after maceration of the tissue (t= 0 min); intensity expressed on a relative scale.
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Fig. 4. Time course release of methylbutanals after disruption of the tissue in presence of exogenous linoleic (18:2) and linolenic acid (18:3). Data are the mean of five replicates; error bars are 9SD.
Fig. 5. Time course release of hexanal after disruption of the tissue in presence of exogenous linoleic (18:2) and linolenic acid (18:3). Data are the mean of five replicates; error bars are 9 SD.
the hypothesis suggested by Riley and coworkers (Riley and Thompson, 1998) that hexenal has a greater reactivity than hexanal for acetal condensation with sugars (into a non-volatile form), thereby reducing its headspace concentration. The results from these maceration studies showed similar release patterns to those obtained in vivo previously (Brauss et al., 1998). Therefore, this model system may have applications in mimicking what happens in the mouth.
3.4. Addition of fatty acids The temporal release patterns of hexanal, hexenal and hexenol from tomatoes blended with buffer alone, or blended with buffer containing either linoleic (18:2) or linolenic (18:3) acids or a mixture of both are shown in Figs. 4–7. To ensure that addition of emulsions did not affect the release properties of the volatiles, the effect of exogenous fatty acids on the release of a non-lipid
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oxidation product, exemplified by the methylbutanals, was studied (Fig. 4). Monitoring was carried out for 15 min after disruption of the tissue to study the long-term effects after maceration but no significant differences in release were seen in the presence or absence of the fatty acid emulsion, suggesting that release properties were unaffected. The same trends were observed for the other five preformed volatiles monitored (isobutylthiazole, methylbutanols, 6-methyl-5hepten-2-one, ethanol, acetaldehyde; data not shown). When linoleic acid was added, hexanal levels
(Fig. 5) were elevated 12 times above control levels alone. Given that 18:2 is the precursor of hexanal, the results are not totally unexpected. Addition of extra 18:3 had no significant effect on hexanal production while a mixture of 18:2 and 18:3 elicited an intermediate response which was somewhat lower than expected despite there being the same amount of 18:2 present in the mixture compared to the 18:2 sample alone. Interpretation of this result on the data available cannot be unequivocal. Although it is attractive to suggest competition between the 18:2 and 18:3 substrates, the emulsion for the mixture also contained twice
Fig. 6. Time course release of hexenals after disruption of the tissue in presence of exogenous linoleic (18:2) and linolenic acid (18:3). Data are the mean of five replicates; error bars are 9 SD.
Fig. 7. Time course release of hexenols after disruption of the tissue in presence of exogenous linoleic (18:2) and linolenic acid (18:3). Data are the mean of five replicates; error bars are 9 SD.
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as much emulsifier (Tween 20) and, as shown below, the extra emulsifier may be the primary reason for the decrease. However, overall, the results suggest that the production of hexanal is limited by the availability of 18:2 substrate, rather than through a lack of enzyme activity. Fig. 6 presents the data for hexenals. Again the natural precursor (18:3) produced substantially more hexenals than the control or the sample with extra 18:2 added. Around 2 min after maceration, there was about 14 times more hexenals in the sample with added 18:3. Again the mixture of 18:2 and 18:3 showed intermediate behaviour and the same interpretation, presented above, applies. Volatile aldehyde production is described as a response to wounding or tissue disruption (Galliard et al., 1977; Buttery and Ling, 1993). Previous studies with tomatoes have shown that C6 aldehydes accumulate over time following tissue maceration. This was shown using a purge and trap technique of the volatile followed by GC analysis (Riley and Thompson, 1998). Nevertheless, a measurement in ‘real time’ allows a more detailed study of the effect of disruption on the formation of C6 aldehydes and alcohols. These results confirm that C6 aldehydes and alcohols are formed during the disruption of the fruit as no sign of them can be seen immediately prior to maceration and their presence in the headspace suggests de novo synthesis. A rapid chain of enzymatic reactions occurs in the fruit, leading to the formation of hexanal within seconds after the disruption. There were some subtle differences in the rates of formation for hexanal and hexenals which may reflect different specificities of the enzymes for the two products. Hexenals reached their maximum intensity (Imax) in the control sample 3 min after maceration, whereas the Imax for hexanal was obtained only after 6 min (data not shown). The results in Figs. 5 and 6 show that fatty acids are rapidly oxidized and cleaved by hydroperoxide lyase to give the corresponding aldehydes. These two enzymes are believed to work closely together, maybe because they are located in the same part of the cell (Smith et al., 1997). The conversion of aldehydes to alcohols in tomato macerates however, seems less straightfor-
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ward (Prestage et al., 1999). Fig. 7 shows the headspace concentration of hexenols after maceration in the presence and absence of added fatty acids. From Fig. 6, one might expect the peak of hexenols concentration to occur after the peak of hexenal (i.e. around 5 min) but Fig. 7 shows an elevated level in the first few minutes of the time course and, thereafter, levels not substantially different from the control. From these results, it seems that the production of the C6 alcohols is not greatly affected by the amount of precursor aldehyde. In Fig. 7, the sample containing 18:2 should show the same behaviour as the control but actually shows a steady increase with time. This can be explained as the ion monitored for hexenal (m/z 83) is derived from dehydration of the molecular ion (M-H2O+ H)+. Hexanal has the same molecular weight as hexenol and also undergoes some dehydration to yield an ion with the same m/z value. Normally the m/z signal from hexanal is very low but, in this case, with large amounts of hexanal (see Fig. 5), the contribution of the hexanal-derived m/z 83 ion is significant and masks the true behaviour of hexenols.
3.5. Effect of surfactants For the experiments described above, Tween 20 was used as the surfactant to emulsify the fatty acids so they could be transported and assimilated by the enzymes in the tomato macerate. Tween 20 has been widely used for this purpose but is also known as an inhibitor of some enzymes (Srinivasulu and Rao, 1993). To assess the effect of Tween 20 on the enzymes producing the C6 compounds, and on the general physicochemical environment, Fig. 8 shows the maximum amounts of hexanal and methylbutanals formed after 3 min in the absence and presence of three different emulsifiers/stabilisers. It is clear that Tween 20 reduced the amount of hexanal released into the headspace compared to lecithin and glycerol monostearate. The most likely explanation for this behaviour was inhibition of one of the steps in the lipoxygenase pathway leading to C6 compound formation. The effects on the release of methylbutanals into the headspace were less clear cut with no significant differences between the
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Fig. 8. Effect of emulsifiers on the release of hexanal and methylbutanals (Imax is the maximum concentration over a 3 min incubation period after maceration).
Fig. 9. Time course release of hexanal after disruption of the tissue in presence of exogenous linoleic (18:2) and linolenic acid (18:3) using lecithin as emulsifier. Data are the mean of five replicates; error bars are 9SD.
four samples; again this strongly suggests that Tween 20 interferes with the lipoxygenase pathway specifically, rather than through some general effect. A further potential cause of differences is that the emulsions may affect partition of the volatiles produced between the aqueous and gas phases. To test this, the amount of hexanal released from tomatoes macerated in the presence of a lecithin/18:0 emulsion was compared with release from tomatoes macerated with 18:0 alone. There was no significant difference between the samples (data not shown) adding credence to the
suggestion above that Tween 20 affects the lipoxygenase pathway directly.
3.6. Release with lecithin as fatty acid emulsifier Release curves for hexanal and hexenal, comparing maceration with buffer alone (control) and with fatty acids in buffer are presented in Figs. 9 and 10. A similar pattern of behaviour was observed for the lecithin samples compared to the Tween 20 samples when fatty acids were added. However, the concentrations measured for the C6
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compounds generated from the lecithin emulsions were higher than with Tween 20 (compare Figs. 5 and 9 (hexanal) and Figs. 6 and 10 (hexenal)). Direct comparison between the figures should be subject to caution because the lecithin and Tween 20 experiments were carried out on different batches of tomatoes and that might account for some of the differences in this instance. However, the data in Fig. 8 show clearly that, even on the same batch of tomatoes, there were substantial differences in C6 volatiles generated from Tween 20 and lecithin emulsions. Since many assays for lipoxygenase have been carried out using Tween 20, some of the published data may underestimate the true activity.
3.7. Addition of enzymes from the lipoxygenase pathway To study the lipoxygenase pathway further, commercial sources of the various enzymes were obtained and added to macerates as solutions or as emulsions. The release of the C6 compounds was then monitored. However, the addition of lipase, phospholipases, lipoxygenase or alcohol dehydrogenase did not significantly change the volatile release profile of the tomatoes for any of the samples (data not shown). Previous work has suggested that some lipid oxidation enzymes are membrane bound which may explain why the addition of exogenous enzymes was ineffective in
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changing the course of the reaction. In Arabidopsis, multiple forms of phospholipase D are involved in the wounding response (Wang et al., 2000) and there appear to be interactions between phospholipase, lipoxygenase and jasmonic acid production. The data from the present paper and from Wang, shows that enzymatic manipulation of the reaction postharvest to modify the flavour characteristics of the fruits is not facile.
4. Conclusions Using this maceration device connected to the API-MS, the measurement of volatiles released from tomatoes upon disruption was rapid and consistent. The rapidity of the measurement allowed analyses of individual fruits (the basis on which consumers often assess tomato quality) while giving detailed information on the generation of preformed and macerate-formed volatiles. The nine selected compounds gave a good fingerprint of the overall aroma profile of the fruit. Information obtained on the dynamics of the reactions allowed the biochemical origin of volatile compounds, (preformed in the ripening fruit or generated during the maceration process) to be identified. Using this system also provided opportunities to study the lipid oxidation pathway in vivo by adding extra substrate although the direct addition of enzymes was not as success-
Fig. 10. Time course release of hexenals after disruption of the tissue in presence of exogenous linoleic (18:2) and linolenic acid (18:3) using lecithin as emulsifier. Data are the mean of five replicates; error bars are 9 SD.
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ful. There are also drawbacks. If the total amount formed in the liquid phase needs to be calculated, various assumptions about mass transfer between the liquid and gas phases during maceration must be made as measurements were all made in the gas phase. It may be possible to calibrate the system by carrying out measurement of total and headspace volatiles and producing a calibration. However, the main use of this technique is as a rapid tool to compare flavour quality of large batches of fruits and offers many possibilities for further studies on tomato flavour.
Acknowledgements We gratefully acknowledge the financial support of the European Commission (Marie Curie Research Training Grant ERB4001GT974094).
References Baldwin, E.A., Nisperos-Carriedo, M.O., Moshonas, M.G., 1991. Quantitative analysis of flavor and other volatiles and for certain constituents of two tomato cultivars during ripening. J. Am. Soc. Hortic. Sci. 116, 265 –269. Brauss, M.S., Linforth, R.S.T., Taylor, A.J., 1998. Effect of variety, time of eating, and fruit-to-fruit variation on volatile release during eating of tomato fruits (Lycopersicon esculentum). J. Agric. Food Chem. 46, 2287 –2292. Buttery, R.G., 1993. Quantitative and sensory aspects of flavour of tomato and other vegetables and fruits. Flavor Science. Acree, T.E., Teranishi, R., American Chemical Society, Washington DC, 259 –285. Buttery, R.G., Ling, L., 1993. Enzymatic production of volatiles in tomatoes. Progress in flavour precursor studies. In: Schreier, P., Winterhalter, P. (Eds.), Proceedings of the International Conference, Wu¨ rzburg, Germany 1992. Allured Publishing Corp., Carol Stream, IL. Buttery, R.G., Teranishi, R., Ling, L.C., 1987. Fresh tomato aroma volatiles: a quantitative study. J. Agric. Food Chem. 35, 540 – 544. Buttery, R.G., Teranishi, R., Flath, R.A., Ling, L.C., 1989. Fresh tomato volatiles-composition and sensory studies. Flavor Chemistry: New Trends and Developments. Buttery, R.G., Shahidi, F., Teranishi, R., ACS symposium Series 388, American Chemical Society: Washington, DC, 213 – 222. Eriksson, C.E., 1979. Review of biosynthesis of volatiles in fruits and vegetables since 1975. Progress in Flavour Research. Land, D.G., Nursten, H.E., Applied Science, London, 159 – 174.
Fisher, C., Scott, T.R., 1997. Food Flavours-Biology and Chemistry. RSC Paperbacks, The Royal Society of Chemistry, 1 – 12. Galliard, T., Matthew, J.A., Wright, A.J., Fishwick, M.J., 1977. The enzymatic breakdown of lipids to volatile and non volatile carbonyl fragments in disrupted tomato fruits. J. Sci. Food Agric. 28, 863 – 868. Gardner, H.W., 1995. Biological roles and biochemistry of the lipoxygenase pathway. Hortscience 30, 197 – 205. Gray, D.A., Prestage, S., Linforth, R.S.T., Taylor, A.J., 1999. Fresh tomato specific fluctuations in the composition of lipoxygenase-generated C6 aldehydes. Food Chem. 64, 149 – 155. Griffiths, A., Prestage, S., Linforth, R., Zhang, J.L., Taylor, A., Grierson, D., 1999. Fruit-specific lipoxygenase suppression in antisense-transgenic tomatoes. Postharvest Biol. Technol. 17, 163 – 173. Kazeniac, S.J., Hall, R.M., 1970. Flavor chemistry of tomato volatiles. J. Food Sci. 35, 519 – 530. Linforth, R.S.T., Ingham, K.E., Taylor, A.J., 1996. Time course profiling of volatile release from foods during the eating process. Flavour Science: Recent developments. Mottram, D.S., Taylor, A.J., Royal Society of Chemistry, UK, 361– 368. Linforth, R.S.T., Savary, I., Pattenden, B., Taylor, A.J., 1994. Volatile compounds found in expired air during eating of fresh tomatoes and in the headspace above tomatoes. J. Sci. Food Agric. 65, 241 – 247. Linforth, R.S.T., Taylor, A.J., 1999. Apparatus and methods for the analysis of trace constituents of gases. US Patent. 5,869,334. Petro-Turza, M., 1986 – 1987. Flavor of tomato and tomato products. Food Rev. Int. 2, 309 – 351. Prestage, S., Linforth, R.S.T., Taylor, A.J., Lee, E., Speirs, J., Schuch, W., 1999. Volatile production in tomato fruit with modified alcohol dehydrogenase activity. J. Sci. Food Agric. 79, 131 – 136. Riley, J.C.M., Thompson, J.E., 1998. Ripening-induced acceleration of volatile aldehyde generation following tissue disruption in tomato fruit. Physiol. Plant 104, 571 – 576. Rychlik, M., Schieberle, P., Grosch, W., 1998. Compilation of odor thresholds, odor qualities and retention indices of key food odorants. Munich, Deutsch Forschungsanstalt fur Lebensmittelchemie. Smith, J.J., Linforth, R., Tucker, G.A., 1997. Soluble lipoxygenase isoforms from tomato fruit. Phytochemistry 45, 453 – 458. Srinivasulu, S., Rao, A.G.A., 1993. Kinetic and structural studies on the interaction of surfactants with lipoxygenase L1 from soybeans (Glycinemax). J. Agric. Food Chem. 41, 366 – 371. Stern, D.J., Buttery, R.G., Teranishi, R., Ling, L., Scott, K., Cantwell, M., 1994. Effect of storage and ripening on fresh tomato quality.1. Food Chem. 49, 225 – 231. Taylor, A.J., 1996. Volatile flavor release from foods during
F. Boukobza et al. / Posthar6est Biology and Technology 23 (2001) 117–131 eating. Crit. Rev. Food Sci. Nutr. 36, 765 – 784. Taylor, A.J., Linforth, R.S.T., Harvey, B.A., Blake, A., 2000. Atmospheric pressure chemical ionisation mass spectrometry for in vivo analysis of volatile flavour release. Food Chem. 71, 327 – 338. Van Gemert, L.J., Nettenbreijer, A.H., 1977. Compilation of
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odour threshold values in air and water. Zeist, National Institute for Water Supply. Wang, C., Zien, C.A., Afitlhile, M., Welti, R., Hildebrand, D.F., Wang, X., 2000. Involvement of phospholipase D in wound-induced accumulation of jasmonic acid in Arabidopsis. The Plant Cell 112, 2237 – 2246.
Measurement of lipid oxidation-derived volatiles in fresh tomatoes Fabienne Boukobza a, Patrick J. Dunphy b, Andrew J. Taylor a,* a
Di6ision of Food Sciences, School of Biosciences, Uni6ersity of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK b Unile6er Research Colworth Laboratory, Colworth House, Sharnbrook, Bedford MK44 1LQ, UK Received 21 February 2001; accepted 2 May 2001
Abstract A rapid, low-shear, reproducible maceration device was developed for tomato fruit and coupled to an Atmospheric Pressure Chemical Ionisation-Mass Spectrometer (API-MS) so that real time, simultaneous measurement of nine key volatile compounds released upon disruption of the tomato tissue was achieved. The minimum detection level of the volatiles was around the odour threshold concentrations of the compounds and variation between release from macerated tomatoes was between 10 and 25%. Based on their temporal differences in release, the compounds were classified into two groups: the preformed compounds already present in the ripe fruit (isobutylthiazole, 6-methyl-5hepten-2-one, methylbutanal, methylbutanol, ethanol and acetaldehyde) and the enzyme generated compounds formed only upon maceration (hexanal, hexenal and hexenol). The addition of exogenous linoleic and linolenic acids (2 mg/g FW) to tomatoes prior to maceration influenced the amount of C6 aldehydes produced. Hexanal was increased 12-fold in the presence of linoleic acid, while hexenal was increased 14-fold in the presence of linolenic acid. Addition of both fatty acids gave intermediate behaviour. Addition of enzymes from the lipid oxidation pathway (lipase, phospholipase, lipoxygenase and alcohol dehydrogenase) at various concentrations, in buffer or emulsion and with or without fatty acids caused no significant changes in the release profile of tomatoes. Results highlighted the inhibitory effect of Tween 20 used as surfactant in emulsions. Alternative surfactants (lecithin and glyceryl monostearate) were tested and did not influence volatile generation. The paper demonstrates that the maceration device coupled to the API-MS can be used for rapid screening of tomato fruit as well as for studying the factors that influence the lipoxygenase pathway. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Lipoxygenase; Flavour; APCI; Lipid oxidation; Fatty acid substrate
1. Introduction
* Corresponding author. Tel.: + 44-115-9516144; fax: + 44115-9516154. E-mail address: [email protected] (A.J. Taylor).
Fresh tomato flavour is determined by a relatively small number of volatile aroma compounds and non-volatile taste compounds (Petro-Turza, 1986 –1987). Among the volatile components, sixcarbon (C6) compounds play a major role giving
0925-5214/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S0925-5214(01)00122-3
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tomato its fresh ‘top-note’. These C6 volatile compounds are formed upon breakdown of the fruit (Galliard et al., 1977). Tissue disruption results in the degradation of endogenous lipids to fatty acids, then oxidation to yield a hydroperoxide followed by selective lyase cleavage to form hexanal and hexenal from the 13-hydroperoxides of linoleic and linolenic acids, respectively (Galliard et al., 1977). Conversion of the C6 aldehydes to the corresponding alcohols may also occur through alcohol dehydrogenase (ADH). The series of reactions is often termed the lipoxygenase pathway and has been extensively studied (Galliard et al., 1977; Eriksson, 1979; Buttery, 1993; Riley and Thompson, 1998; Gray et al., 1999; Griffiths et al., 1999). Its general importance in plants (Gardner, 1995) as well as its specific role during the ripening of tomatoes has been described (Baldwin et al., 1991; Buttery and Ling, 1993; Riley and Thompson, 1998). The lipoxygenase pathway is involved in the production of flavour compounds as well as in plant defence, plant development and plant communication. Recent studies of the pathway have shown that it is more complex than originally postulated by Galliard, as different isoforms of the lipoxygenase enzyme exist and appear to have different selectivity and activity towards fatty acid substrates (Gray et al., 1999). A transgenic approach was used to determine the relative importance of lipoxygenase (Griffiths et al., 1999) and of alcohol dehydrogenase enzyme (Prestage et al., 1999) in the formation of the C6 aldehydes and alcohols in tomato fruits. These studies demonstrated both the complexity of this system and highlighted the necessity for rapid assays so that representative samples of modified fruits could be assayed for their ability to generate the different C6 compounds. Previous analyses of tomato aroma volatiles have used solvent extraction of tomato fruit macerates (Buttery et al., 1987, 1989; Buttery, 1993), followed by Gas Chromatography-Mass Spectrometry (GC-MS) to identify and quantify the key compounds responsible for tomato aroma. However, extraction, followed by GC-MS is timeconsuming and limits the number of samples that can be analysed in a day (typically 5– 8 in our lab)
as well as making it difficult to follow the dynamics of the reaction. It should also be remembered that tomato fruit is a living organism whose composition varies rapidly with time. It is therefore desirable to analyse tomato fruits at the same developmental stage, but, due to the unpredictable ripening pattern of tomato fruit, this results in an uneven supply of samples for analysis, and can sometimes overwhelm analyses based on extraction/GC-MS. An alternative method of volatile analysis is to analyse the headspace above tomato macerate (Baldwin et al., 1991; Linforth et al., 1994) and concentrate volatiles on some form of trapping material prior to GC-MS analysis. This simplifies the extraction step but provides an indirect measure of the volatiles present in the tomato itself. Depending on the method of maceration and headspace sampling, it is possible to estimate total amounts by assuming the volatile is in equilibrium between the air and liquid phases and then applying a correction for the air–liquid partition coefficient and the trapping efficiencies of each compound. Alternatively the results can be used to compare the volatile compositions of tomatoes providing the assay conditions (and the tomato composition) do not vary significantly. However, GC-MS is still necessary to identify and quantify the volatile components in the headspace, which limits the number of samples that can be analysed in a day. With the advent of direct headspace analyses like Atmospheric Pressure Ionisation Mass Spectrometry (API-MS) (Linforth et al., 1996; Taylor, 1996; Taylor et al., 2000) it is now possible to measure mixtures of volatiles in headspace directly with a millisecond time delay. Using carefully controlled conditions in the ion source (Taylor et al., 2000), quantitative, real-time analysis of volatile mixtures can be obtained at concentrations around 10 ppbv (nl of vapour in a litre of air). The technique has been used for a preliminary study of volatile release from tomatoes by sampling air directly from the nose of a subject eating the fruits to demonstrate the effect of variety and fruit-to-fruit variation (Brauss et al., 1998). Measurement in vivo is desirable in that it reflects the volatile profile reaching the olfactory receptors and therefore relates well with sensory
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perception. Mastication in vivo is variable, however, and, for a rapid, reproducible biochemical assay for volatile products of lipid oxidation, mechanical maceration of tomato is preferred. Released volatiles are then removed from the headspace by a controlled gas flow and direct analysis of the headspace achieved by API-MS. This paper describes real-time monitoring of the C6 compounds (and other volatile compounds) in the headspace of tomatoes using controlled maceration to mimic tomato breakdown in mouth. A further aim of this work is to study the lipoxygenase pathway in tomato macerate by the addition of enzymes and substrates.
2. Material and methods
2.1. Tomato fruits Batches of tomatoes (Lycopersicon esculentum L., cv. Flavia) were purchased from the local
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supermarket throughout the season. Each batch was stored at room temperature (21 °C) for a maximum of 3 days and used for experiments when fruits were all fully red. The average weight was 89 9 4 g. To avoid excessive variation in raw material composition, each batch of tomatoes was used to generate data for a single factor, e.g. maceration optimisation (see Section 2.2 and Section 2.3), effect of enzymes (see Section 2.4) or effect of substrate (see Section 2.5).
2.2. Tomato maceration Tomatoes were blended in the maceration device (volume 355 ml) made from a commercial food blender (Phillips, type HR2810/A; Fig. 1). Three Swagelok bulkhead fittings were bolted into the blender lid. The left and right fittings were connected to PTFE lines (4 mm OD) for flushing air through the blender. The centre aperture was fitted with a septum so that additional substrates and enzymes could be added via a syringe (Fig.
Fig. 1. Schematic of the maceration device and connections to the APCI-MS.
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1). The intact fruit was placed inside the blender, which was then sealed. The headspace within the device was continually flushed with air (170 ml/ min) so that volatiles formed were removed rapidly (total volume of device 355 ml, so one change of air every 2 min). There was dilution of the headspace by the incoming air, but it was constant. On the outlet side, a portion of the airflow was continuously sampled into the API-MS at 11.5 ml/min through a heated transfer line (0.53 mm id fused silica tube held at 100 °C). The excess of air flow was vented to atmosphere (Fig. 1). The headspace was initially monitored for about 30 s to obtain a baseline measure of volatiles above the intact fruits, then the fruit was blended (2– 5 s) and monitoring of the headspace above the macerate carried out for a further 3– 15 min to follow the release of the selected volatile compounds as described below.
2.3. Sensiti6ity and reproducibility of maceration de6ice Sensitivity for the nine compounds was estimated by first calibrating the API-MS interface with a solution of each compound in hexane (Taylor et al., 2000). The gas phase concentration for a macerated tomato sample was then calculated (Cg) and the signal to noise ratio (SNc) for that concentration determined using the Masslynx software on the API-MS (Micromass, Altrincham, UK). The minimum detectable gas phase concentration of volatile (Cgmin) was estimated (assuming a signal to noise ratio of 5:1 is necessary for detection) using the following calculation: Cgmin = (Cg × 5)/SNc. Reproducibility of the device was tested using three large beef-type tomatoes (about 230 g), which were divided into three portions (77 g each) and macerated. It was assumed that each portion of the tomato was identical in terms of composition and release behaviour. Portions of tomato were macerated as described in Section 2.2 and volatile release monitored as described in Section 2.7. The headspace concentrations of the nine selected compounds were determined at 2 min after maceration and variation expressed as the coefficient of variation (standard deviation× 100/mean).
2.4. Addition of enzymes Enzymes from the lipid oxidation pathway were injected individually through the septum onto the top of the intact tomato (about 90 g) before maceration. Enzyme type and amounts introduced per tomato were as follows: phospholipase A2 from porcine pancreas (120, 240, and 480 units/ tomato), phospholipase D from Streptomyces chromofuscus (800 or 3500 units/tomato), lipase from Mucor meihei (20000 or 26000 units/tomato), lipoxygenase from soybean (1500, 5000 or 10000 units/tomato) and alcohol dehydrogenase from bakers yeast (3300 or 6000 units/tomato), all purchased from Sigma. Enzymes were added either in a sodium phosphate buffer alone (pH 7), in buffer with emulsifier (Tween 20) or in an emulsion prepared with an equal amount of Tween 20 and linoleic acid. Solutions and emulsions were freshly prepared (10 ml) and homogenised with an Ultraturrax blender (10 min). For each experiment, an aliquot (1 ml) was added to the tomato fruit.
2.5. Addition of free fatty acids Emulsions were prepared by mixing linoleic or linolenic acid (1 g) with Tween 20 (1 g) in sodium phosphate buffer (60 ml) with an Ultraturrax blender (10 min) at pH 7. When both fatty acids were added together (1 g each), they were mixed with an equal amount of Tween 20 (2 g). Emulsion (10 ml) was injected onto the intact tomato, then the fruit was blended (2–5 s), resulting in efficient mixing of the macerated tissue and the emulsion. This macerated mixture was incubated for 15 min and volatile compounds sampled and analysed as in Section 2.7. The final amount of fatty acid added was around 2 mg/g FW. This corresponds to an excess of free fatty acid. The estimated concentrations of 18:2 and 18:3 in tomato calculated from previous experiments (Gray et al., 1999) are 0.223 and 0.05 mg/g FW, respectively. Therefore, 10–40 times more fatty acids were added compared to the endogenous situation. Blank runs verified that no volatiles were released from the emulsion on its own. As a further control, tomatoes were macerated with the
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same volume of buffer (10 ml) mixed with Tween 20 but containing no enzymes.
2.6. Effect of emulsifier on lipid oxidation pathway The following surfactants/emulsifiers were compared: Tween 20, lecithin and glyceryl monostearate (GMS). Each compound was added individually (1 g) in sodium phosphate buffer (pH 7; 60 ml) and homogenised using an Ultraturrax blender (10 min). Tomatoes (89 g) were macerated with aliquots (2 ml; equivalent to 33 mg of surfactant) of each type of surfactant/emulsifier in buffer.
2.7. API-MS A Platform Quadrupole mass spectrometer (Micromass, Altrincham, UK) operating in the API positive ion mode was fitted with a custombuilt air-sampling interface (Linforth and Taylor, 1999). The following m/z values and cone voltages (CV) were used: isobutylthiazole (m/z 142; CV 37), 6-methyl-5-hepten-2-one (m/z 127; CV 20), hexanal (m/z 101; CV 12), hexenal (m/z 99; CV 15), methylbutanal (m/z 87; CV 18), hexenol (m/z 83; CV 18), methylbutanol (m/z 71; CV 15), ethanol (m/z 47; CV 15) and acetaldehyde (m/z 45; CV 15). Calibration was achieved using authentic standards as described previously (Taylor et al., 2000). Concentrations of the samples were expressed as mg volatile compound/m3 air. Although data collection occurred on a millisecond scale, data points were extracted every 0.2 min so that statistical variation could be more easily calculated at regular time points and to reduce the data file size for easier handling.
3. Results and discussion
3.1. Maceration de6ice The maceration device was designed to macerate individual tomato fruits (up to 100 g) rapidly and in a consistent manner, but without breaking the tomato fruit seeds, which contain lipid and
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can contribute to the products of lipid oxidation. Since this does not usually occur in vivo, it is possible to create artefacts by high shear homogenisation of tomato fruits. The short maceration time proved effective for the ripe fruits used in this study but not for hard, unripe fruit, which were poorly macerated. The rapidity of the technique (and the use of several maceration devices) enabled a typical sample to be run in 3 min with less than 1 min delay for changeover. Total time per sample was less than 5 min. It was therefore possible to analyse around 100 tomato samples per day, allowing measurement of fruit-to-fruit variation as well as minimising the time to analyse a batch of fruit, as it is known that the rapid metabolic changes in ripe fruit can result in a change in flavour quality (Stern et al., 1994). For some experiments, longer incubation times (15 min after maceration) were used to study the appearance of certain products (e.g. C6 alcohols). Analysis by API-MS in the Selected Ion Mode was chosen so as to monitor only the compounds of interest and to increase the sensitivity of detection. Because resolution was by mass alone, the system was unable to differentiate between positional isomers or stereoisomers. Therefore, the signal for ‘methylbutanals’ represented the sum of the two isomers 2-methylbutanal and 3-methylbutanal; similarly, the signal for ‘hexenals’ represented the sum of the isomers (E)-2-hexenal and (Z)-3-hexenal. The sensitivity depends on the type of volatile molecule analysed, with ionisation efficiency and the amount of chemical noise at that mass determining overall sensitivity (Taylor et al., 2000). Table 1 shows the sensitivity of the APIMS for compounds above the tomato macerate and the odour thresholds of those compounds as this is the analytical limit of interest; i.e. if the compounds are present below the odour threshold, they are unlikely to contribute to tomato aroma unless there is synergism between flavour compounds (Fisher and Scott, 1997). The minimum detectable limits are estimates, based on a signal to noise calculation. The odour threshold data in the literature are very variable due to differences in methods of testing and differences between isomeric forms. This is particularly true for the hexenals where the value of 0.09 mg/m3 is
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Table 1 Comparison of the minimum detectable gas phase concentration (described in Section 2.3) for each volatile compound to the corresponding odour threshold Compound
Minimum detectable concentration (Cgmin) (mg/m3)
Isobutylthiazole 2.7 6-Methyl-5-hepten 1.9 -2-one Hexanal 420.5 12.3 Hexenal Methylbutanal 161.2 Hexenol 16.7 Methylbutanol 1.0 Ethanol 1.7 Acetaldehyde 2.0 a b
Odour threshold (mg/m3)
No data 300–500b 40a 0.09–480a 3–6a 4–16a 100–200b 20–76 000b 41a
Data are from (Rychlik et al., 1998). Data are from (Van Gemert and Nettenbreijer, 1977).
Table 2 Variation in release from the nine compounds from three replicates from each of three tomatoes Compound
Tomato 1
Isobutylthia-z Not detected ole 6-Methyl-5-he 3.39 pten-2-one Hexanal 7.81 Hexenal 4.62 Methylbu-tana 7.74 l Hexenol 7.55 Methylbu-tano 1.69 l Ethanol 9.69 Acetaldehyde 3.10
Tomato 2
Tomato 3
Not detected
Not detected
13.30
6.61
17.95 19.27 13.01
27.93 26.24 9.11
16.21 15.13
25.70 12.23
25.18 10.40
12.38 9.81
Values are the percentage coefficients of variation (CV = SD× 100/mean) for the release at 2 min after maceration. Isobutylthiazole was not present in these fruits.
the odour threshold for (Z)-3-hexenal and 480 mg/m3 is the value for (E)-2-hexenal. The isomers of hexenol and methylbutanol do not show such large differences (Table 1). For most compounds, the API-MS is detecting the volatile compounds below, or around, their odour thresholds despite the dilution effect from the maceration vessel and
the data are therefore comparable to the aroma detection of humans when they consume tomatoes. The reproducibility of the maceration device was tested using three replicates cut from a large beef tomato. The amounts of the nine compounds liberated after 2 min were measured and variation expressed as the percentage coefficient of variation (Table 2). For the three tomatoes tested, no isobutylthiazole could be detected and variation for the other compounds ranged from under 10% (tomato 1) to around 20% (tomatoes 2 and 3).
3.2. Fruit-to-fruit 6ariation Individual tomato fruits from one batch were macerated and release of the nine compounds followed. The time course of release of hexanal and methylbutanal is given in Fig. 2 and these traces are typical of the other seven compounds. Data represent the average concentration for five replicates and error bars correspond to the standard deviation for each data point. Error bars were relatively small for hexanal for the first minute and then increased with time as the concentration built up. The corresponding coefficients of variation (SD×100/mean) calculated for these data were all below 25% (data not shown). The variation was less than that noted by Brauss et al. (1998) who measured the concentration in the nasal expired air of people during eating of tomatoes. The in vivo experiments had variation in the range 27 to 61%, which suggests that the eating process was variable or it may reflect the previously reported wide range of fruit-to-fruit variation in tomatoes (Kazeniac and Hall, 1970). For experiments to study the biochemistry of the lipid oxidation pathway in macerated tomato fruits, 25% variation was considered acceptable and further experiments utilised five replicates.
3.3. Temporal differences in release Typical traces showing the release patterns obtained for the nine selected compounds are presented in Fig. 3. Fruits were blended at t= 0 min and monitoring carried out for 3 min after macer-
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ation. The maceration device was disconnected from the API-MS around 3.5 min, which accounted for the rapid drop in volatile concentrations for all compounds around this time (Fig. 3). Care must be taken in interpreting these traces as they represent the net concentration in the headspace which is a function of: (1) partition coefficient; (2) dilution and removal by the air flow; and (3) possible synthesis or degradation of the compounds. However, the temporal differences in release suggest different origins for these compounds. Two clear types of release behaviour can be differentiated in Fig. 3: some compounds showed rapid release immediately after maceration, reaching maximum concentration within the first 30 s while the concentration of other compounds increased at a steady rate, reaching a maximum concentration after 2 min. Isobutylthiazole, 6-methyl-5-hepten-2-one, methylbutanal, methylbutanol and acetaldehyde belonged to the former group while the C6 compounds hexenal, hexanal and hexenol comprised the latter group. The rapid release behaviour suggested these compounds were preformed in fruit prior to maceration. The release pattern observed was indicative of release into the headspace, followed by
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dilution and depletion of the compounds from the tomato matrix. Methylbutanal and, to a lesser extent, acetaldehyde and methylbutanol intensity decreased much faster than the other compounds, which may have been due to their relative volatility or indicative of further degradative processes. The initial concentration of ethanol released during the first 30 s after disruption increased steadily for a further 2 min. This suggests the presence of some ethanol in the intact fruit followed by a slow partition into the gas phase. The second type of release behaviour involved compounds, which were formed only after tissue disruption and were derived from the lipid oxidation pathway (Galliard et al., 1977). Closer examination revealed differences in the release patterns for the C6 aldehydes. Hexanal was produced slowly and reached its maximum intensity after 3 min (typically around 5–6 min, then slowly decreased, data not shown), whereas hexenal exhibited a rapid increase during the first 2 min and reached its maximum intensity around 2–3 min before starting to decrease. Given the various mechanisms that determine headspace concentration of these compounds, precise interpretation of the curves was not possible. It is interesting to note that the decrease in hexenal may be linked to
Fig. 2. Time course release of hexanal and methylbutanal after maceration of tomatoes. Data represent the mean of five replicates; error bars are 9 SD.
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Fig. 3. Time course release of nine different volatile compounds from tomato after maceration of the tissue (t= 0 min); intensity expressed on a relative scale.
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Fig. 4. Time course release of methylbutanals after disruption of the tissue in presence of exogenous linoleic (18:2) and linolenic acid (18:3). Data are the mean of five replicates; error bars are 9SD.
Fig. 5. Time course release of hexanal after disruption of the tissue in presence of exogenous linoleic (18:2) and linolenic acid (18:3). Data are the mean of five replicates; error bars are 9 SD.
the hypothesis suggested by Riley and coworkers (Riley and Thompson, 1998) that hexenal has a greater reactivity than hexanal for acetal condensation with sugars (into a non-volatile form), thereby reducing its headspace concentration. The results from these maceration studies showed similar release patterns to those obtained in vivo previously (Brauss et al., 1998). Therefore, this model system may have applications in mimicking what happens in the mouth.
3.4. Addition of fatty acids The temporal release patterns of hexanal, hexenal and hexenol from tomatoes blended with buffer alone, or blended with buffer containing either linoleic (18:2) or linolenic (18:3) acids or a mixture of both are shown in Figs. 4–7. To ensure that addition of emulsions did not affect the release properties of the volatiles, the effect of exogenous fatty acids on the release of a non-lipid
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oxidation product, exemplified by the methylbutanals, was studied (Fig. 4). Monitoring was carried out for 15 min after disruption of the tissue to study the long-term effects after maceration but no significant differences in release were seen in the presence or absence of the fatty acid emulsion, suggesting that release properties were unaffected. The same trends were observed for the other five preformed volatiles monitored (isobutylthiazole, methylbutanols, 6-methyl-5hepten-2-one, ethanol, acetaldehyde; data not shown). When linoleic acid was added, hexanal levels
(Fig. 5) were elevated 12 times above control levels alone. Given that 18:2 is the precursor of hexanal, the results are not totally unexpected. Addition of extra 18:3 had no significant effect on hexanal production while a mixture of 18:2 and 18:3 elicited an intermediate response which was somewhat lower than expected despite there being the same amount of 18:2 present in the mixture compared to the 18:2 sample alone. Interpretation of this result on the data available cannot be unequivocal. Although it is attractive to suggest competition between the 18:2 and 18:3 substrates, the emulsion for the mixture also contained twice
Fig. 6. Time course release of hexenals after disruption of the tissue in presence of exogenous linoleic (18:2) and linolenic acid (18:3). Data are the mean of five replicates; error bars are 9 SD.
Fig. 7. Time course release of hexenols after disruption of the tissue in presence of exogenous linoleic (18:2) and linolenic acid (18:3). Data are the mean of five replicates; error bars are 9 SD.
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as much emulsifier (Tween 20) and, as shown below, the extra emulsifier may be the primary reason for the decrease. However, overall, the results suggest that the production of hexanal is limited by the availability of 18:2 substrate, rather than through a lack of enzyme activity. Fig. 6 presents the data for hexenals. Again the natural precursor (18:3) produced substantially more hexenals than the control or the sample with extra 18:2 added. Around 2 min after maceration, there was about 14 times more hexenals in the sample with added 18:3. Again the mixture of 18:2 and 18:3 showed intermediate behaviour and the same interpretation, presented above, applies. Volatile aldehyde production is described as a response to wounding or tissue disruption (Galliard et al., 1977; Buttery and Ling, 1993). Previous studies with tomatoes have shown that C6 aldehydes accumulate over time following tissue maceration. This was shown using a purge and trap technique of the volatile followed by GC analysis (Riley and Thompson, 1998). Nevertheless, a measurement in ‘real time’ allows a more detailed study of the effect of disruption on the formation of C6 aldehydes and alcohols. These results confirm that C6 aldehydes and alcohols are formed during the disruption of the fruit as no sign of them can be seen immediately prior to maceration and their presence in the headspace suggests de novo synthesis. A rapid chain of enzymatic reactions occurs in the fruit, leading to the formation of hexanal within seconds after the disruption. There were some subtle differences in the rates of formation for hexanal and hexenals which may reflect different specificities of the enzymes for the two products. Hexenals reached their maximum intensity (Imax) in the control sample 3 min after maceration, whereas the Imax for hexanal was obtained only after 6 min (data not shown). The results in Figs. 5 and 6 show that fatty acids are rapidly oxidized and cleaved by hydroperoxide lyase to give the corresponding aldehydes. These two enzymes are believed to work closely together, maybe because they are located in the same part of the cell (Smith et al., 1997). The conversion of aldehydes to alcohols in tomato macerates however, seems less straightfor-
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ward (Prestage et al., 1999). Fig. 7 shows the headspace concentration of hexenols after maceration in the presence and absence of added fatty acids. From Fig. 6, one might expect the peak of hexenols concentration to occur after the peak of hexenal (i.e. around 5 min) but Fig. 7 shows an elevated level in the first few minutes of the time course and, thereafter, levels not substantially different from the control. From these results, it seems that the production of the C6 alcohols is not greatly affected by the amount of precursor aldehyde. In Fig. 7, the sample containing 18:2 should show the same behaviour as the control but actually shows a steady increase with time. This can be explained as the ion monitored for hexenal (m/z 83) is derived from dehydration of the molecular ion (M-H2O+ H)+. Hexanal has the same molecular weight as hexenol and also undergoes some dehydration to yield an ion with the same m/z value. Normally the m/z signal from hexanal is very low but, in this case, with large amounts of hexanal (see Fig. 5), the contribution of the hexanal-derived m/z 83 ion is significant and masks the true behaviour of hexenols.
3.5. Effect of surfactants For the experiments described above, Tween 20 was used as the surfactant to emulsify the fatty acids so they could be transported and assimilated by the enzymes in the tomato macerate. Tween 20 has been widely used for this purpose but is also known as an inhibitor of some enzymes (Srinivasulu and Rao, 1993). To assess the effect of Tween 20 on the enzymes producing the C6 compounds, and on the general physicochemical environment, Fig. 8 shows the maximum amounts of hexanal and methylbutanals formed after 3 min in the absence and presence of three different emulsifiers/stabilisers. It is clear that Tween 20 reduced the amount of hexanal released into the headspace compared to lecithin and glycerol monostearate. The most likely explanation for this behaviour was inhibition of one of the steps in the lipoxygenase pathway leading to C6 compound formation. The effects on the release of methylbutanals into the headspace were less clear cut with no significant differences between the
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Fig. 8. Effect of emulsifiers on the release of hexanal and methylbutanals (Imax is the maximum concentration over a 3 min incubation period after maceration).
Fig. 9. Time course release of hexanal after disruption of the tissue in presence of exogenous linoleic (18:2) and linolenic acid (18:3) using lecithin as emulsifier. Data are the mean of five replicates; error bars are 9SD.
four samples; again this strongly suggests that Tween 20 interferes with the lipoxygenase pathway specifically, rather than through some general effect. A further potential cause of differences is that the emulsions may affect partition of the volatiles produced between the aqueous and gas phases. To test this, the amount of hexanal released from tomatoes macerated in the presence of a lecithin/18:0 emulsion was compared with release from tomatoes macerated with 18:0 alone. There was no significant difference between the samples (data not shown) adding credence to the
suggestion above that Tween 20 affects the lipoxygenase pathway directly.
3.6. Release with lecithin as fatty acid emulsifier Release curves for hexanal and hexenal, comparing maceration with buffer alone (control) and with fatty acids in buffer are presented in Figs. 9 and 10. A similar pattern of behaviour was observed for the lecithin samples compared to the Tween 20 samples when fatty acids were added. However, the concentrations measured for the C6
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compounds generated from the lecithin emulsions were higher than with Tween 20 (compare Figs. 5 and 9 (hexanal) and Figs. 6 and 10 (hexenal)). Direct comparison between the figures should be subject to caution because the lecithin and Tween 20 experiments were carried out on different batches of tomatoes and that might account for some of the differences in this instance. However, the data in Fig. 8 show clearly that, even on the same batch of tomatoes, there were substantial differences in C6 volatiles generated from Tween 20 and lecithin emulsions. Since many assays for lipoxygenase have been carried out using Tween 20, some of the published data may underestimate the true activity.
3.7. Addition of enzymes from the lipoxygenase pathway To study the lipoxygenase pathway further, commercial sources of the various enzymes were obtained and added to macerates as solutions or as emulsions. The release of the C6 compounds was then monitored. However, the addition of lipase, phospholipases, lipoxygenase or alcohol dehydrogenase did not significantly change the volatile release profile of the tomatoes for any of the samples (data not shown). Previous work has suggested that some lipid oxidation enzymes are membrane bound which may explain why the addition of exogenous enzymes was ineffective in
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changing the course of the reaction. In Arabidopsis, multiple forms of phospholipase D are involved in the wounding response (Wang et al., 2000) and there appear to be interactions between phospholipase, lipoxygenase and jasmonic acid production. The data from the present paper and from Wang, shows that enzymatic manipulation of the reaction postharvest to modify the flavour characteristics of the fruits is not facile.
4. Conclusions Using this maceration device connected to the API-MS, the measurement of volatiles released from tomatoes upon disruption was rapid and consistent. The rapidity of the measurement allowed analyses of individual fruits (the basis on which consumers often assess tomato quality) while giving detailed information on the generation of preformed and macerate-formed volatiles. The nine selected compounds gave a good fingerprint of the overall aroma profile of the fruit. Information obtained on the dynamics of the reactions allowed the biochemical origin of volatile compounds, (preformed in the ripening fruit or generated during the maceration process) to be identified. Using this system also provided opportunities to study the lipid oxidation pathway in vivo by adding extra substrate although the direct addition of enzymes was not as success-
Fig. 10. Time course release of hexenals after disruption of the tissue in presence of exogenous linoleic (18:2) and linolenic acid (18:3) using lecithin as emulsifier. Data are the mean of five replicates; error bars are 9 SD.
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ful. There are also drawbacks. If the total amount formed in the liquid phase needs to be calculated, various assumptions about mass transfer between the liquid and gas phases during maceration must be made as measurements were all made in the gas phase. It may be possible to calibrate the system by carrying out measurement of total and headspace volatiles and producing a calibration. However, the main use of this technique is as a rapid tool to compare flavour quality of large batches of fruits and offers many possibilities for further studies on tomato flavour.
Acknowledgements We gratefully acknowledge the financial support of the European Commission (Marie Curie Research Training Grant ERB4001GT974094).
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