E. T. Contis et al.

(Editors)
Food Flavors: Formation, Analysis and Packaging Influences
© 1998 Elsevier Science B.V. All rights reserved 667

Changes in the flavour of monoterpenes during their autoxidation under

storage conditions

J. Pokomy, F. Pudil, J. Volfovd and H. Valentovd

Department of Food Chemistry and Analysis, Prague Institute of Chemical Technology,

Technickd 5, CZ-16628 Prague 6, Czechia

Abstract
Limonene and linalool were oxidized at 40 and 60°C at limited access of oxygen, and their
oxidative changes were determined by gas chromatography - mass spectroscopy (GC/MS).
The respective oxides belonged to important oxidation products. The changes were compared
with those of sensory odour profile. Citrus odour notes slowly disappeared, while the intensity
of woody, acidic and heavy odour notes increased. Heavy note reminded higher esters,
tropical fruits and flowers. Dihydropyridine antioxidants acted as inhibitors of medium
activity; their presence slightly influenced the composition of oxidized products and the
sensory character.

1. INTRODUCTION

Monoterpenes are important components of many essential oils, particularly citrus oils,
which consists of 80-95% monoterpenes, mainly limonene. Limonene is easily oxidized via
free radicals, which are converted into a mixture of six unstable hydroperoxides by reaction
with oxygen [1], in a manner similar to the structurally related carvomenthene [2], a-terpineol
and a-pinene [3,4]. Similar reaction proceeds under intensive ultraviolet irradiation even in
absence of photosensibilizers [5-7]; they are oxidized into hydroperoxides, however, their
composition is different. Carvone and carveol [5] and 1,2-limonene oxide [1] belong to
important oxidation products that deteriorate the sensory character. Then* precursors are two
mesomeric free radicals formed on the 6th carbon atom of limonene [8]. In addition to 1,2-
epoxide, the 8,9-isomer is produced. The epoxides and hydroperoxides are converted into the
respective hydroxylic derivatives [8]. The relationship between the changes of the chemical
composition of terpenes undergoing autoxidization and changes of their sensory character
were the object of this study.

2. MATERIALS AND CHEMICALS

Monoterpenes, (S)-(-)-limonene (96%), (±)-linalool (97%) and various reference

substances, were produced by Aldrich. Dihydropyridine antioxidants, Diludine (2,6-dimethyl-
3,5-6/5-ethoxycarbonyl-l,4-dihydropyridine) and OSI 7284 (2,6-dimethyl-3,5-6w-
butoxycarbonyl-l,4-dihydropyridine), were synthetized at the Institute of Organic Synthesis,
Riga, Latvia.
668

3. ANALYTICAL METHODS

3.1. Solid phase microextraction (SPME)

Volatiles were adsorbed on a 65 |im Carbowax-divinylbenzene fiber for a manual holder

(Supelco, USA). The extraction time was 10 nun at 40°C, the desorption 2 min at 220^*0; the
cleaning time 30 min at 220°C.

3.2. Gas liquid chromatography (GLC)

The GC8000 Series Fisons gas chromatograph was equipped with a headspace autosampler
HS800 and a 60 m x 0.32 mm Supelcowax 10 (layer thickness 0.25 mm) capillary column
(Supelco, USA). Column temperature was programmed from 50°C (2 min), heating rate
2°C/min to 220''C (30 min). The injector temperature was 220°C, the flame ionization
detector (FID) temperature 250°C; helium carrier gas pressure was 100 kPa, and the
input/split ratio 1:25. Retention indices were calculated using a mixture of ^t-alkanes as
reference substances. For the GC-mass spectrometry (GC/MS) analysis, the MSD8000 mass
spectrometer was used; the ionizing energy was 70 eV. Internal standards were used for the
calculation of absolute amounts of components (A2-decane).

33, Sensory analysis

Sensory analysis was performed according to the international standard [9] in a test room
provided with six standardized test booths [10]. The assessor panel consisted of 12 selected
and trained persons [11] with at least 6 months experience in sensory profiling of. A 100 mg
sample was placed into a 250 mL wide-neck ground-glass bottle, and the odour intensity was
evaluated by sniffing. For the analysis of stabilized samples, 100 ^L of 1% methanol solution
of the antioxidant was added, and the solvent evaporated. The sample was then added, the
bottle closed, and shaken. Odour acceptability was determined using an unstructured graphical
scale: straight lines 100 mm long [13] (0% = rather bad, 100% = excellent). The sensory
profile [12] consisting of 24-36 descriptors (see the spider-web diagrams in Figure 2), was
evaluated using unstructured graphical scales, i. e. straight lines 100 mm long [13] (0% =
imperceptible, 100% = very strong). Two samples were served per session. A total of 24
responses were used for the calculation of means. The standard deviation of means varied
between 2-6% of the scale.

4. OXTOATION PROCEDURE

A portion of 100 mg of a terpenic substance was placed into a 10 mL glass vial, and 5 mg
of A2-decane (internal standard) were added. The vial was sealed and the mixture aged at 40^C
in a thermostat. Volumes of 100 |LIL vapor phase were injected into the GC injector using a
gas-tight Hamilton syringe. For sampling into the GC-MS, the solid phase microextraction
(SPME) sampling technique was used. In experiments with stabilized samples, 100 mL of a 1
% methanol solution was added, the solvent was evaporated, the sample of terpenes was
 669

added, the vial sealed, and the antioxidant dissolved by shaking, stored at 40 or 60''C,
respectively.

5. RESULTS AND DISCUSSION

5.1. Oxidation of limonene

Limonene was oxidized both at 40 and 60°C in the presence of insufficient air in the
headspace for complete oxidation, simulating the actual conditions in packed flavoured foods
and beverages. Pure limonene was added at the beginning of storage, but after several hours at
40°C, several major oxidation products were formed simultaneously; at least 52 components
were detected after 20 hours of storage. An example of limonene oxidized at 60^C for 20
hours is shown in Figure 1, and the list of substances identified in the oxidized mixture is
shown in Table 1.

too
4 • 7

75

50-

f
1
1 <
25
1 tl i« 11
It
19 I t 1

iliililijl
It i i 1

^j
L . L lllilL II LiijLilJIJ
10 20 70
TdbM CalA.]

Figure 1. GC profile of limonene oxidized at 60°C for 20 h

670

Table 1
Identification of reaction products of limonene oxidation
Peak Retention Retention Identified
No. time [min] index compounds
1 6.57 822 acetone
2 7.50 890 methanol
3 8.31 933 ethanol
4 10.05 1000 «-decane
5 19.91 1209 limonene
6 21.93 1249 1,3,8-p-menthatriene
7 23.79 1277 p-cymene
8 34.85 1448 cw-limonene oxide
9 35.66 1462 /raw^-limonene oxide
10 46.46 1616 menthadienol
11 47.63 1626 ascaridol
12 49.09 1639 2,8-menthadien-l-ol
13 50.13 1647 citral
14 53.02 1670 carvone
15 58.85 1833 cw-carveol
16 59.69 1848 p-cymenol
17 60.58 1863 /rcms-carveol
18 67.77 1990 menthadienol
19 68.23 1999 7-hydoxylimonene
Peak numbers refer to Figure 1; n-decane was used as inner standard (retention index RI
1000).

Hydroperoxides, the primary reaction products, were destroyed under conditions of GC

separation so that only tiie more stable oxidation products were detected, such as cis- and
/raMs-limonene oxides, carvone, acetone, citral, cis- and /raAis-6,8-menthadien-2-ols
(carveols), 2,8-menthadien-l-ol, l,8-menthadien-7-ol (7-hydroxy limonene), /7-cymenol and
ascaridol (a cyclic peroxide, which is relatively stable). During the interval studied, limonene
remained the most prominent compound present; the reason was that the availability of
oxygen in the sealed sample was low so that only small part of the original amount could be
oxidized. Numerous compounds were formed in the beginning of oxidation (most likely
produced by decomposition of hydroperoxides), but were decomposed during further storage.
Other compounds, such as cw-limonene oxide and menthadienol, were formed in larger
amounts only after more than 60 hours of oxidation, probably being only tertiary reaction
products. Examples are given m Table 2.
 671

Table 2
Time development of some limonene reaction products during storage at 60°C
Time [h] Acetone 1,3,8-p-Men- cw-Limonene Carvone cw-Carveol
thatriene oxide
2.0 1.29 25.14 0.92 0.26 0.88
10.5 1.12 19.10 3.29 1.68 2.01
19.5 1.62 16.44 4.95 2.56 3.79
28.0 2.77 13.15 5.34 3.23 4.11
34.5 3.67 11.16 5.40 3.03 3.49
41.0 2.90 9.67 5.66 3.09 3.16
47.5 7.88 7.69 5.01 3.31 3.31
56.0 11.38 3.15 6.07 3.69 3.42
60.5 11.35 1.18 7.38 2.65 2.49
65.0 11.76 0.69 12.73 4.85 3.84
67.0 15.86 0.73 18.24 7.84 5.73
69.0 23.42 0.71 27.59 17.99 13.66
Peak areas are expressed in % total peak area

Sensory profiles obtained at defined times of oxidation are shown in Figure 2; relative
values are given as they are more suitable for comparison. The lemon odour note decreased.
lemon juice
heavy-^ ^ I uamnn peel

pungentx V i \ ^ ^'\ X orange juice

fresh^-^..^^ /^3K!L2\ XK^^Si-.^^ orange peel

menthol y—-^ ^^^^^^^^^^ — 1 fruity

terpenic"^ / f \ \ ^floral

woody^ / A ^acidic
anise^ ^spicy

* « — 0 hours >-o-48 hours ~^—66 hours 1

-o—161 hours —e—232 hours

Figure 2. Sensory profile of stored limonene

while woody, acidic and heavy odour notes became stronger during storage. Some ratios of
odour intensities are given in Table 3. Odour notes reminding higher esters (10-14 carbon
atoms), tropical fruits and tropical flowers are denoted as heavy (the term is used in the
fragrance industry).
672

Table 3
Effect of storage time of oxidizing limonene on the ratio of intensities of some odor notes in
the sensory profile
Storage time Ratio
woody:lemon woodyiorange acidic:lemon heavyifresh
0 0.35 0.54 0.35 0.53
48 0.50 0.67 0.69 0.42
66 0.49 0.53 0.77 0.52
161 1.05 2.62 0.70 1.18
232 3.29 3.83 1.71 3.25
Defmitions of odour notes: woody = associated with oak lactones; lemon = associated with
lemon juice; acidic = associated with acetic acid; heavy = associated with odour of higher
esters (10-14 carbon atoms). Tropical fruits or tropical flowers; fresh = associated with
meadow flowers of temperate climate, with low molecular weight esters (3-5 carbon atoms).

5.2. Oxidation of linalool

Linalool was oxidized under the same conditions as limonene, but, because of its lower
stability, results were obtained at 40°C. An example of the GC separation of the reaction
mixture obtained after 700 h at 40°C is shown in Figure 3, and the identification of some
prominent peaks is shown inTable 4. Contrary to limonene, linalool was

Figure 3. GC profile of oxidizing linalool stored at 40**C for 700 hours

 673

Table 4
Identification of compounds in stored oxidizing linalool
Peak Retention Retention Compound
No. time [min] index identified
1 5.68 709 acetaldehyde
2 6.49 824 acetone
3 7.34 887 crotonaldehyde
4 8.21 933 methyl vinyl ketone
5 8.64 953 pentanone (probably 2-pentanone)
6 11.13 1038 pentenone (probably 4-penten-2-one)
7 15.02 1131 methyl pentenone (probably 4-methyl-3-penten-2-one)
8 18.98 1203 2,3-metiiyldihydro-4-methylfiiran
9 23.67 1284 3-hydroxy-3-methyl-2-butanone
10 24.05 1289 hydroxypropanone
11 25.09 1304 hydroxybutanone
12 31.52 1404 a furan derivative
13 32.34 1418 a pyran derivative
14 34.07 1445 m-linalool oxide (furan derivative)
15 34.91 1459 acetic acid
16 35.93 1474 /ra«s-linalool oxide (fiiran derivative)
17 39.98 1537 3,7-dimethyl-l-octen-3-ol
18 40.94 1553 linalool
19 43.29 1588 4,4-dimethyl-2-butenolide
20 45.01 1608 hotrienol
21 48.78 1641 5-ethenyldihydro-5-methyl-2(3H)-furanone
22 51.62 1661 5-ethyldihydro-5-methyl-2(3H)-fiiranone
23 52.67 1672 cu-linaiool oxide (pyran derivative)
24 54.09 1683 /ra/u-linalool oxide (pyrane derivative)
25 64.42 1939 3,7-dimethyl-l ,5-octadien-3,7-diol
26 73.95 2103 8-hydroxylinalool

Peaks correspond to those in Figure 3; «-decane was used as the internal standard (RI = 1000)

decomposed at a uniformly constant reaction rate during the storage. It was decomposed not
only by oxidation but also by other reactions, such as retroaldolization. Among the most
prominent reaction products were four isomeric linalool oxides were identified as cisr- and
/ra«5-5-ethenyl-tetrahydro-a,a,5-trimethyl-c/.s-furanmethanols and cis- and trans-S-
ethyltetrahydro-2,2,6-trimethyl-pyran-3-ols. Several aliphatic products were also identified.
Linalool oxides of the pyran type (contrary to those of thefiirantype) do not increase in their
intensitiesfiromthe beginning of reaction, but rather increase as an exponential fimction of
time (Table 5).
 674

Table 5
Changes of relative intensities of linalool oxides during the oxidative storage
Time[h] cw-Linalool trans-Lmalool cw-Linalool tranS'Lmalool
oxide (fiiran) oxide (fiiran) oxide (pyran) oxide (pyran)
17 0.0014 0.0022 0.00008 0.00008
161 0.0369 0.0402 0.00064 0.00061
234 0.0727 0.0657 0.00115 0.00105
330 0.1266 0.1065 0.00229 0.00191
424 0.1925 0.1565 0.00416 0.00329
519 0.2856 0.2303 0.00711 0.00569
687 0.6293 0.5245 0.03299 0.02717
Peak have structure as given in Table 4; relative intensities are expressed on the basis of
linalool =100

5.3. Effect of dihydropyridine antioxidants on the oxidation of limonene and linalool

Dihydropyridine antioxidants inhibited the oxidation of limonene, but also affected the rate
of formation of various products at different degrees, e.g. they inhibited the formation of
carvone (Figure 4), but enhanced the formation of limonen oxidesfi-omlimonene (Figure 5).
On the contrary, they inhibited the formation of linalool oxide firom linalool (Figure 6). The
sensory profile of limonene was influenced by the presence of 1,4-dihydropyridines, both
Diludine and OSI7284 (Figure 7).

+ 0.2
8
a*
0.15
JS
0.1

4-0.06

50 100 150 250

Exposure time [h]

Figure 4. Effect of antioxidants on the formation of carvone in autoxidizing limonene

 675

120 -Rosemary extract

-Oiludine
-OSI 7284
100 I
-pure

I
I
100 150

Exposure time [h|

Figure 5. Effect of antioxidants on the fonnation of limonene oxides in autoxidizing limonene

0.035
-Rosemary extract
-Oiludine
0.03 -I
-OSI 7284
-pure
0.025
<
0.02

0.015

0.01 •{

0.005

0
100 200 300 400 500 600
Exposure time [h]

Figure 6. Effect of antioxidants on the formation of linalool oxides in autoxidizing linalool

676

lemon juioe

heavy^ I lamonoaal

\ 50/- ^ \ xoiangejulce
pungentx V /

fresh /— /\ ^^^^jof / ifC ^^Sta^--^ orange peel

menthol \——"^ >5^Wi V x ' ^ ^ — ^ '"^

terpente^ / I'Sf \ ^floral

woody^ / \ ^acidic
•nise spicy

-•-Limonene pure (fresh] -o-Diludlne -ili-OSI7284 -S-Rosemary

Figure 7, Sensory profiles of limonene oxidized at 60°C for 222h

6. SUMMARY

During storage of flavoured foods, terpenes are oxidized even in presence of low content
of oxygen. Limonene and linalool, typical components of citrus oils, were used as model
substrates. Nonoxidative reactions of terpenes and secondary reactions of oxidation products
proceed, especially, after exhaustion of oxygen. Intensities of typical citrus flavour notes
decrease and off-odours imparted by oxidation products increase, particularly woody, heavy
and acidic notes. Antioxidants inhibit the flavour deterioration, rosemary extract being more
active than 1,4-dihydropyridines. The compositions of stored limonene and linalool are
affected differently by antioxidants used, which influences the respective sensory profiles. The
effect of the particular antioxidant on flavour changes should ne taken into account when they
are applied to foods and beverages.

?• ACKNOWLEDGEMENTS

The project was supported by COPERNICUS CIPA-CT94/0121; Prof G. Dubursfromthe

Institute of Organic Synthesis, Riga, Latvia, prepared the samples of dihydropyridine
antioxidants.

7. REFERENCES
1 S. Anandaraman and G. A. Reineccius, Food Technol, 40 (1986) 11, 88.
2 P. Schieberle, W. Maier, J. Firl and W. Grosch, J. High Resolution Chromatog., 10
(1987) 588.
 677

3 C. S. Foote, S. Wheeler and W. Ando, Tetrahedron Lett., 46 (1965) 4111.

4 G. O. Schenck, K. Golhiick, G. Buchwald, S. Schroeter and G. Ohloff, Liebigs Ann.
Chem.,674(1964)93.
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(1965) 26.
9 ISO 6658: Sensory analysis - Methodology - General guidance. ISO, Geneva, 1985.
10 ISO 8589: Sensory analysis - General guidance for the design of test rooms. ISO,
Geneva, 1988.
11 ISO 8586: Sensory analysis - General guidance for the selection, training andmonitoring
of assessors. ISO, Geneva, 1989.
12 ISO 6564: Sensory analysis - Flavour profile. ISO, Geneva, 1985.
ISO 4121: Sensory analysis - Grading of food products by methods using scale
categories. ISO, Geneva, 1988.