W.L.P. Bredie and M.A.

Petersen (Editors) 193

Flavour Science: Recent Advances and Trends
9 2006 Elsevier B.V. All rights reserved.

Characterisation of the odour volatiles in Citrus

aurantifolia Persa lime oil from Vietnam

Nguyen Thi Lan Phi a, Nguyen Thi Minh Tu b, Chieko Nishiyama a

and Masayoshi Sawamura a

aDepartment of Bioresources Science, Faculty of Agriculture, Kochi

University, B-200 Monobe, Nankoku, Kochi 783-8502, Japan," bHanoi
University of Technology, Institute of Biological and Food Technology, 1
Dai Co Viet, Hanoi, Vietnam

ABSTRACT

Essential oil of C. aurantifolia Persa (lime) from Vietnam was isolated by a cold-
pressing method with a yield of 0.02% of the flesh fruit. The odour-active constituents
were investigated by capillary GC, GC-MS, GCO and AEDA. Ninety-six compounds
were detected and 92 compounds were identified in the lime oil. Limonene (73.5%) was
the major component, followed by geranial (8.4%), neral (4.9%), myrcene (2.1%) and
[3-bisabolene (1.6%). Eight compounds, limonene, geranial, neral, myrcene, geranyl
acetate, a-pinene, Gt-terpineol and [3-bisabolene were evaluated as the odour-active
compounds, showing high FD values (FD-factor > 6). From the GC-sniffing results,
neryl acetate, [3-bisabolene, 1-carvone, geranyl acetate, Gt- and [3-citronellol, cumin
aldehyde, perillaldehyde, nerol, tridecanal, germacrene B, geraniol, dodecyl acetate,
caryophyllene oxide and perillyl alcohol were suggested as contributors to the
characteristic aroma of this Vietnamese lime.

1. I N T R O D U C T I O N

Lime is one of the principal citrus fruits in the world. The two species of lime that have
been studied mostly are Citrus aurantifolia Swingle (Mexican, Key or West Indian lime)
and Citrus latifolia Tanaka (Tahiti, Persian or Bears lime). The composition of lime oil
has been reported by many researchers [ 1,4-9]. C. aurantifolia Persa (lime) is one kind of
sour citrus fruits, very important and popular in Vietnam, as well as Yuzu (C junos Sieb.
ex Tanaka) in Japan or lemon in other countries in the world. It has been used as a folk
medicine in curing sore throat, cough or hair care [2]. As a result of different geographical
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origins, climate and ancient habits and practices in planting, lime species has become very
abundant. However, there have been very few studies reporting on the composition and no
studies have been undertaken to characterise the aroma volatiles of Vietnamese lime. The
purpose of this study was to elucidate the characteristic components of the essential oil
from Vietnam lime, and to properly evaluate the quality, origin or genuineness.

2. M A T E R I A L S AND M E T H O D S

2.1. Sample preparation

Lime (Citrus aurantifolia Persa) fruits were obtained in September 2004 from Institute
of Fruit and Vegetable Research, Hanoi, Vietnam. The peel oil was extracted by a hand
pressing method as previously reported [3].

2.2. Essential oil GC analysis

The analysis of the oil composition was carried out using a Shimadzu GC-17A equipped
with an FID and a DB-Wax column (60 m • 0.25 mm i.d., film thickness of 0.25 gm,
Folsom, CA). The column temperature was programmed from 70 ~ (2 min) to 230 ~
(20 min) at 2 ~ The peel oil of 0.5 gl were injected directly and the split ratio of
the injector being 1:50. The quantitative analysis was done in triplicate using 1-hexanol
and nonadecane as internal standards. A Shimadzu GC-17A linked to a Shimadzu QP-
5050 MS was used for identifying the detected volatiles.

2.3. Essential oil GCO analysis

A Shimadzu GC-17A equipped with a DB-Wax column (60 m • 0.53 mm i.d., film
thickness 1 gm, Folsom, CA) connected to a humidifier ODO II (SGE, Japan) was used
for GC-olfactometry (GCO). The cold-pressed lime oil was stepwise diluted three-fold
with acetone based on AEDA technique which was developed by Grosch [10].

2.4. Similarity testing of reconstructed essential oil

Aroma model samples of the lime were prepared using authentic compounds on the
basis of the results of chemical and sensory analyses. Propylene glycol was used as a
solvent for the aroma models. A sensory test was performed to evaluate the similarity
between the scents of cold-pressed lime oil and the model samples.

3. R E S U L T S AND DISCUSSIONS

The yield of the essential oil was 0.02% of the flesh fruit. GC-MS identified 92
compounds (some compounds are shown in Table 1) among the 96 compounds detected
by GC-FID which constituted approximately 97.9% of the lime oil. The quantitative
analyses were done in triplicate. There was no significant difference between replicated
datasets (p<0.05).
 195

Table 1. Volatile aroma components o f Citrus aurantifolia Persa (lime) essential oil.

Peak w/w b
no. Compound RI a (%) Odour description c Nd RFA e Identification f
1 a-Pinene 1029 0.3 Green, woody 7 6.6 RI, MS
3 [3-Pinene 1117 ** Green, herbal 5 l 1.0 RI, MS
4 Sabinene 1127 0.1 Woody, fruity 6 8.5 RI, MS
6 Myrcene 1162 2.1 Citrus, balsamic 8 2.6 RI, MS
9 Limonene 1214 73.5 Herbal, citrus, minty 10 0.6 RI, MS
10 ~-Phellandrene 1219 0.2 Citrus, minty 6 6.0 RI, MS
12 cis-~-Ocimene 1235 0.6 Herbal, woody 6 3.7 RI, MS
13 7-Terpinene 1252 0.9 Fresh, mushroom 5 2.6 RI, CoGC
15 Octanal 1289 0.3 Grassy, flowery 6 5.1 RI, MS
17 Nonanal 1392 0.4 Green, waxy 5 3.8 RI, MS
22 (E)-Limonene oxide 1462 * Grassy, floral 4 27.8 RI, CoGC
24 8-Elemene 1472 0.1 Sweet, spicy 5 6.4 RI, MS
25 Citronellal 1478 0.1 Fresh, floral 5 6.4 RI, MS
26 Decanal 1496 0.6 Fatty, floral 6 3.6 RI, MS
29 Linalool 1545 0.5 Floral, spicy, pungent 6 4.0 RI, MS
39 cis-Verbenol g 1628 ** Grassy, dusty 4 22.4 MS
49 Neral 1682 4.9 Citrus, floral 9 1.9 RI, MS
53 a-Terpineol 1696 0.1 Citrus, floral, bitter 7 12 RI, MS, CoGC
55 Germacrene D 1709 0.1 Grassy, citrus 6 11.1 RI, MS
56 Neryl acetate 1726 ** Lime-like, pungent 4 9.0 RI, MS
57 [3-Bisabolene 1731 1.6 Lime-like, pungent 7 2.6 RI, MS, CoGC
58 Geranial 1734 8.4 Citrus, grassy 10 1.6 RI, MS
59 /-Carvone 1739 * Cool, lime-like 4 61.1 RI, CoGC
60 Geranyl acetate 1753 0.1 Lime-like 8 13.1 RI, MS
61 a-Citronellol 1755 * Lime-like 3 51.1 RI, MS, CoGC
62 [3-Citronellol 1764 * Lime-like 4 36.5 RI, MS, CoGC
63 ~-Selinene g 1771 ** Grassy, citrus 5 14.2 MS
64 Cumin aldehyde 1779 ** Lime-like, pungent 4 26.1 RI
65 Perillaldehyde 1783 ** Lime-like, pungent 4 12.0 RI, CoGC
66 Nerol 1797 ** Lime-like, fresh 5 12.5 RI, MS
67 Tridecanal 1811 ** Lime-like, sour 5 12.1 RI, MS
68 Germacrene B 1830 ** Lime-like, floral 5 14.5 RI, MS
69 Geraniol 1845 ** Lime-like, fresh 5 12.4 RI, MS, CoGC
70 Dodecyl acetate 1882 * Lime-like 3 34.7 RI, MS
71 Tetradecanal 1918 ** Citrus, herbal, cool 6 14.0 RI, MS
73 Caryophyllene oxide 1989 ** Lime-like, cool, floral 4 16.3 RI, MS
74 Perillyl alcohol 2004 * Lime-like, floral 4 28.5 RI, CoGC
75 Pentadecanal g 2024 0.1 Cool, sour, citrus 5 10.6 MS
84 Cedryl acetate 2170 ** Green, citrus 5 13.5 RI, CoGC
87 a-Bisabolol 2216 0.1 Lemon-like, flowery 4 6.4 RI, MS
92 Cinnamyl alcohol 2283 ** Citrus, oily 4 8.8 RI, MS, CoGC
aRetention index on D B - W a x column, b ' W e i g h t percent less than 0.005%; **Weight percent
between 0.005% and 0.05%. COdour description at the GC-sniffing port during GCO. dN: l o g 3 ( F D -
factor), eRFA: log (FD-factor)/S 1/2, where S is the weight percentage, fIdentification method:
retention index (RI), mass spectrum (MS) and co-injection with authentic standards (CoGC).
gTentatively identified.
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Hydrocarbons including 13 monoterpenes (77.8%) and 19 sesquiterpenes (3.3%), 19

aldehydes (15.4%), 25 alcohols (1.2%), 9 acids and esters (0.2%), 3 ketones and 4
oxides in trace amount were determined. The most abundant compound was limonene
(73.5%), followed by geranial (8.4%), neral (4.9%), myrcene (2.1%) and 13-bisabolene
(1.6%). A series of the 12 aliphatic aldehydes ranging from heptanal to nonadecanal,
except heptadecanal, were also identified. Germacrene B and D were detected in this
lime oil at the concentration of 0.03% and 0.1%, respectively.
The lime oil aroma was evaluated as fresh, sour and green-citrus by the assessors. As
summarised in Table 1, limonene, geranial, neral, myrcene, geranyl acetate, ~-pinene,
a-terpineol and [3-bisabolene were the higher odour-active compounds (FD factor > 6).
1-Carvone in a relatively small amount, showed the highest RFA value (61.1). Geranial
and neral were described as having citrus and grassy/floral aroma. The description from
GC-olfactometry revealed that neryl acetate, 13-bisabolene, l-carvone, geranyl acetate,
a- and [3-citronellol, cumin aldehyde, perillaldehyde, nerol, tridecanal, germacrene B,
geraniol and dodecyl acetate would contribute to the characteristic aroma of lime.
A subsequent study looked at the sensory effects of blending of pure odorants. The
aroma models were compared with the original lime oil in respect to their sensory
characteristics. Various combinations and concentrations of authentic compounds were
examined. The final blend, which had the odour rather similar to that of lime had a
composition (%) as follows: limonene (50.00), neral (2.25), geranial (7.75), myrcene
(0.33), geranyl acetate (0.67), ~-citronellol (0.32), [3-citronellol (0.32), nerol (0.06),
tridecanal (0.07), geraniol (0.07), dodecyl acetate (0.04), [3-bisabolene (0.67), neryl
acetate (0.27), cumin aldehyde (0.13), caryophyllene oxide (0.07), perillaldehyde (0.13),
perillyl alcohol (0.04) and 1-carvone (0.13). The similarity of this aroma model to the
real peel oil from the scoring test was 6.8 +0.7.

References
1. B. Lawrence, Perfum. Flavor., 21 (1996) 62.
2. D.T. Loi, Vietnamese medicinal plants, Hanoi, Vietnam (1986) (in Vietnamese).
3. M. Sawamura, K. Shichiri, Y. Ootani and X. H. Zheng, Agric. Biol. Chem., 55 (1991)
2571.
4. M. Sawamura, J. Agric. Food Chem., 4 (2000) 131.
5. S.M. Njoroge, H. Ukeda, H. Kusunose and M. Sawamura, Flavour Fragrance J., 11 (1996)
25.
6. I.D. Morton and A.J. MacLeod (eds.), Food flavours, Amsterdam, The Netherlands (1990)
93.
7. N.T. Minh Tu, L.X. Thanh, A. Une, H. Ukeda and M. Sawamura, Flavour Fragrance J., 17
(2002) 169.
8. W. Feger, H. Brandauer and H. Ziegler, Flavour Fragrance J., 15 (2000) 81.
9. P. Dugo, L. Mondello, G. Lamonica and G. Dugo, J. Agric. Food Chem., 45 (1997) 3608.
10. H. Guth and W. Grosch, Flavour Fragrance J., 8 (1993) 173.