Changes in the Concentrations of Key Fruit Odorants Induced by

Chapter 8

Changes in the Concentrations of Key Fruit Odorants Induced by Mastication A . Buettner and P. Schieberle

Downloaded by UNIV OF CINCINNATI on May 25, 2016 | http://pubs.acs.org Publication Date: September 7, 2000 | doi: 10.1021/bk-2000-0763.ch008

Deutsche Forschungsanstalt fuer Lebensmittelchemie, Garching, Germany

Recently, the potent odorants of fresh, hand-squeezed orange juices were characterized by aroma dilution techniques. Quantification using stable isotope dilution assays (SIDA) and calculation of the odor activity values revealed ethyl butanoate, (R)-limonene and (Z)hex-3-enal among the most potent odorants of the fresh juices. Based on these results a novel approach is proposed to observe the concentration changes of single key odorants occuring during mastication of orange juice. Changes in the concentrations of key odorants were measured by comparing their amounts before and after in-mouth "reactions". This method, designated as S O O M -concept(spit-off odorant measurement) determines the amounts of single odorants lost by certain in-mouth events such as release, adsorption, or chemical modification. Results obtained for orange juice and also model solutions indicated a strong influence of the chemical structure on substance-specific losses.

Up to now, different approaches have been applied to study the processes occurring when foods are eaten. For example, headspace techniques involving Tenax-trapping or direct MS-analysis have been used to determine the odorants being exhaled through the nose or the mouth breath-by-breath, supplying data on the degree of flavor release during consumption of food (1,2,3). Recently, the APCI-MS technique was also applied for the investigation of odorants released during consumption of orange juice and orange slices (4). However, very little is known about certain processes in the mouth, such as the decrease of odorants in food during mastication or the amounts of odorants remaining in the mouth itself due to effects like adsorption to the mucous membrane. When considering such factors, the following physiological conditions in humans have to be regarded: Before swallowing, the base of the tongue is in contact with the soft palate above

© 2000 American Chemical Society

Roberts and Taylor; Flavor Release ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

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88 the tongue (5). During the mastication of food, normal respiration continues thus showing that the oral cavity is mainly closed off from the trachea by the soft palate. This prevents ingress of food material into the trachea. Up to now, the question could not be sufficiently answered to what extent odorants are transferred to the olfactory epithelium before swallowing of the food material, even before just swallowing saliva. However, it has been proposed that the main amount of odorants released during eating are transferred retronasally when the food material is swallowed. Land (1994) showed that the act of swallowing is always immediately followed by the expiration of a 5 to 15 mL volume of air mainly consisting of the gas phase above the food material in the oral cavity (6). When the tongue presses the masticated food against the soft palate above the tongue and, thereby, pushes it forward into the throat this pulse of air, enriched with odorants, is transferred to the olfactory epithelium and then perceived retronasally. Based on these physiological prerequisites, the aim of our study was to determine the decrease of important orange odorants occurring during mastication in-mouth without swallowing. Care was also taken not to swallow saliva. Therefore, the "SOOM-concept" (spit-off odorant measurement) was developed, involving the quantification of the odorants remaining in the food material after mastication by stable isotope dilution analysis. This methodology was previously applied by Hofmann and Schieberle for the determination of concentration changes of odorants occurring during the mastication of strawberries (7). They observed significant differences in the decrease of several odorants during mastication taking into account possible enzymic degradation of the odorants investigated. Our studies were focused on several key orange aroma compounds (8,9). The odorants and their F D factors obtained previously by A E D A are given in Table I.

Table I. F D factors of important odorants of fresh, hand-squeezed juice of Valencia late-orange (modified data from [9]).

Odorant ethyl butanoate ethyl hexanoate ethyl 3-hydroxyhexanoate (fl)-limonene acetaldehyde hexanal octanal decanal (Z)-hex-3-enal

a

FD factor ) 1024 32 64 64 16 32 64 16 512 b)

a) The Flavour dilution (FD) factor was determined in etheral extracts containing the juice volatiles. Analyses were performed by two assessors in duplicates. b) The Flavour dilution (FD) factor was determined by static headspace-olfactometry of fresh, hand-squeezed orange juice.


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Experimental Procedures


Materials Fresh oranges (Citrus sinensis (L.) Osbeck; cultivar Valencia late, grown in Argentina) were purchased from a local market and were used within two days. Fresh orange juice was obtained by hand-squeezing of the fruits in a kitchen juicer immediately before use for the mastication experiments. Ethyl butanoate 99 %, ethyl hexanoate 99+ %, ethyl octanoate 99+ %, ethyl 3hydroxyhexanoate 98+ %, acetaldehyde 99 %, hexanal 98 %, octanal 99 %, decanal 95 %, CK)-limonene 97 % and myrcene >90 % were obtained from Aldrich (Steinheim, Germany) and were purified prior to analysis by distillation. Aqueous solutions of single reference aroma compounds at four different concentration levels were prepared in water. In-mouth experiments are restricted by the small amount of food that can be masticated. In these studies 25 m L of orange juice or model solution were rinsed in the mouth so the actual amount of odorants in the mouth was only 2.5 μg of odorant for the lowest concentrated model solution applied. Therefore, concentrations had to be determined by stable isotope dilution assays (10).

Quantification of Flavour Compounds Determination of odorants in solvent extracts Juice was masticated for a certain period of time in the mouth, then expectorated into saturated C a C l solution in order to inhibit enzymic reactions, spiked with stable isotope labeled standards and stirred for equilibration. The solution was extracted with diethylether (30 mL, five times, total volume 150 mL) and the combined organic phases were dried over anhydrous N a S 0 . The volatile fraction was subsequently isolated by high vacuum transfer and the obtained aroma extract concentrated by careful distillation (9). A t least four replicates were performed. Quantification of the volatiles was performed by multidimensional gas chromatography (MD-HRGC) with the MS-system ITD-800 running in the Cl-mode with methanol as the reagent gas as described previously (7/). Myrcene was used as internal standard for the quantification of limonene (9). 2

2

4

Determination of odorants by static headspace analysis For the determination of acetaldehyde, the masticated material was expectorated into saturated C a C l solution, the vessel sealed immediately with a septum and spiked with known amounts of [l,2- C ]-acetaldehyde. After stirring for 30 min to reach equilibration, aliquots of the headspace were withdrawn with a gastight syringe and injected for MS-analysis using the system described previously (12). A t least four replicates were performed. 2

13

2


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Results and Discussion Orange juice In a first series of experiments, the SOOM-concept was applied to observe the concentration changes of seven orange odorants during mastication for 1 min of fresh, hand-squeezed orange juice in comparison to fresh juice. The results given in Figure 1 indicated that the amounts of odorants remaining in the mouth differed significantly depending on the structure of the odorant. For example, on average about 30 % more of the initial amount of ethyl hexanoate was retained in the mouth compared to ethyl butanoate. The recovery of structurally related odorants like the two aldehydes (Z)-hex-3-enal and hexanal differed by about 10 % after mastication. Furthermore, the recovery of the odorants varied significantly for some compounds e.g. ethyl hexanoate and hexanal, while others such as ethyl 3-hydroxyhexanoate and (2)-hex-3-enal were much more consistent.

Figure 1. Remaining quantity of selected odorants (values are the means of four replicates; the error bars show the standard deviations) in spitted off orange juice after mastication for I min in the mouth.



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Model experiments To gain more systematic insights into the processes in mouth, the influence of the chemical structure of the odorants on their release in mouth was investigated using appropriate aqueous model solutions of reference odorants. First, the decrease of the aldehydes hexanal, octanal and decanal, during 1 min of mastication in the mouth was determined using different concentrations (100 μg, 1 mg, 10 mg to 100 mg of aldehyde per liter water). The amounts of aldehydes remaining in the solution after mastication in the mouth are displayed in Figure 2. In all concentrations, the three aldehydes were discriminated according to their chain length, that means according to their polarity. The most polar of the three aldehydes (hexanal) was the least reduced after mastication with nearly 100% recovery for the highest concentration. In contrast to this, for octanal a decrease of 14 % was determined in the highest concentration while the amount of decanal was even lower (29 % less than the initial concentration). Comparing the highest and the lowest concentrations for each of the aldehydes the difference in the decrease in mouth is obvious. For example, in contrast to the 100 % recovery of hexanal in the highest concentration, only 66 % of hexanal were recovered after mastication when the lowest concentration was applied. Similar results were obtained for octanal and decanal showing a decrease of 14 % and 29 %, respectively, for the highest concentration while in the lowest concentration the aldehydes were reduced to about 50 %. It is interesting to note that the difference for octanal and decanal leveled out in the lower concentration ranges.

10080604020-

1

1

1

1

1—

100

1000

100000

10000

initial concentration of odorant

water]

Figure 2. Remaining quantity of aldehydes (values are the means of four replicates; the error bars show the standard deviations) in spitted off aqueous solutions after mastication for 1 min in the mouth.



92 Similar experiments were performed using reference esters that are important in orange juice aroma. To gain additional data on the influence of the chain length, ethyl octanoate was studied as a further odorant. The data obtained for ethyl butanoate, ethyl hexanoate, ethyl octanoate and ethyl 3-hydroxyhexanoate are displayed in Figure 3. The results were very similar to the findings obtained for the aldehyde solutions. Again a discrimination for esters of different chain length was observed with the highest reduction for the least polar compound ethyl octanoate. The fact that the decrease of an odorant in mouth is directly related to its polarity is also demonstrated by comparison of the curves of ethyl 3-hydroxyhexanoate and ethyl hexanoate (top and third curve down in the diagram). The hydroxyester was significantly retained in the aqueous phase obviously due to the hydroxygroup. So, an increase in the polarity of an odorant results in a lower retardation in the mouth. In accordance with the findings of the aldehyde experiments the reduction of all esters investigated after mastication was significantly higher when solutions containing lower concentrations were applied. So, in comparison to the highest concentrations about 10 to 20 % less of the initial concentrations of all esters were present in the lowest concentrated solutions after mastication.

lOO-i

80 H

60H

—•— ethyl butanoate — · — ethyl hexanoate —*— ethyl octanoate

40 H

— τ — ethyl 3-hydroxyhexanoate • • ' • I

100

1000

10000

100000

initial concentration of odorant [^g/L water]

Figure 3. Remaining quantity of esters (values are the means offour replicates; the error bars show the standard deviations) in spitted off aqueous solutions after mastication for 1 min in the mouth.



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Two important odorants showing very high concentrations in fresh orange juice are limonene (50 to 100 mg/kg) and acetaldehyde (5 to 10 mg/kg). The decrease of both odorants in model solutions during mastication in mouth was investigated in the concentrations naturally found in orange juice (see Figure 4). For limonene, at the higher concentration of 100mg/L only 2 0 % remained in the mouth, while in the lower concentration (10mg/L) 4 5 % of the odorant was retained. The difference observed for acetaldehyde was not as drastic between the highest and the lowest concentration applied. A decrease of 25 % was found for the lowest concentration and 15 % for the highest concentration after mastication. Again a difference between the more polar acetaldehyde and the less polar limonene is observed, corroborating the assumption that polar groups present in odorants lead to less significant losses during mastication compared to non-polar odorants.

Figure 4. Remaining quantity of acetaldehyde and limonene (values are the means of four replicates; the error bars show the standard deviations) in spitted off aqueous solutions after mastication for 1 min in the mouth. In Figure 5 the data for all esters and aldehydes in the masticated model solutions of the lowest concentration (100 μg/L water) are compared. The discrimination according to the polarity is obvious. The increasing retardation in mouth can be clearly related to the increasing chain length in both groups of compounds.



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Figure 5. Remaining quantity of odorants (initial concentration 100 μ&Σ water; values are the means of three replicates) in spitted off aqueous solutions after mastication for 1 min in the mouth. Now the question arises whether the time of mastication influences the retardation of odorants in mouth. When we eat orange slices they usually remain for a while in the mouth till the fruit pieces are small enough for swallowing. However, orange juice is normally in contact with the mouth just for a few seconds prior to swallowing. So, the same experiments were performed using the model solutions but, this time, the duration of mastication was only 5 s. The results obtained for the long-time and the short-time mastication of the aldehydes are shown in Figure 6. The mastication experiment of 5 s revealed a significantly lower decrease of the three odorants with a recovery of about 90 % of the initial concentration. Furthermore the discrimination of the aldehydes according to their chain length leveled out in comparison to the long-time mastication data, only the amount of decanal was slightly reduced. Similar results were obtained for the short-time mastication of the esters (see Figure 7), with a significantly higher recovery for all esters of about 90 % in comparison to the long-time mastication and also no distinct discrimination between the odorants. These data indicate that the amount of odorants remaining in the mouth is significantly influenced by the duration of mastication.



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Figure 6. Influence of the duration of mastication (1 min or 5 s) on the remaining quantity of aldehydes (initial concentration 100 μg/L water; values are the means of four replicates) in spitted off aqueous solutions.

Figure 7. Influence of the duration of mastication (1 min or 5 s) on the remaining quantity of esters (initial concentration 100 μg/L water; values are the means of four replicates) in spitted off aqueous solutions.



96 Application of model solutions is a first approach to study the interactions occurring between odorants and the real mouth system when no other compounds (except water) are present. However, real food systems are much more complex, containing different matrix compounds which could influence these interactions of odorants and mouth to a major extent. A comparison of the recovery of the esters after mastication of fresh orange juice and model solutions, respectively, is given in Figure 8a. Displayed are the data of the model solutions for similar concentrations as they were determined in the fresh juice. Additionally the variance of the quantitative data is given with error bars. Between both systems, the fresh juice and the model solutions, the decrease of the esters did not differ to a major extent after 1 min of mastication. However, the variance between the samples of the fresh juices was a little bit higher then in the model solutions. Regarding the aldehydes (see Figure 8b) the differences between orange juice and model solution were higher, with a significantly lower decrease especially of octanal and decanal after mastication of the fresh juice. In this case interactions of the aldehydes with matrix compounds of the juice seem to take place thereby reducing the extent of flavour release in mouth in comparison to the release determined for the model solutions in water. Again a slightly higher variance in the samples of the juices were observed, especially for hexanal. When studying real food systems one always has to keep in mind interactions occurring with matrix compounds. Furthermore the possibility of enzymic reactions has to be considered. When fruit cells are crushed, odorants can be produced or destroyed by enzymic activity. Recently we showed (in cooperation with the group of Andy Taylor in Nottingham) that the concentrations of some of the investigated odorants, e.g. hexanal and (Z)-3-hexenal increase two-or three-times their initial amounts due to enzymic activity one hour after the preparation of the juice (unpublished data). In contrast to this, the concentrations of ethyl butanoate and acetaldehyde did not change significantly (4).

Conclusions The SOOM-concept is a novel approach for the exact quantification of flavour changes occuring during mastication even in relatively low concentration ranges. Our investigations showed that the structure of an odorant influences significantly its retardation in mouth being directly related to the polarity of the odorant. Furthermore it was found that the proportion of retardation in mouth increases when lowering the concentration of an odorant. This effect was observed for the first time and is especially interesting for extremely odor-active volatiles which are present in very low concentrations in food. The results indicate that the SOOM-concept can also be a useful tool for studying interactions of odorants with matrix compounds under realmouth conditions. Combination of this methodology with other techniques like nosespace-analyses offers the possibility to balance the amounts of odorants being effective in flavour perception.



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Figure 8. Remaining quantity of a) esters and b) aldehydes (values are the means of four replicates; the error bars show the standard deviations) in spitted off aqueous model solutions and in spitted off orange juice after mastication for 1 min in mouth.


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References


1 2 3 4. 5.

Soeting, W.J.; Heidema, J. Chem. Senses 1988, 13, 607-617. Linforth, R.S.T.; Taylor, A.J. Food Chem. 1993, 48, 115-120. Ingham, K . E . ; Linforth, R.S.T.; Taylor, A.J. Food Chem. 1995, 54, 283-288. Buettner, Α.; Baek, I.; Linforth, R.; Schieberle, P.; Taylor, Α., in preparation. Taschenatlas der Anatomie: innere Organe; Kahle, W.; Leonhardt, H.; Platzer, W., Eds.; Georg Thieme Verlag, Stuttgart, 1984, pp. 198-199. 6 Land, D.G. In Flavor-Food Interactions; McGorrin, R. J., Leland, J.V., Eds.; ACS-Symp. Ser. 633, pp. 2-11. 7 Hofmann, T.; Schieberle, P. In Interaction of food matrix with small ligands influencing flavour and texture. Cost 96, Volume 3, pp. 191-194. 8. Hinterholzer, Α.; Schieberle, P. Flav. Fragr. J. 1998, 13, 49-55. 9 Buettner, Α.; Schieberle, P. in preparation. 10 Buettner, Α.; Hofmann, T.; Schieberle, P.; in preparation. 11. Guth, H . J. Agric. Food Chem. 1997, 45, 3027-3032. 12. Guth, H . ; Grosch, W. J. Agric. Food Chem. 1994, 42, 2862-2866.


Changes in the Concentrations of Key Fruit Odorants Induced by

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