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THE EFFECT OF PROCESSING ON CHIRAL AROMA COMPOUNDS IN FRUITS AND ESSENTIAL OILS
THE EFFECT OF PROCESSING ON CHIRAL AROMA COMPOUNDS IN FRUITS AND ESSENTIAL OILS
B.D. Baigrie,* M G Chisholmt and D.S. Mottram§
*Reading Scientific Services Limited, Lord Zuckerman Research Centre, The University of Reading, Whiteknights, Reading, RG6 6LA, U.K. tThe Pennsylvania State University, Division of Science, Behrend College, Erie, PA 16563, U.S.A. §The University of Reading, Department of Food Science, and Technology, Whiteknights, Reading, RG6 6AP, U.K. 1 INTRODUCTION The determination of the enantiomeric distribution of chiral compounds found in fruits and essential oils has become increasingly important in determining the origin of natural and processed foodstuffs. The method depends in part on a knowledge of the enantiomeric distributions found in nature (and their consistency) and also the changes that may occur with the extraction and processing methods used. The enantiomeric distribution of linalool in nature demonstrates the variation which may be found for chiral compounds. In bergamot oil and basil herb, linalool occurs as the almost pure R-(-) isomer; in lavender flowers it occurs as 95%-98% of the R-(-) isomer ' and in bitter orange oil it occurs as 79%-87% of the R-(-) isomer. However in sweet orange oil the 5-(+) isomer predominates, occurring as 94%-96% of the total, ' and in coriander oil 86%-87% is found as the S-(+) isomer. ' In passion fruit and apricot, linalool occurs as the racemate. Another group of compounds that may occur naturally in the racemic form are the yand 5-lactones. Their enantiomeric distribution in many fruits has been extensively studied and summarized by Casabianca et al The enantiomeric distributions of y- and 5-lactones vary among different fruits and with the origin for the same fruit. It has been noted that despite its relatively high abundance in many fruits, y-hexalactone is useless for assessing the origin of flavours because of the wide range of enantiomeric distributions found. Racemates also occur naturally when fermentation takes place in the production of the foodstuff. Racemic linalool and a-terpineol are both found in bergamot tea, and in wine, a-terpineol has been found in the racemic form. This is the result of fermentation which occurs in the production of both tea and wine. The R-(+) form of a-terpineol occurs frequently in essential oils, ' but it rarely occurs in the enantiomerically pure form. Naturally occurring racemates have been reported in geranium oil ' and yellow passion fruit. Since a-terpineol lies further along the biosynthetic pathway than linalool, and it is also an artefact whose abundance depends upon the extraction and processing methods used, a wide range of enantiomeric distributions would be expected. 1
2
2 3
4
5 6
2 5
5
7-9
10
11
12
13
14 15
14 16
6
Flavour Science: Recent Developments
152
Racemic compounds may occur in essential oils and foods as a result of the extraction and processing methods used in their isolation or production. Partial racemisation may also occur depending on the procedures used for chiral analysis. It is well known that linalool and oc-terpineol are very labile in acidic media, ' so it is important to know the pH at which the oil was isolated, before making an assessment of its origin. Varying tendencies towards the racemisation of limonene, linalool and a-terpineol have been reported for lavender oils, depending upon the method of extraction, and the pH of steam distillation. In contrast, model studies on linalool under simultaneous distillation/extraction conditions at pH 7 showed no racemisation. Kreis et al found up to 8% racemisation of linalool isolated from Flores Lavandulae by hydrodistillation at pH 5 over differing time periods. Solvent extraction produced negligible racemisation. These results, together with the variation in enantiomeric distribution of key monoterpenes in different fruits, which could be used to determine the origin of essential oils and foods, provide a complex picture when using chirospecific analysis as a tool to determine authenticity. A preliminary investigation of changes in the enantiomeric distribution of limonene, linalool and a-terpineol in cherries caused by canning, showed significant shifts towards racemisation for all three compounds. To investigate the effect of processing conditions on the racemisation of chiral aroma compounds further, a simpler investigation was undertaken, which examined the effect of temperature and pH used in the extraction of some selected citrus oils from different regions. Pinene (a- and (3-), limonene, linalool and a-terpineol were the chiral aroma compounds selected for analysis. 17 18
15
5
19
20
2 MATERIALS AND METHODS 2.1 Samples Bergamot (Citrus aurantium (L.) Bergamia), lemon (Citrus limon, (L.) Burman) and Persian lime (Citrus latiofolia, Tanaka) were obtained as tree fruit from several regions of the world. Commercial oils from the same fruits were supplied by two flavour houses in the U.K. either in the expressed or in the distilled forms. 2.2 Sample Preparation 2.2.1 Hydrodistillation. Samples of the expressed commercial oils were hydrodistilled for an hour at pH 2 and 6. The pH of 2 was achieved by carrying out the distillation in a 5% solution of citric acid. 2.2.2 Extraction of Fruit. The skin of each fruit was scraped with a zesting tool to remove a thin layer of the oil-rich material. Care was taken to keep the skin separated from the pulp and juice by ensuring that the fruit was not broken. The zest from an individual fruit was stirred with 30 ml of a 1:1 mixture of pentane-ether for 30 min. The solvent was filtered from the peel residue and diluted by a factor of 10 with pentane for chiral analysis. 2.2.3 Simulation of Extraction in Contact with Juice. Authentic bergamot oil (1 ml) was stirred with a 5% citric acid solution for 72 hours at room temperature. Samples were removed at regular intervals and prepared for chiral analysis. 2.3 Chiral Analysis using Multidimensional Gas Chromatography (MDGC) MDGC was carried out on a dual oven linked system comprised of a Carlo Erba 5160 Mega Series gas chromatograph (GC 1) containing the pre-column and a Carlo Erba Fractovap 4200 series GC (2) containing the analytical column. Heart cutting was achieved
a
30.9 31.2 34.4
22.7 33.7 33.9 34.0
70.4 29.6 69.0 31.1 69.1 30.9
69.1 68.8 65.6
77.3 66.3 66.2 66.0
1.5 1.0 1.3
3.7 3.3 4.6
3.4 3.8 5.2
Wt °/o
8.5 8.8 9.4
8.8 8.8 9.4
2.9 7.6 7.4 7.6
91.5 91.2 90.6
91.2 91.2 90.6
97.1 92.4 92.6 92.4
fi-Pinene
6.8 5.7 6.9
15.8 15.1 14.8
11.6 16.7 18.9
wt %
1.9 1.8 1.8
7.9 8.0 8.8
7.5 1.3 1.3 1.4
98.1 98.2 98.2
92.1 92.0 91.2
92.5 98.7 98.7 98.6
RW
Limonene
31.4 25.2 27.2
38.5 38.1 38.7
66.3 50.1 51.0
wt%
Quantity of compound (both enantiomers) expressed as % total as measured by GC.
Cold pressed Hydrodist. pH 6 Hydrodist. pH 2
Bergamot
Cold expressed Hydrodist. pH 6 Hydrodist. pH 2
Lime
Distilled Cold expressed Hydrodist. pH 6 Hydrodist. pH 2
Lemon
*(-)
a-Pinene
77.7 73.3 63.7
63.0 62.8 54.1
47.9 32.8 34.3 40.0
23.3 26.7 36.3
37.0 37.0 45.9
52.2 67.2 65.7 60.0
Linalool
16.7 22.5 16.0
0.31 0.35 0.90
0.22 0.44 0.43
wt%
38.0 35.1 49.6
74.6 70.5 62.0
57.7 60.7 60.5 45.1
62.0 64.9 50.4
25.4 29.5 38.0
42.3 39.3 39.5 54.9
RW
0.31 1.54 6.74
0.56 0.63 1.19
0.33 0.69 0.70
wt%
a-Terpineol
Table 1 The Effect of Distillation on the Enantiomeric Distribution of Chiral Aroma Compounds. All Samples are Commercial. The Values are the Average of at least Two Runs
Chirality and Flavour 153
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154
by using a Multiple Switching Intelligent Controller (MUSIC) system (Chrompack U.K. Ltd.) which is based on the Deans pressure switching concept. The heart cut sample was trapped and cooled by liquid nitrogen. Injector temperature: 200 °C; detector temperature: 250 °C (both); injection mode: on-column; sample size 2 ul; solution concentration: 0.2% in pentane. 2.3.1 Precolumn. Stabilwax-DA chemically bonded 30 m x 0.53 mm internal diameter, 1.0 um film thickness (Restek Corp). Carrier gas: He 10 ml min flow rate; temperature programme: 30 °C for 30 s, 30 °C to 60 °C at 40 °C min" , 60 °C to 200 °C at 3 °C min" . 2.3.2 Analytical Column. Chirasil Dex CB chemically bonded (heptakis-(2,3,6-tri-0methyl)-p-cyclodextrin in 10% OV 1701) 25 m x 0.25 mm, 0.25 um film thickness (Chrompack, U.K. Ltd.). Carrier gas: 0.88 bar He; temperature programme: a- and Ppinene: 40 °C for 15 min then 4 °C min" ; limonene and linalool: 70 °C for 15 min then 4 °C min" ; a-terpineol: 120 °C for 15 min then 4 °C min" . 2.3.3 Identification of Enantiomers. The order of elution for each compound was assigned by comparison to standards of known optical purity. The order of elution was found to be: a-pinene: S-(-), R-(+)\ P-pinene: R-{+), S-{-)\ limonene: S-(-), R-(+); linalool: £_(+); a-terpineol: R-(+). 21
-1
1
1
1
1
1
3 RESULTS AND DISCUSSION The monoterpene hydrocarbons underwent little change in their enantiomer distribution upon hydrodistillation in neutral or acid solution, as shown in Table 1, in agreement with previous work. ' Linalool showed only a slight change in neutral solution, but significant racemisation occurred at pH 2. Kreis et al. found a 5% change for lavender oil under similar conditions and Weinreich reported a 2%-15% change. a-Terpineol is the least stable of these monoterpenes in acid solution, and underwent the most racemisation upon hydrodistillation at pH 2. Since it is formed as an artefact in citrus oils upon heating at pH 2, then the higher level of racemisation is not unexpected. The racemisation of linalool which occurred while bergamot oil was in contact with cold 5% citric acid, shown in Table 2, suggests that if good manufacturing procedures permit the extraction of citrus oils from the whole fruit, then in the case of bergamot oil, linalool may not be present in the pure R-(-) form. Under hydrodistillation conditions, most of the oil is removed from the vicinity of the acid in the first few minutes, so prolonged distillation under these conditions will not increase the amount of racemisation found in Table 1. The variation in enantiomeric distribution that occurs with fruits from different regions are shown in Table 3. These results point to some of the difficulties in using known values for determining the origin and processing methods used in the production of citrus oils. The range of values for linalool in lemon oil, where the enantiomeric excess ranges from 0% to 5 15
19
15
22
Table 2 The Effect of Acid Contact at pH 2 at 25 °C on the Enantiomeric Distribution of Linalool in Bergamot Oil Enantiomer
Cold pressed
2 hours
4 hours
24 hours
72 hours
R(-)
100.0 0.0
92.2 7.8
93.0 7.0
79.9 20.1
53.0 47.0
S(+)
Chirality and Flavour
155
Table 3 Enantiomeric Distribution of Chiral Aroma Compounds for Authentic Citrus Oils a-Pinene
p-Pinene
RW S(-j
sf-j
Limonene
Linalool
sr-;
a-Terpineol
sr-;
Lemon Cyprus Israel U.S.A. 1 U.S.A. 2
60.1 72.2 66.4 73.3
39.9 27.9 33.6 26.7
7.7 6.2 6.7 4.7
92.3 93.8 93.3 95.3
1.2 1.4 1.3 1.6
98.9 98.6 98.7 98.4
39.7 45.0 50.8 72.0
60.3 55.0 49.2 28.0
71.2 80.0 78.8 88.8
70.1 68.6
29.9 31.4
10.0 8.9
90.0 91.1
2.7 2.2
97.3 97.8
62.1 64.6
37.7 35.4
79.8 20.2 77.2 22.8
71.2 68.2
28.8 31.7
8.2 9.4
91.8 90.6
2.3 1.7
97.7 98.3
98.3 98.7
1.7 1.3
61.3 38.7 37.4 62.6
28.8 20.0 21.2 11.2
Lime Cuba Mexico Bergamot U.K. unripe U.K. ripe
Table 4 Enantiomeric Distributions of Chiral Compounds in Commercial Citrus Oils. All oils were cold pressed except those marked" a-Pinene
fl-Pinene
Limonene
Linalool
a-Terpineol
s(-) R(+)
R(+) S(-)
S(-) R(+)
R(-) S(+)
S(-) R(+) 80.4 77.6 78.5 76.8 77.2 60.7 57.7
Lemon Sicily California Spain Argentina Unknown 1 Unknown 2 Distilled*
69.2 70.2 69.8 69.4 66.3 79.7 77.3
30.8 29.8 30.2 30.6 33.7 20.3 22.7
6.1 93.4 5.1 94.9 6.1 93.9 5.1 94.9 7.5 92.5 4.9 95.1 2.9 97.1
1.5 1.4 1.6 1.3 1.3 1.2 7.8
98.5 98.6 98.4 98.7 98.7 98.8 92.2
61.0 58.9 48.3 46.8 51.8 32.8 47.9
39.0 41.1 51.7 53.2 48.2 67.2 52.1
19.6 22.4 21.5 23.2 22.8 39.3 42.3
69.1 30.9 70.9 29.1
8.8 3.5
91.2 96.5
7.9 7.4
92.1 92.6
63.0 37.0 47.7 52.3
74.6 25.4 51.5 48.5
68.0 69.2 69.8 68.8 63.0 70.7
8.6 8.5 8.5 8.3 8.7 5.2
91.4 91.5 91.5 91.7 91.3 94.8
1.6 4.9 1.9 1.8 1.3 1.3
98.4 95.1 98.1 98.2 98.7 98.7
70.7 100 77.7 68.6 100 64.2
23.9 51.4 38.0 23.3 64.0 36.5
Lime Unknown Distilled* Bergamot Italy 1 Italy 2 Italy 3 Sicily Argentina 'Compound'*
32.0 30.8 30.2 31.2 37.0 29.3
29.3 0.0 23.3 31.4 0.0 35.8
76.1 48.6 62.0 76.7 36.0 63.5
Flavour Science: Recent Developments
156
45%, makes linalool a poor choice as an indicator compound for lemon oil production. The range of values found for a-terpineol show a narrower range for lemon and lime oils than those of linalool, but the values for bergamot oil show no pattern with both the and the S-(-) isomer being present in excess, which is further supported by the data for the commercial oils shown in Table 4. a-Terpineol is clearly not a good indicator compound to determine the origin of an oil, particularly for bergamot oil. However, linalool and aterpineol are minor components in lemon and lime oils, although they are major odourants. The enantiomeric distribution of limonene shows good consistency for all the authentic citrus oils examined, and agrees with reported values, and lies within the range shown by a wide range of citrus oils. ' The commercial samples shown in Table 4, which contain more than 3 % of S-(-) limonene (both lime, the distilled lemon and the Italian No. 2 bergamot samples), clearly lie outside the expected range for citrus oils and suggest an anomaly in their production. The one good indicator compound that emerges from these data is linalool in bergamot oil. If the oil is cold pressed, and has been isolated without juice contact, then close to 100% of the R-(~) isomer should be present. However, the commercial bergamot oil samples in Table 4 show an additional dilemma. The Italian No. 2 sample has acceptable values for linalool, but those of both limonene and a-terpineol lie outside the expected ranges for a cold pressed citrus oil isolated without juice contact. The samples from Sicily and Italy No. 1 show a high percentage of S-(-)-linalool for a cold pressed oil, but if good manufacturing processes permit juice contact, then this value may be acceptable. The sample from Argentina appears to be an authentic cold pressed bergamot oil, on the basis of the enantiomeric distribution data. 23 24
1
4 CONCLUSION The data in Tables 1-4 show that it would be almost impossible to use chiral analysis alone, particularly on a single compound, to specify the origin of citrus oils. The range of values found for authentic citrus oil samples together with the loose definition of 'good manufacturing procedures' make the task of establishing acceptable values for enantiomeric distributions very difficult. The best way to assess the origin of a natural product would seem to be to adopt a more comprehensive approach and use chiral analysis in conjunction with quantitative analysis of chiral indicator compounds and other instrumental methods such as isotope ratio mass spectrometry or site-specific natural isotope fractionation nuclear magnetic resonance spectroscopy (SNIF-NMR). In all cases a comprehensive data base of acceptable values is needed to effectively establish the origin of a natural product. REFERENCES 1. 2. 3. 4.
A. Crotoneo, I. Stagno d'Alcontres and A. Trozzi, Flavour Fragr. J., 1992, 7, 15. V. Schubert and A. Mosandl, Phytochem. Anal, 1991, 2, 171. P. Kreis and A. Mosandl, Flavour Fragr. J., 1992, 7,187. P. Dugo, L. Mondello, E. Cogliandro, A. Verzera and G. Dugo, J. Agric. Food Chem., 1996, 44, 544. 5. A. Bernreuther and P. Schreier, Phytochem. Anal, 1991, 2, 167. 6. P. Werkoff, S. Brennecke, W. Bretschneider, M. Giintert, R. Hopp and H. Surburg, Z Lebensm. Unters. Forsch., 1993, 196, 307. 7. A. Bernreuther, N. Christoph and P. Schreier, J. Chromatogr., 1989, 481, 363.
Chirality and Flavour 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
18. 19.
20.
21. 22. 23. 24.
157
S. Nitz, H. Kollmansberger and F. Drawert, Chem. Mikrobiol. Technol. Lebensm., 1989, 12, 75. E. Guichard, A. Kustermann and A. Mosandl, J. Chromatogr., 1990, 498, 396. H. Casabianca, J.-B. Graff, P. Jame and C. Perrucchietti, J. High Resol. Chromatogr., 1995, 18, 279. A. Mosandl, J. Chromatogr., 1992, 624, 267. H. Casabianca and J.-B. Graff, HRC J. High Resolut. Chromatogr, 1994, 17, 184. C. Askari, U. Hener, H. Schmarr, A. Rapp and A. Mosandl, Fresenius J. Anal. Chem., 1991,340, 768. U. Ravid, E. Putievsky and I. Katzir, Flavour Fragr. 1, 1995, 10, 281. B. Weinreich and S. Nitz, Chem. Mikrobiol. Technol. Lebensm., 1992, 14, 117. P. Kreis and A. Mosandl, Flavour Fragr. J., 1993, 8, 161. G. Schmaus and K.H. Kubeczka, in 'Essential Oils and Aromatic Plants', eds. A. Baerheim-Svendsen and J.J. Scheffer, Nijhoff/Junk Publ., Dordrecht, Netherlands, 1985, p. 127. T.S. Chamblee, B.C. Clarke, Jr., G.B. Brewster, T. Radford and G.A. Iacobucci, J. Agric. Food Chem., 1991, 39, 162. P. Kreis, R. Braundorf, A. Dietrich, U. Hener, B. Maas and A. Mosandl, in 'Progress in Flavour Precursor Studies', eds. P. Schreier and P. Winterhalter, Allured Publishing Co., Carol Stream, IL, 1993, p. 77. B.D. Baigrie, D.S. Mottram and K.J. Pierce, in 'Chemical Markers for Processed and Stored Foods', eds. T.C. Lee and H.J. Kim, American Chemical Society, Washington, D.C., 1996, A.C.S. Symposium Series 631, in press. D R . Deans, Chromatographia, 1968, 5-6, 187. B.C. Clark, Jr. and T.S. Chamblee, in 'Off-flavors in Foods and Beverages' ed. G. Charalambous, Elsevier Science Publishers, Amsterdam, 1992, p. 229. A. Mosandl, U. Hener, P. Kreis and H.-G. Schmarr, Flavour Fragr. J., 1990, 5, 193. U. Hener, A. Hollnagel, P. Kreis, B. Maas, H.-G. Schmarr, V. Schubert, K. Rettinger, B. Weber and A. Mosandl, in 'Flavour Science and Technology', eds. Y. Bessiere and A.F. Thomas, John Wiley and Sons, Chichester, 1990, p. 25.
B.D. Baigrie,* M G Chisholmt and D.S. Mottram§
*Reading Scientific Services Limited, Lord Zuckerman Research Centre, The University of Reading, Whiteknights, Reading, RG6 6LA, U.K. tThe Pennsylvania State University, Division of Science, Behrend College, Erie, PA 16563, U.S.A. §The University of Reading, Department of Food Science, and Technology, Whiteknights, Reading, RG6 6AP, U.K. 1 INTRODUCTION The determination of the enantiomeric distribution of chiral compounds found in fruits and essential oils has become increasingly important in determining the origin of natural and processed foodstuffs. The method depends in part on a knowledge of the enantiomeric distributions found in nature (and their consistency) and also the changes that may occur with the extraction and processing methods used. The enantiomeric distribution of linalool in nature demonstrates the variation which may be found for chiral compounds. In bergamot oil and basil herb, linalool occurs as the almost pure R-(-) isomer; in lavender flowers it occurs as 95%-98% of the R-(-) isomer ' and in bitter orange oil it occurs as 79%-87% of the R-(-) isomer. However in sweet orange oil the 5-(+) isomer predominates, occurring as 94%-96% of the total, ' and in coriander oil 86%-87% is found as the S-(+) isomer. ' In passion fruit and apricot, linalool occurs as the racemate. Another group of compounds that may occur naturally in the racemic form are the yand 5-lactones. Their enantiomeric distribution in many fruits has been extensively studied and summarized by Casabianca et al The enantiomeric distributions of y- and 5-lactones vary among different fruits and with the origin for the same fruit. It has been noted that despite its relatively high abundance in many fruits, y-hexalactone is useless for assessing the origin of flavours because of the wide range of enantiomeric distributions found. Racemates also occur naturally when fermentation takes place in the production of the foodstuff. Racemic linalool and a-terpineol are both found in bergamot tea, and in wine, a-terpineol has been found in the racemic form. This is the result of fermentation which occurs in the production of both tea and wine. The R-(+) form of a-terpineol occurs frequently in essential oils, ' but it rarely occurs in the enantiomerically pure form. Naturally occurring racemates have been reported in geranium oil ' and yellow passion fruit. Since a-terpineol lies further along the biosynthetic pathway than linalool, and it is also an artefact whose abundance depends upon the extraction and processing methods used, a wide range of enantiomeric distributions would be expected. 1
2
2 3
4
5 6
2 5
5
7-9
10
11
12
13
14 15
14 16
6
Flavour Science: Recent Developments
152
Racemic compounds may occur in essential oils and foods as a result of the extraction and processing methods used in their isolation or production. Partial racemisation may also occur depending on the procedures used for chiral analysis. It is well known that linalool and oc-terpineol are very labile in acidic media, ' so it is important to know the pH at which the oil was isolated, before making an assessment of its origin. Varying tendencies towards the racemisation of limonene, linalool and a-terpineol have been reported for lavender oils, depending upon the method of extraction, and the pH of steam distillation. In contrast, model studies on linalool under simultaneous distillation/extraction conditions at pH 7 showed no racemisation. Kreis et al found up to 8% racemisation of linalool isolated from Flores Lavandulae by hydrodistillation at pH 5 over differing time periods. Solvent extraction produced negligible racemisation. These results, together with the variation in enantiomeric distribution of key monoterpenes in different fruits, which could be used to determine the origin of essential oils and foods, provide a complex picture when using chirospecific analysis as a tool to determine authenticity. A preliminary investigation of changes in the enantiomeric distribution of limonene, linalool and a-terpineol in cherries caused by canning, showed significant shifts towards racemisation for all three compounds. To investigate the effect of processing conditions on the racemisation of chiral aroma compounds further, a simpler investigation was undertaken, which examined the effect of temperature and pH used in the extraction of some selected citrus oils from different regions. Pinene (a- and (3-), limonene, linalool and a-terpineol were the chiral aroma compounds selected for analysis. 17 18
15
5
19
20
2 MATERIALS AND METHODS 2.1 Samples Bergamot (Citrus aurantium (L.) Bergamia), lemon (Citrus limon, (L.) Burman) and Persian lime (Citrus latiofolia, Tanaka) were obtained as tree fruit from several regions of the world. Commercial oils from the same fruits were supplied by two flavour houses in the U.K. either in the expressed or in the distilled forms. 2.2 Sample Preparation 2.2.1 Hydrodistillation. Samples of the expressed commercial oils were hydrodistilled for an hour at pH 2 and 6. The pH of 2 was achieved by carrying out the distillation in a 5% solution of citric acid. 2.2.2 Extraction of Fruit. The skin of each fruit was scraped with a zesting tool to remove a thin layer of the oil-rich material. Care was taken to keep the skin separated from the pulp and juice by ensuring that the fruit was not broken. The zest from an individual fruit was stirred with 30 ml of a 1:1 mixture of pentane-ether for 30 min. The solvent was filtered from the peel residue and diluted by a factor of 10 with pentane for chiral analysis. 2.2.3 Simulation of Extraction in Contact with Juice. Authentic bergamot oil (1 ml) was stirred with a 5% citric acid solution for 72 hours at room temperature. Samples were removed at regular intervals and prepared for chiral analysis. 2.3 Chiral Analysis using Multidimensional Gas Chromatography (MDGC) MDGC was carried out on a dual oven linked system comprised of a Carlo Erba 5160 Mega Series gas chromatograph (GC 1) containing the pre-column and a Carlo Erba Fractovap 4200 series GC (2) containing the analytical column. Heart cutting was achieved
a
30.9 31.2 34.4
22.7 33.7 33.9 34.0
70.4 29.6 69.0 31.1 69.1 30.9
69.1 68.8 65.6
77.3 66.3 66.2 66.0
1.5 1.0 1.3
3.7 3.3 4.6
3.4 3.8 5.2
Wt °/o
8.5 8.8 9.4
8.8 8.8 9.4
2.9 7.6 7.4 7.6
91.5 91.2 90.6
91.2 91.2 90.6
97.1 92.4 92.6 92.4
fi-Pinene
6.8 5.7 6.9
15.8 15.1 14.8
11.6 16.7 18.9
wt %
1.9 1.8 1.8
7.9 8.0 8.8
7.5 1.3 1.3 1.4
98.1 98.2 98.2
92.1 92.0 91.2
92.5 98.7 98.7 98.6
RW
Limonene
31.4 25.2 27.2
38.5 38.1 38.7
66.3 50.1 51.0
wt%
Quantity of compound (both enantiomers) expressed as % total as measured by GC.
Cold pressed Hydrodist. pH 6 Hydrodist. pH 2
Bergamot
Cold expressed Hydrodist. pH 6 Hydrodist. pH 2
Lime
Distilled Cold expressed Hydrodist. pH 6 Hydrodist. pH 2
Lemon
*(-)
a-Pinene
77.7 73.3 63.7
63.0 62.8 54.1
47.9 32.8 34.3 40.0
23.3 26.7 36.3
37.0 37.0 45.9
52.2 67.2 65.7 60.0
Linalool
16.7 22.5 16.0
0.31 0.35 0.90
0.22 0.44 0.43
wt%
38.0 35.1 49.6
74.6 70.5 62.0
57.7 60.7 60.5 45.1
62.0 64.9 50.4
25.4 29.5 38.0
42.3 39.3 39.5 54.9
RW
0.31 1.54 6.74
0.56 0.63 1.19
0.33 0.69 0.70
wt%
a-Terpineol
Table 1 The Effect of Distillation on the Enantiomeric Distribution of Chiral Aroma Compounds. All Samples are Commercial. The Values are the Average of at least Two Runs
Chirality and Flavour 153
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by using a Multiple Switching Intelligent Controller (MUSIC) system (Chrompack U.K. Ltd.) which is based on the Deans pressure switching concept. The heart cut sample was trapped and cooled by liquid nitrogen. Injector temperature: 200 °C; detector temperature: 250 °C (both); injection mode: on-column; sample size 2 ul; solution concentration: 0.2% in pentane. 2.3.1 Precolumn. Stabilwax-DA chemically bonded 30 m x 0.53 mm internal diameter, 1.0 um film thickness (Restek Corp). Carrier gas: He 10 ml min flow rate; temperature programme: 30 °C for 30 s, 30 °C to 60 °C at 40 °C min" , 60 °C to 200 °C at 3 °C min" . 2.3.2 Analytical Column. Chirasil Dex CB chemically bonded (heptakis-(2,3,6-tri-0methyl)-p-cyclodextrin in 10% OV 1701) 25 m x 0.25 mm, 0.25 um film thickness (Chrompack, U.K. Ltd.). Carrier gas: 0.88 bar He; temperature programme: a- and Ppinene: 40 °C for 15 min then 4 °C min" ; limonene and linalool: 70 °C for 15 min then 4 °C min" ; a-terpineol: 120 °C for 15 min then 4 °C min" . 2.3.3 Identification of Enantiomers. The order of elution for each compound was assigned by comparison to standards of known optical purity. The order of elution was found to be: a-pinene: S-(-), R-(+)\ P-pinene: R-{+), S-{-)\ limonene: S-(-), R-(+); linalool: £_(+); a-terpineol: R-(+). 21
-1
1
1
1
1
1
3 RESULTS AND DISCUSSION The monoterpene hydrocarbons underwent little change in their enantiomer distribution upon hydrodistillation in neutral or acid solution, as shown in Table 1, in agreement with previous work. ' Linalool showed only a slight change in neutral solution, but significant racemisation occurred at pH 2. Kreis et al. found a 5% change for lavender oil under similar conditions and Weinreich reported a 2%-15% change. a-Terpineol is the least stable of these monoterpenes in acid solution, and underwent the most racemisation upon hydrodistillation at pH 2. Since it is formed as an artefact in citrus oils upon heating at pH 2, then the higher level of racemisation is not unexpected. The racemisation of linalool which occurred while bergamot oil was in contact with cold 5% citric acid, shown in Table 2, suggests that if good manufacturing procedures permit the extraction of citrus oils from the whole fruit, then in the case of bergamot oil, linalool may not be present in the pure R-(-) form. Under hydrodistillation conditions, most of the oil is removed from the vicinity of the acid in the first few minutes, so prolonged distillation under these conditions will not increase the amount of racemisation found in Table 1. The variation in enantiomeric distribution that occurs with fruits from different regions are shown in Table 3. These results point to some of the difficulties in using known values for determining the origin and processing methods used in the production of citrus oils. The range of values for linalool in lemon oil, where the enantiomeric excess ranges from 0% to 5 15
19
15
22
Table 2 The Effect of Acid Contact at pH 2 at 25 °C on the Enantiomeric Distribution of Linalool in Bergamot Oil Enantiomer
Cold pressed
2 hours
4 hours
24 hours
72 hours
R(-)
100.0 0.0
92.2 7.8
93.0 7.0
79.9 20.1
53.0 47.0
S(+)
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Table 3 Enantiomeric Distribution of Chiral Aroma Compounds for Authentic Citrus Oils a-Pinene
p-Pinene
RW S(-j
sf-j
Limonene
Linalool
sr-;
a-Terpineol
sr-;
Lemon Cyprus Israel U.S.A. 1 U.S.A. 2
60.1 72.2 66.4 73.3
39.9 27.9 33.6 26.7
7.7 6.2 6.7 4.7
92.3 93.8 93.3 95.3
1.2 1.4 1.3 1.6
98.9 98.6 98.7 98.4
39.7 45.0 50.8 72.0
60.3 55.0 49.2 28.0
71.2 80.0 78.8 88.8
70.1 68.6
29.9 31.4
10.0 8.9
90.0 91.1
2.7 2.2
97.3 97.8
62.1 64.6
37.7 35.4
79.8 20.2 77.2 22.8
71.2 68.2
28.8 31.7
8.2 9.4
91.8 90.6
2.3 1.7
97.7 98.3
98.3 98.7
1.7 1.3
61.3 38.7 37.4 62.6
28.8 20.0 21.2 11.2
Lime Cuba Mexico Bergamot U.K. unripe U.K. ripe
Table 4 Enantiomeric Distributions of Chiral Compounds in Commercial Citrus Oils. All oils were cold pressed except those marked" a-Pinene
fl-Pinene
Limonene
Linalool
a-Terpineol
s(-) R(+)
R(+) S(-)
S(-) R(+)
R(-) S(+)
S(-) R(+) 80.4 77.6 78.5 76.8 77.2 60.7 57.7
Lemon Sicily California Spain Argentina Unknown 1 Unknown 2 Distilled*
69.2 70.2 69.8 69.4 66.3 79.7 77.3
30.8 29.8 30.2 30.6 33.7 20.3 22.7
6.1 93.4 5.1 94.9 6.1 93.9 5.1 94.9 7.5 92.5 4.9 95.1 2.9 97.1
1.5 1.4 1.6 1.3 1.3 1.2 7.8
98.5 98.6 98.4 98.7 98.7 98.8 92.2
61.0 58.9 48.3 46.8 51.8 32.8 47.9
39.0 41.1 51.7 53.2 48.2 67.2 52.1
19.6 22.4 21.5 23.2 22.8 39.3 42.3
69.1 30.9 70.9 29.1
8.8 3.5
91.2 96.5
7.9 7.4
92.1 92.6
63.0 37.0 47.7 52.3
74.6 25.4 51.5 48.5
68.0 69.2 69.8 68.8 63.0 70.7
8.6 8.5 8.5 8.3 8.7 5.2
91.4 91.5 91.5 91.7 91.3 94.8
1.6 4.9 1.9 1.8 1.3 1.3
98.4 95.1 98.1 98.2 98.7 98.7
70.7 100 77.7 68.6 100 64.2
23.9 51.4 38.0 23.3 64.0 36.5
Lime Unknown Distilled* Bergamot Italy 1 Italy 2 Italy 3 Sicily Argentina 'Compound'*
32.0 30.8 30.2 31.2 37.0 29.3
29.3 0.0 23.3 31.4 0.0 35.8
76.1 48.6 62.0 76.7 36.0 63.5
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45%, makes linalool a poor choice as an indicator compound for lemon oil production. The range of values found for a-terpineol show a narrower range for lemon and lime oils than those of linalool, but the values for bergamot oil show no pattern with both the and the S-(-) isomer being present in excess, which is further supported by the data for the commercial oils shown in Table 4. a-Terpineol is clearly not a good indicator compound to determine the origin of an oil, particularly for bergamot oil. However, linalool and aterpineol are minor components in lemon and lime oils, although they are major odourants. The enantiomeric distribution of limonene shows good consistency for all the authentic citrus oils examined, and agrees with reported values, and lies within the range shown by a wide range of citrus oils. ' The commercial samples shown in Table 4, which contain more than 3 % of S-(-) limonene (both lime, the distilled lemon and the Italian No. 2 bergamot samples), clearly lie outside the expected range for citrus oils and suggest an anomaly in their production. The one good indicator compound that emerges from these data is linalool in bergamot oil. If the oil is cold pressed, and has been isolated without juice contact, then close to 100% of the R-(~) isomer should be present. However, the commercial bergamot oil samples in Table 4 show an additional dilemma. The Italian No. 2 sample has acceptable values for linalool, but those of both limonene and a-terpineol lie outside the expected ranges for a cold pressed citrus oil isolated without juice contact. The samples from Sicily and Italy No. 1 show a high percentage of S-(-)-linalool for a cold pressed oil, but if good manufacturing processes permit juice contact, then this value may be acceptable. The sample from Argentina appears to be an authentic cold pressed bergamot oil, on the basis of the enantiomeric distribution data. 23 24
1
4 CONCLUSION The data in Tables 1-4 show that it would be almost impossible to use chiral analysis alone, particularly on a single compound, to specify the origin of citrus oils. The range of values found for authentic citrus oil samples together with the loose definition of 'good manufacturing procedures' make the task of establishing acceptable values for enantiomeric distributions very difficult. The best way to assess the origin of a natural product would seem to be to adopt a more comprehensive approach and use chiral analysis in conjunction with quantitative analysis of chiral indicator compounds and other instrumental methods such as isotope ratio mass spectrometry or site-specific natural isotope fractionation nuclear magnetic resonance spectroscopy (SNIF-NMR). In all cases a comprehensive data base of acceptable values is needed to effectively establish the origin of a natural product. REFERENCES 1. 2. 3. 4.
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