FLAVOUR AND FRAGRANCE JOURNAL

Flavour Fra`r[ J[ 1999^ 04] 212Ð217

Quantitative determination and characteristic flavour of

daidai (Citrus aurantium L. var. cyathifera Y. Tanaka) peel
oil
H. S. Song,1 M. Sawamura,1 T. Ito,1 A. Ido1 and H. Ukeda1
1
Department of Bioresources Science, Faculty of Agriculture, Kochi University, B-200 Monobe, Nankoku, Kochi 783-8502, Japan

Received 07 February 1999

Revised 00 April 1999
Accepted 14 April 1999

ABSTRACT] Daidai "Citrus aurantium L[ var[ cyathifera Y[ Tanaka# peel oil was extracted by cold!pressing[
The ~avour components of daidai were quantitatively determined using two internal standards with GC and
GCÐMS[ The characteristic ~avour components of daidai were examined by GCÐolfactometry[ The percentages
by weight of hydrocarbons "05 compounds#\ esters "01 compounds#\ alcohols "00 compounds# and aldehydes "09
compounds# were approximately 85[3)\ 1[0)\ 9[2) and 0[0)\ respectively[ Geranial\ octyl acetate\ geraniol\
octanal\ cedrol\ cis!limonene oxide\ trans!1!decenal\ linalyl acetate and b!elemol were identi_ed as characteristic
~avour components of daidai[ Terpenes were the major components of daidai peel oil\ but they contributed little
to the characteristic ~avour of daidai[ Linalyl acetate and b!elemol had the highest ~avour dilution factors[ The
results of the omission test showed geraniol\ octanal\ linalyl acetate and cis!limonene oxide to be the active
components of the daidai ~avour[ Copyright Þ 1999 John Wiley + Sons\ Ltd[

KEY WORDS] daidai^ Citrus aurantium L[ var[ cyathifera Y[ Tanaka^ quantitative analysis^ weight percent^
sni.ng!test^ characteristic ~avour^ aromagram^ GCÐMS^ cold!pressed oil^ GCÐolfactometry "GCÐO#^ omission
test

Introduction threshold values to determine relative ~avour activity[

Daidai\ a sour Japanese Citrus fruit\ has been used in
food products such as sauces and dressings because of
its desirable ~avour[ There are few studies of daidai
essential oils including oils extracted by solvent!dis!
tillation and cold!pressing[0\1 It has been reported that
limonene\ octanal\ linalol and linalyl acetate are the
major volatile ~avour compounds of daidai[0\1 Flavour
analysis by means of gas chromatography "GC#\ gas
chromatographyÐmass spectrometry "GCÐMS# and\
recently\ gas chromatography olfactometry "GCÐO#
has been carried out to determine the ~avour
components[ In particular\ GCÐO\ including aroma
extract dilution analysis "AEDA# and CharmAnalysis\
has been used to identify the potent odorants in foods[2\3
Flavour dilution "FD# factors expressed as a result of
GCÐO ratings give us information about the charac!
teristic ~avour di}erences in foods[4 Moreover\ GCÐO
correlates concentration with FD factors equivalent to
 Correspondence to] M[ Sawamura\ Department of Bioresources Science\ Fac!
ulty of Agriculture\ Kochi University\ B!199 Monobe\ Nankoku\ Kochi 672!
7491\ Japan[ E!mail] sawamuraÝcc[kochi!u[ac[jp[
Contract:grant sponsor] Sasakawa Scienti_c Research Grant\ Japan Science
Society[
Contract:grant number] 09!014[
However\ the FD factor does not always correlate with
the characteristic ~avour\ because it sometimes depends
on the content[ The relative ~avour activity\ in which
both FD factor and content are considered\ may be a
good index to the ~avour characteristics[5
In the present study\ quantitative determination of
volatile components of daidai peel cold!pressed oil was
carried out by means of two internal standards and their
characteristic ~avour components were examined by
GCÐO and by sensory analysis[
Experimental
Materials
Mature daidai fruits were obtained from the Kochi
Fruit Experimental Station\ Kochi\ Japan[ The peel oils
were extracted by hand!pressing of the ~avedo and col!
lected in brine solution on ice in the same way as pre!
viously reported[6
Quantitative Determination
Quantitative analysis of citrus cold!pressed oil was
reported recently[6 The volatile components were ana!

Copyright Þ 1999 John Wiley + Sons\ Ltd[

213 H[ S[ SLONG ET AL[
lysed with a Shimadzu GC!03A gas chromatograph[
Peak areas were integrated with a Shimadzu C!R5A
Chromatopack integrator[ One ml Cold!pressed oil was
injected into a fused!silica capillary column "49 m×9[14
mm i[d[\ _lm thickness 9[14 mm coated with Thermon
599T "Shimadzu Co[ Ltd[\ Kyoto#\ which is equivalent
to PEG 19M[ The ~ow rate of nitrogen carrier gas
was 0[9 ml:min[ The split ratio was 0 ] 49[ The column
temperature was programmed from 69>C to 129>C at a
rate of 1>C:min[ The injector and detector were kept
at 149>C[ A non!polar column was also used for GC
analysis\ which was DB!4 "29 m×9[14 mm i[d[\ _lm
thickness 9[14 mm\ J+W Scienti_c\ USA#[
GC–O and AEDA
Sni} testing and AEDA were carried out following the
method of Grosch[5 The FD factors of the ~avour com!
ponents were determined by AEDA[ A Shimadzu GC!
7A gas chromatograph equipped with FID was used[ A
semibore capillary column "29 m×9[42 mm i[d[\ _lm
thickness 49 mm# coated with Supelcowax 09 "Supelco\
PA\ USA# was used for e}ective separation of the
peaks[2\4\6 The GC conditions were the same as described
above[ The ~ow rate of nitrogen carrier gas was 49
ml:min and the split ratio was 04 ] 24[
GC–MS Conditions
A Shimadzu GC!06A coupled with a Shimadzu QP!
4999 was used[ The column was the same as the GC!
03A[ The split ratio of injector was 0 ] 23[ The other
GCÐMS was a GC HP5989 with a JMS!599W "JEOL\
Tokyo#[ A column with HP!4 "crosslinked 4) PHME
Siloxane\ 29 m×9[21mm i[d[\ _lm thickness 9[14 mm#
was used[ The carrier gas was helium[ MS conditions
for both were as follows] ionization voltage\ 69 eV^ ion
source temperature\ 149>C[ The column temperature
was programmed in the same way as the GCÐO[ Both
mass spectra and their GC retention data of the indi!
vidual components were identi_ed by comparison with
those of authentic compounds previously analysed and
stored in the data system[ Peak enrichment on co!injec!
tion was also compared with authentic standards wher!
ever possible[ The Kovats indices for the two di}erent
columns were also determined for all volatile constitu!
ents\ using a homologous series of n!alkanes[7\8
Omission Test
The model samples of daidai ~avour were made with
authentic compounds according to the results of sni}
testing and AEDA[09\00 The sensory analysis panel was
composed of 09 assessors[ The closest match with daidai
~avour was assigned 8 points\ and the least close 0 point[
The statistical analysis device was the t!test[01
Copyright Þ 1999 John Wiley + Sons\ Ltd[
Results and Discussion
A typical chromatogram of daidai peel oil is shown in
Figure 0 "top#[ Percentage weight of ~avour com!
ponents of daidai peel oil are given in Table 0[ Hydro!
carbons "05 compounds# such as limonene\ myrcene
and a!pinene accounted for about 85[3)\ esters "01
compounds#\ such as linalyl acetate and geranyl acetate\
for about 1[0)\ alcohols "00 compounds#\ such as lin!
alol and nerolidol\ for about 9[2)\ and aldehydes "09
compounds# such as decanal and octanal\ for about
0[0)[ The monoterpene hydrocarbon contents of lim!
onene and myrcene were as high as 81[4) and 1[1)\
respectively[ Linalyl acetate "0[3)# and octanal "9[5)#
were abundant daidai ~avour components\ followed by
limonene[ Linalyl acetate was predominant in bergamot
oils\ giving freshness to citrus ~avour[1\02 A high lim!
onene content was detected in the essential oil of daidai\
grapefruit\ sweet orange and mandarin[03
The aromagram derived from the gas chromatogram
of daidai peel oil is shown in Figure 0 "bottom#[ Thirty!
one odorants\ their FD factors and relative ~avour
activities in the extract of daidai peel oil are shown in
Table 1[ Nine compounds\ such as octanal\ linalyl acet!
ate and geranial were detected as daidai!characteristic
~avour components by means of the sni} testing[ The
sum of those components accounted for 1[0) by
weight[ Limonene was found to be the major ~avour
component of daidai peel oil\ and occupied the lowest
relative ~avour activity\ despite its high FD factor "2#[6
Myrcene\ linalol and octanal showed results similar to
those for limonene in the aspects of content\ relative
~avour activity and FD factor[ From these results\ the
contents of the ~avour components were shown to
in~uence the FD factors\ even though they were not
characteristic odorants of daidai[ In this case\ their con!
tribution to the characteristics of daidai essential oil
were reconsiderable from their relative ~avour activi!
ties[ Cedrol\ p!cymene\ cis!limonene oxide\ b!elemol\
octyl acetate and trans!1!decenal showed high relative
~avour activity[ Except for p!cymene\ these components
were described as daidai!like[ p!Cymene was seen as an
important ~avour component because of its high rela!
tive ~avour activity\ even though its odorant description
was di}erent from that of daidai!like aroma[ The rela!
tive ~avour activity of linalyl acetate was quite low\
which was one of the compounds having the highest
FD!factors[
The null model of daidai ~avour was constructed
with authentic compounds\ as shown in Table 2[ Nine
compounds\ evaluated as daidai!like ~avour\ were used
for the omission tests from the results of sni} testing[
We tried to prepare the daidai ~avour model with the
same concentration as that found in the essential oil[
However\ it was judged that the model solution smelled
too strongly and had a quite di}erent ~avour to daidai[
Flavour Fra`r[ J[ 1999^ 04] 212Ð217
 PEEL OIL OF CITRUS AURANTIUM 214

Figure 1. Gas chromatogram (top) and aromagram (bottom) of volatile flavour components of daidai peel oil

Copyright Þ 1999 John Wiley + Sons\ Ltd[ Flavour Fra`r[ J[ 1999^ 04] 212Ð217
215 H[ S[ SLONG ET AL[

Table 1. Volatile flavour components of daidai (Citrus aurantium L. var. cyathifera Y. Tanaka) peel oil
Peak no[ Component RI "non!polar# RI "polar# Peak ")# "w:w# Reference no[a
0 Ethyl acetate 770 9[2 1
1 a!Pinene 814 0915 9[5 1\06 a
2 Camphene 845 0001 tr 1\06 a
3 b!Pinene 867 0054 9[3 1\06 a
4 Sabinene 862 0066 9[1 1\06
5 Myrcene 875 0198 1[1 1\06 a
6 Limonene 0922 0107 81[4 1\06 a
7 cis!b!Ocimene 0926 0145 tr 1\06 a
8 trans!b!Ocimene 0938 0155 9[2 1
09 p!Cymene 0171 tr 1\06 a
00 Terpinolene 0958 0176 tr 1\06 a
01 Octanal 0991 0185 9[5 0\1\06 a
02 Octyl acetate 0262 tr 1a
03 Nonanal 0096 0281 tr 1\06 a
04 cis!Limonene oxide 0027 0326 tr 1\06
05 trans!Limonene oxide 0032 0338 tr 1\06 a
06 b!Terpineol 0077 0350 tr 04
07 Citronellal 0043 0362 9[0 1\06 a
08 Decanal 0129 0383 9[1 1\06 a
19 Camphor 0408 tr 1\06 a
10 Linalol 0985 0433 9[1 0\1\06
11 Linalyl acetate 0958 0445 0[3 0\1\06 a
12 Nonyl acetate 0293 0460 tr 1a
13 b!Elemene 0274 0466 9[0 1\06 a
14 Caryophyllene 0304 0477 tr 1\06 a
15 Isomenthol 0484 tr 05 a
16 Undecanal 0294 0487 tr 1\05 a
17 Thujyl alcohol 0505 tr 1
18 trans!1!Decenal 0518 tr 06 a
29 a!Humullene 0335 0532 tr 1\06 a
20 trans!b!Farnesene 0345 0544 tr 1\06 a
21 Decyl acetate 0398 0555 9[0 1a
22 Neral 0562 tr 1a
23 Terpinyl acetate 0230 0572 9[0 1
24 Germacrene D 0374 0588 9[0 1\06
25 Neryl acetate 0253 0693 tr 1a
26 Geranial 0153 0604 9[0 1
27 Bicyclogermacrene 0620 tr 1
28 trans!Carvone 0132 0625 tr 1\05 a
39 Gernayl acetate 0270 0636 9[1 1
30 Perillyl aldehyde 0169 0647 9[0 1\06 a
31 p!Menth!0!en!8!yl!acetate 0678 tr 1
32 Tridecanal 0687 tr 1
33 Gernayl propionate 0702 tr 1\06 a
34 Geraniol 0158 0726 tr 1a
35 trans!Carveol 0731 tr 04\06
36 Dodecyl acetate 0509 0758 tr 1a
37 Perillyl acetate 0770 tr 1
38 Nerolidol 0422 1929 9[0 1\06
49 b!Elemol 0438 1947 tr 1a
40 Cedrol 1029 tr 1a
41 Unknown 1028 tr
42 Thymol 1292 tr 1\06
43 a!Bisabolol 0574 1365 tr 1\06
tr\ trace "less than 9[94)#\ a\ authentic standards used for identi_cation and co!injection^ a Reference no[\ where identi_ed earlier[

Therefore\ dilution was carried out without alteration
of the ratio of concentration[ Secondly\ FD factors were
also considered in the dilution ratio "DR# in order to
make the daidai ~avour model as similar as possible[
The index of FD factor 25 was decided as dilution stan!
dard[ On the basis of this dilution standard\ DR was
decided as shown in Table 2[
Water09 was considered as a dilution solvent[ Lim!
onene and linalol were also considered as dilution sol!
vents because of their abundance in citrus essential oils[
Several full models for trial containing these solvents
were prepared and o}ered to panels[ The panel pre!
ferred the full model containing limonene as the dilution
solvent[ Limonene acts as a solvent to enable dissolution
of citrus essential oil components in nature\ while con!
tributing little to the characteristic ~avour of daidai\
but it is likely to be important as a background ~avour
of citrus essential oils[ Sensory tests were performed

Copyright Þ 1999 John Wiley + Sons\ Ltd[ Flavour Fra`r[ J[ 1999^ 04] 212Ð217
 PEEL OIL OF CITRUS AURANTIUM 216

Table 2. Aroma description of volatile flavour components of daidai peel oil

Peak no[ Component Odorant description FD factor "2n# Relative ~avour activitya
1 a!Pinene Pine!like 5 2[7
3 b!Pinene Pine!like 0 9[7
4 Sabinene Bitter\ Pleasant 1 1[9
5 Myrcene Earthy\ Mushroom!like 6 1[2
6 Limonene Sour\ Lemon!like 6 9[2
8 trans!b!Ocimene Mushroom!like 2 1[5
09 p!Cymene Sour\ Sweet 4 64[3
01 Octanal Daidai!like\ Aldehyde 5 2[7
02 Octyl acetate Daidai!like\ Sweet 3 35[6
03 Nonanal Sweet\ Fragrant 4 02[1
04 cis!Limonene oxide Daidai!like\ Citrusy 6 47[4
07 Citronellal Sweet\ Fragrant 6 01[7
08 Decanal Sweet\ Sour 5 6[0
10 Linalol Fragrant 6 6[0
11 Linalyl acetate Daidai!like 7 2[2
13 b!Elemene Sweet\ Pleasant 6 01[5
18 trans!1!Decenal Daidai!like\ Pleasant 5 26[3
22 Neral Sour\ Pleasant 3 09[6
23 Terpinyl acetate Grassy\ Acidic 2 3[2
24 Germacrene D Grassy\ Pleasant 3 4[6
25 Neryl acetate Sour\ Citrusy 5 03[5
26 Geranial Daidai!like\ Sour 2 2[6
28 trans!Carvone Bitter 3 20[2
39 Geranyl acetate Sweet\ Sour 2 2[4
34 Geraniol Daidai!like\ Pleasant 4 07[1
35 trans!Carveol Sour\ Bitter 4 05[4
36 Dodecyl acetate Sour 4 15[3
37 Perillyl acetate Sweet\ Fragrant 5 00[2
38 Nerolidol Sour\ Citrusy 4 6[8
49 b!Elemol Daidai!like\ Sweet 7 37[4
40 Cedrol Daidai!like\ Sweet 5 76[3

n\ the index of FD!factor "2n#[ a Relative ~avour activitylog 2n:S9[4\ where 2n is the FD factor and S is the peak percentage weight[

Table 3. Concentrations of flavour compounds in the model of daidai flavour

Compounds FD factor "2n# DRa Conc[ in daidai peel oil ")# Conc[ in mixed solution ")#

Geranial 2 0 9[049 9[0499

Octyl acetate 3 4:5 9[991 9[9906
Geraniol 4 3:5 9[906 9[9002
Octanal 5 2:5 9[470 9[9994
Cedrol 5 2:5 9[990 9[9994
trans!1!Decenal 5 2:5 9[996 9[9924
cis!Limonene oxide 6 1:5 9[992 9[9909
Linalyl acetate 7 0:5 0[253 9[1162
b!Elemol 7 0:5 9[995 9[9909

A solution of 9[0) limonene in 09) methanol was used for dilution[ a DR is dilution ratio[

to decide the proper concentration of limonene in the
sample solutions for omission tests[ Thus\ the dilution
solvent was decided to be 9[0) limonene solution in
09) methanol[ Methanol a}ected the sensory test less
than ethanol[00 The volume of the compound withdrawn
was replaced with 9[0) limonene solution[
The results of omission tests are summarized in Table
3[ The full model was composed of all the nine com!
pounds blended in the concentration described above\
and used as the standard for scoring test[ The full model\
whose mean score was 5[3\ presented daidai!like ~avour[
Omission tests were performed by removing one or
more compounds from the full model in each prep!
aration[ Tested samples 0Ð2 indicate that geranial
played a signi_cant role in the daidai ~avour\ but the
absence of octyl acetate did not signi_cantly a}ect its
~avour[ It seems that geraniol had no signi_cant di}er!
ence from the result of the forth and _fth samples[
Samples 5Ð00 indicate that octanal\ cedrol and trans!1!
decenal\ with FD factors of 25\ were not signi_cant in
the daidai ~avour[ Removal of cis!limonene oxide and
linalyl acetate\ with high FD factors\ signi_cantly low!
ered the similarity to the model of the daidai ~avour\
according to samples 02Ð05[ The results of samples 7
and 8\ and 02 and 03\ suggested that both geranial and
octyl acetate were important in the presence of other
~avour!related components such as octanal\ cis!lim!
onene oxide and linalyl acetate[ From these experiments

Copyright Þ 1999 John Wiley + Sons\ Ltd[ Flavour Fra`r[ J[ 1999^ 04] 212Ð217
217 H[ S[ SLONG ET AL[

Table 4. Sensory analysis for model samples of daidai aroma

Sample no[ Compounds omitted from the full model sample Mean score
Full model 5[3

0 Geranial\ octyl acetate 4[5

1 Geranial 3[0
2 Octyl acetate 4[4
3 Geranial\ octyl acetate\ geraniol 3[7
4 Geraniol 4[2
5 Geraniol\ octyl acetate\ octanal\ cedral\ trans!1!decenal 3[8
6 Geraniol\ octyl acetate\ cedrol\ trans!1!decenal 4[0
7 Geraniol\ octyl acetate\ octanal 3[3
8 Octanal 5[1
09 trans!1!Decenal 4[4
00 Octanal\ cedrol\ trans!1!decenal 4[4
01 Geranial\ octyl acetate\ octanal\ cis!limonene oxide 3[7
02 cis!Limonene oxide\ linalyl acetate\ b!elemol 4[9
03 Geranial\ octyl acetate\ cis!limonene oxide\ linalyl acetate\ b!elemol 2[7
04 Geranial\ octyl acetate\ cis!limonene oxide 3[9
05 Geranial\ octyl acetate\ linalyl acetate 3[1
06 Geranial\ octyl acetate\ b!elemol 3[8
07 Geranial\ cis!limonene oxide\ linalyl acetate\ b!elemol 3[6
The composition of the full model sample is shown in Table 2[ Sensory analysis] scoring test "the scores between 8 and 0#[  and  Signi_cant
di}erence in favour between the full model and its insu.cient sample by t!test " p ³ 9[94\  p ³ 9[90#[

it is concluded that those nine compounds comprising
the full model contribute signi_cantly to the charac!
teristic ~avour and are the essential minimum when the
~avour of daidai is reproduced arti_cially[
Acknowled`ements * This work was partly supported by a Sasakawa
Scienti_c Research Grant\ No[ 09!014\ from The Japan Science
Society[
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Copyright Þ 1999 John Wiley + Sons\ Ltd[ Flavour Fra`r[ J[ 1999^ 04] 212Ð217