E. T. Contis et al.

(Editors)
Food Flavors: Formation, Analysis and Packaging Influences
© 1998 Elsevier Science B.V. All rights reserved 679

Effect of rosemary and 1,4-dihydropyridines on oxidative and flavour

changes of bergamot oil

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

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

Technicka5,CZ-166 28 Prague 6, Czechia

Abstract
Bergamot oil was oxidized in the temperature range of 40-60°C at restricted oxygen
levels. The course of oxidation was investigated by gas chromatography (GLC) and gas
chromatogaphy with mass spectrometric detection (GC/MS). Flavour acceptabilities and
sensory profiles were determined at the same time. The sensory acceptability decreases on
storage, and sensory profiles change, too. Limonene oxides and carvone, which were
identified as main oxidation products, have particular sensory profiles. An addition of
rosemary extracts inhibits the oxidation, but it influences the ratios of various components of
stored oil. Therefore, it changes moderately the sensory profile. The sensory profile changes
are more pronounced, when 1,4-dihydropyridine antioxidants are used. Both oxidative and
sensory changes depend on the structure of dihydropyridines, too.

1. INTRODUCTION

Citrus oils are widely used as flavorings for food and cosmetic products. Bergamot oil,
producedfi'ombitter orange {Citrus aurantium subsp. Bergamie Risso et Poiteau Engler) were
applied for various foods and beverages, such as fiuit juices [1], soft drinks [2], liqueurs [3],
firuit jams [4],flavouredtea [5] and even flying oil [6].
The disadvantage of bergamot oil is its high content of monoterpenes [7], particularly
limonene, linalyl acetate and linalool, which decrease resistance against oxidation under
storage. Black tea, flavoured with bergamot oil, was stable only for two months at room
temperature [8]. At 50 or 60°C, the peroxide value of 300 mval/kg was soon reached which
caused deterioration of flavour quality [9]. Stored samples of bergamot oil have high peroxide
values [10], unless they have been stabilized with antioxidants; they are oxylabile even in cold
orfi^ozenstorage [11]. The peroxide value was found a good indicator of sensory quality of
orange oil, but tiie gas-chromatographic profile was preferable [12]. More than 160
components were identified in aged bergamot oil, using GC/MS [13]. Oxidation mechanisms
of terpenic hydrocarbons and their oxygenated compounds were discussed [14].
Oxidation products modify the flavour of essential oils. Therefore, we studied the
relationship between the chemical composition and the sensory profile of stored bergamot oil,
and the effect of antioxidants on the product.
680

2. MATERIALS AND CHEMICALS

Bergamot oil (free of antioxidants, without deterpenation) was supplied by Sigma (UK),
(S)(-)-limonene (96 % purity) and (±)-linalool (97 % purity) by Aldrich (Gemany). Rosemary
extract was produced by extracting 100 g of fresh dried rosemary leaves (grown in Poland, in
1995) four times with 1 L acetone, macerating overnight at room temperature of 22°C,
combining the filtrates, removing the solvent by distillation, and drying on a boiling water
bath. The main active components were identified as camosic acid and camosol. Diludine
(2,6-dimethyl-3,5-diethoxycarbonyl-l,4-dihydropyridine) of 99 % purity and OSI 7284 (2,6-
dimethyl-3,5-dibutoxycarbonyH,4-dihydropyridine) of 98 % purity were prepared in the
Institute of Organic Syntheis (Director: Prof Dr. G. Duburs) in Riga, Latvia.

3. EXPEMMENTAL PROCEDURES

3.1. Oxidation of bergamot oil

A 100 fiL portion of bergamot oil was placed into a 10 mL vial containing an internal
standard (5 mg of «-decane). The vial was sealed and conditioned at 40°C (in some
experiments, at 60'^C) in a thermostat. A volume of 0.1 mL of vapour phase was injected by
gas-tight Hamilton syringe mto the gas- chromatograph (using a headspace autosampler).
Alternatively, the solid phase microextraction (SPME) sampling technique was used. To study
the antioxidant efficiency, 1 mg of the antioxidant dissolved in 100 mL methanol, was added,
and the solvent evaporated. Essential oil was added afterwards, the vial was sealed, and
thoroughly shaken.

3.2. Sotid phase microextraction (SPME)

A 65 pm Carbowax-divinylbenzene fiber for a manual holder (Supelco, USA) was used for
extracting volatiles for GC/MS. The fiber was inserted into the vial containing the stored
sample (see Section 3.1). Extraction time was 10 min at 40°C and desorption time 2 min at
220°C. The fiber was cleaned for 30 min At the same temperature. Details of the apparatus are
given in another chapter in these proceedings[16].

3.3. Gas chromatography (GLC) and GLC with mass spectrometric detection (GC/MS)
The gas chromatograph GC8000 (Fisons Instruments) was equipped with an autosampler
HS800 (injector temperature 220°C) and a 60 m x 0.32 mm column, coated with Supelcowax
10 (fibn thickness 0.25 mm) was used. The column temperature was programmed from 50*^0
(2 min isothermally), heating rate 2°C/min to 220°C (isothermal for 30 min) Tnc fl^me
ionization detector (FID) was used (temperature 250*^0). The c^^T?er gas was helium at an
initial pressure 100 kPa. The input/split ratio 1:25. Retention indices were calculated using a
mixture of «-alkanes as reference substances, as well as for calculation of peak areas. A
MSD8000 mass spectrometer was used for GC/MS; the ionizing energy was 70 eV. Pure
standards (Aldrich, purified by GLC) were used for identification of mass spectra.

3.4. Sensory analysis

Conditions for the sensory analysis were in agreement with the international standard ISO
[17]; the test room was equipped with six standardized test booths [18]. The panel of assessors
consisted of selected, trained and monitored persons [19] with experience in sensory profiling
of at least six months. The amount of 100 mg of sample was placed into a 250-mL wide-neck
 681

ground-glass bottle, and left at least 2-3 hours to equilibriate. The odour intensity was
evaluated by sniffing. For the analysis of stabilized samples, 100 jiL of 1% methanolic
solution were added, the solvent evaporated, the sample was added, the bottle thoroughly
shaken, and left for 2-3 h. For the hedonic rating, the odour acceptability was determined
using an unstructured graphical scale - straight lines 100 mm long [19] (0% = rather bad;
100% = excellent). The sensory profile [20] consisted of 24-36 descriptors (they are shown in
the respective figures in spider-web diagrams); unstructured graphical scales 100 mm long
were used (0% = imperceptible; 100% = very strong). Two samples were served at a session
in random order. The total of 24 responses were used for the calculation of means. The
standard deviation of the means varied between 2-6% of the scale.

4. RESULTS AND DISCUSSION

4.1. Composition of bergamot oil

Bergamot oil was analyzed by GC/MS, and a chromatographic profile is shown in Figure
1. The sample was moderately oxidized (20 h at 40°C) in order to better determine the peak
position of some oxidation products. The chemical structure of identified compounds was
determined by comparison of mass spectra and retention indices with those of authentic
substances; they are listed in Table 1. Linalool, linalyl acetate and limonene were present as
major terpenic components, in agreement with the literature [7, 22, 24], and myrcene, cis- and
/ran^-ocimene, and p-cymene were found in smaller amounts. Caryophyllene was the only
sesquiterpene detected.

3.5]
21

3.0J 141 1<

4
If

S 2.5
r ||

ii
f 1
• 1 11 1 14 1

1.5-1 11
t 1 1
17 1
1 h
k
i.oj 1 3 1
0 1 ^
0,5-]
iMuJLuLjLL^U JLJ
30 40

Time [min]
Figure 1. Chromatographic profile of bergamot oil after storage at 40°C for 20 h. Numbers
above each peak refer to the compound shown in Table 1.
682

Table 1
List of identified substances in the sample of very slightly oxidized bergamot oil (20 h at
40°C, corresponding to Figure 1)
Peak Retention Retention Identified
No. time [min] index substance
1 6.53 840 acetone
2 7.54 910 methanol
3 8.23 946 ethanol
4 16.79 1174 p-myrcene
5 18.65 1209 limonene
6 20.83 1246 c»5-ocimene
7 21.85 1263 trans-ocimeiae
8 23.01 1281 /j-cymene
9 34.04 1453 c/f-linalool oxide
10 34.95 1467 acetic acid
11 35.91 1481 /rans-linalool oxide
12 39.38 1536 3,7-dimethyl-1 -octen-3-ol
13 39.62 1540 y-terpineol
14 40.97 1561 linalool
15 41.68 1571 iinalyl acetate
16 43.15 1594 sesquiterpene (probably »o-caryophyllene)
17 49.36 1648 citral
18 50.13 1654 a-terpineol
19 51.98 1668 neryl acetate
20 52.67 1675 carvone
21 53.78 1681 geranyl acetate
22 66.47 1975 caryophyllene oxide

4.2. Oxidation of bergamot oil

Bergamot oil was oxidized under simulated storage conditions of flavored food products, i.
e. under limited access of oxygen at 40°C and in thin film of about 1 mm. Examples of
chromatographic profiles are shown in Figure 2 where a sample taken near the beginning of
oxidation is compared with a sample stored for about a month, when oxygen has been
consumed and various secondary (non-oxidative) reactions proceeded. Various oxidation
reaction proceeded in the beginning of storage, but when oxygen in the headspace has been
exhausted, various secondary reactions followed. Changes at 60°C were very sunilar,
therefore, they are not presented here.
Limonene was gradually destroyed firom the start of storage and the course followed the
zero order kinetics until complete exhaustion of oxygen. Data for several other compounds are
given in Table 2. Myrcene was oxidized from the beginning, too. On contrary, the degradation
of/?-cymene followed after a distinct lag period. The contents of a- and y-terpineols and 3,7-
dimethyH-octen-3-ol mcreased during llie storage. The two ocimenes reached their maximum
in the beginning of storage. Acetone, a typical oxidation product formed by decomposition of
a hydroperoxide, was produced very rapidly, reaching a maximum after 40 hours. Both cis-
and /ran^-linalool oxides were formed slowly, and attained their maxima when oxygen was
already consumed.
 683

Table 2
Changes of some compounds during the storage of bergamot oil at 40'^C
Peak No. 39 h 185 h 354 h 428 h 612 h 739 h
1 14.60 6.88 5.22 4.38 6.48 4.16
4 148.63 107.45 46.89 31.33 20.52 16.75
6 6.65 8.47 7.69 9.21 6.53 5.34
7 27.59 27.08 18.15 16.62 11.46 9.50
8 216.48 288.96 179.57 40.23 - -
9 4.26 5.46 6.05 6.18 - 6.36
11 3.01 4.60 5.65 5.87 5.73 6.07
12 0.55 0.91 1.31 1.74 1.61 1.82
13 0.50 0.69 1.12 1.27 1.35 1.69
18 2.40 3.50 4.42 6.25 6.03 7.54
Note: Peak numbers are the same as in Table 1. Numbers refer to FID peak area.

4.3. Effect of rosemary extract on the degradation of bergamot oU

Rosemary extract, an efficient natural antioxidant used for the stabilization of fats and oils
[25, 26], was found active in stabilizing limonene and linalool under storage conditions at
40°C. No changes or very small changes were observed in most components for the first 400
hours of storage. The activity was similar to that observed in Citrus hystrix oil [16].
Changes of the composition of bergamot oil stabilized with rosemary extract during the
storage at oxygen are partially shown in Table 3. The contents of acetone, 3,7-dimethyl-l-
octen-3-ol, /7-cymene, P-myrcene, a- and y-terpineols changed similarly during the storage as
in non-stabilized oil. The degradation oicis- and /ra^w-ocimenes was moderately inhibited by
rosemary extract. Both cis- and /ra/w-linalool oxides were formed in inhibited samples at the
same rate at the beginning as in the control containing no antioxidants, however, they were
decomposed at a faster rate at later stages of storage (after 400 h) if rosemary extract was
present.

Table 3
Changes of some components of bergamot oils stored with rosemary extract at 40°C
Peak No. 41 h 187ji 356 h 543 h 713 h
1 20.91 5.10 2.39 1.60 1.34
4 58.78 120.74 57.77 31.89 24.26
6 7.80 8.24 8.04 8.82 7.98
7 30.96 29.36 22.04 17.02 13.83
8 96.29 175.51 90.01 21.67 2.16
9 4.71 5.06 4.95 3.87 2.28
11 3.77 4.07 4.01 3.65 2.33
12 0.79 0.92 1.28 1.56 1.95
13 0.64 0.69 1.11 1.23 1.81
18 3.07 3.54 4.91 5.55 7.59
The structure and numbers of peaks are the same as in Table 1.

Since bergamot oil is a complicated mixture of terpenes, the sensory changes that occur
with storage are difficult to evaluate and compare. Therefore, the effect of rosemary extract
 684

was studied using purified limonene and linaiooi. An example of limonene is shown in Figure
3 (changes occurring in stored linaiooi were very sunilar). Degradation of citrus notes was
very pronounced in the non-stabilized sample, but it was slightly smaller in the stabilized
sample. In presence of rosemary extract, the formation of woody, acidic and heavy odour
notes were suppressed in stored bergamot oil; on contrary, the formation of spice and fresh
odour notes was stimulated. The hedonic rating decreased by 28% of the scale in case of
unstabilized sample and by 14% only in case of stabilized sample, respectively.

70-

60-

SO

a
e <0

0S
30

20-
., 1 1 ^

10-

0-
-1 t • 1 LJLL- IJJi JI II •. .- t
^

Time [min]

Figure 2. Chromatographic profiles of oxidized bergamot oil, A - 40 hours at 40*^0,

B - 740 hours at 40°C.

lemon juice
heavy ^ I _ , _ _ _ ^ lemon peel

pungent ^ V *7 y 1 > orange juice

^^^^"^^^^ /\^^^} r 7 i \ y ^ \ ^ ^ , . ^ - ^ orange peel

menthol ^—^-^ ' 'Z^^^^^i^^^^^^^^^^'*^~^~~~' ^ ' ^

terpenic^ / i ^ \ ^floral
-o-O hours
- o — rosemary
woody f \ acidic
~-^r- dliudlne
anise spicy - • ^ OSI7284

Figure 3. Sensory profiles of limonene stored with and without rosemary extract
 685

4.4. Effect of 1,4-dihydropyridine antioxidants on the degradation of bergamot oil

Dihydropyridine derivatives are used for the stabilization of feeds and phannaceutical
preparations such as carotene supplements [27, 28]. In terpenes they were found less efficient
as inhibitors of oxidation, similarly as in pure terpenes [29], but they influenced the formation
of various oxidation and other degradation products. Changes of several important
components are summarized in Table 4 for oil stabilized with Diludine and in Table 5 for oil
stabilized with OSI 7284, respectively.

Table 4
Changes of some components during the storage of bergamot oil at 40°C in presence of
Diludine
Peak No. 43 h 189 h 358 h 547 h 717 h
1 27.25 21.76 15.71 15.55 15.40
4 142.14 125.20 53.72 29.34 14.17
6 6.77 7.70 6.52 8.60 4.32
7 25.43 23.25 13.41 14.90 7.56
S 266.62 525.62 361.06 21.30 -
9 4.00 6.03 10.36 12.67 9.71
11 2.86 5.45 9.72 11.33 9.25
12 0.60 0.85 1.11 1.57 1.00
13 0.57 0.87 1.11 1.67 0.66
18 2.69 0.35 4.12 5.89 5.24
Compounds and numbers of peaks are the same as in Table 1.

Table 5
Changes of some components during the storage of bergamot oil stabilized with OSI 7284
Peak No. 45 h 207 h 375 h 548 h 721 h
1 40.28 18.01 11.16 6.07 4.05
4 217.14 154.47 70.95 29.77 18.55
6 12.87 10.76 9.43 8.32 6.04
7 45.91 35.87 24.18 15.56 10.47
8 282.24 334.63 207.30 53.46 1.89
9 6.05 6.64 6.95 5.80 4.22
11 4.73 5.46 6.14 5.51 4.17
12 0.98 1.11 1.25 1.70 1.47
13 0.93 1.03 1.25 1.00 1.10
18 3.82 4.44 5.20 6.06 6.12
Compounds and numbers of peaks are the same as in Table 1.

The destruction of myrcene was efficiently inhibited in presence of OSI 7284, but no effect
was observed with Diludine. The maximum contents of cis- and trans—linalool oxides were
higher than in absence of antioxidants or in presence of rosemary extract. The maximum was
particularly high, when Diludine has been added. The content of p-cymene changed similarly.
The maxima of cis- and /ran^-ocimenes were much higher in oil stabilized with OSI 7284
than m oil stabilized with Diludine. During 300-400 h of storage the concentrations of a- and
686

y-terpineols were almost the same in all samples studied. During further storage their content
decreased in presence of dihydropyridines, especially Diludine, while it continued to increase
in non-stabilized oil or in oil stabilized with rosemary extract. Similar differences were
observed for 3,7-dimethyl-l-octen-3-ol. The formation of acetone was also stimulated by
dihydropyridine derivatives. These differences could be explained by presence of a nitrogen
group in dihydropyridines. Their weakly basic character may influence not only the course of
hydroperoxide decomposition, but the course of non-oxidative degradative changes such as
retroaldolization.
From thesedata, it is evident that changes in sensory profiles of stored bergamot oil are not
due only to the inhibition of primary oxidation reactions, but also depend on the structure of
antioxidant added.

5. ACKNOWLEDGEMENTS

Supported by the research grants Copernicus CIPA-CT94/0111 and OK 175 ( M S M T CR).

The rosemary extract was prepared and characterized by Dr. J, Korczak, Agricultural
Univeristy, PoznapPoland. The dihydropyridine antioxidants were preprared and characterized
by prof. Dr. G. Duburs and co-workers. Institute of Organic Synthesis, Riga, Latvia.

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 687

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