Effect of cis/trans isomerism of β-carotene on the ratios of volatile compounds produced during oxidative degradation

Article abstract of DOI:10.1021/jf021000g

β-Carotene is, when cleaved, an important source of flavor and aroma compounds in fruits and flowers. Among these aroma compounds, the main degradation products are β-ionone, 5,6-epoxy-β-ionone, and dihydroactinidiolide (DHA), which are associated by flavorists and perfumers with fruity, floral, and woody notes. These three species can be produced by degradation of β-carotene through an attack by enzyme-generated free radicals and a cleavage at the C9-C10 bond. This study investigated the influence of cis/trans isomerism at the C9-C10 bond on the production of β-carotene degradation compounds, first with a predictive approach and then experimentally with different isomer mixtures. β-Carotene solutions containing high ratios of 9-cis-isomers produced more DHA, suggesting a different pathway than for the transformation of all-trans-β-carotene to ionone and DHA. These results are important in the search for financially viable processes to produce natural carotene-derived aroma compounds.

Full text of DOI:10.1021/jf021000g

1984 J. Agric. Food Chem. 2003, 51, 1984−1987
Effect of cis/trans Isomerism of â-Carotene on the Ratios of
Volatile Compounds Produced during Oxidative Degradation
YVES WACHEÄ ,*,† AUREÄ LIE BOSSER-DERATULD,†,§
JEAN-CLAUDE LHUGUENOT,# AND JEAN-MARC BELIN
†
Laboratoire de Microbiologie UMR UB/INRA 1082 and Laboratoire de Toxicologie alimentaire UMR
UB/INRA 0938, ENSBANA, 1 esplanade Erasme, 21000 Dijon, France
â-Carotene is, when cleaved, an important source of flavor and aroma compounds in fruits and flowers.
Among these aroma compounds, the main degradation products are â-ionone, 5,6-epoxy-â-ionone,
and dihydroactinidiolide (DHA), which are associated by flavorists and perfumers with fruity, floral,
and woody notes. These three species can be produced by degradation of â-carotene through an
attack by enzyme-generated free radicals and a cleavage at the C9-C10 bond. This study investigated
the influence of cis/trans isomerism at the C9-C10 bond on the production of â-carotene degradation
compounds, first with a predictive approach and then experimentally with different isomer mixtures.
â-Carotene solutions containing high ratios of 9-cis-isomers produced more DHA, suggesting a different
pathway than for the transformation of all-trans-â-carotene to ionone and DHA. These results are
important in the search for financially viable processes to produce natural carotene-derived aroma
compounds.
KEYWORDS: â-Carotene isomers; xanthine oxidase; â-ionone; dihydroactinidiolide
INTRODUCTION
Carotenoids are important constituents of foods. Due to their
color and the fact that their degradation leads to the production
of aroma compounds (1), they play a major role in the sensorial
properties of food products. Their structure and related physi-
cochemical properties also result in a role of importance in
biological systems. The biological functions of â-carotene 1 have
been attributed to its ability to scavenge free radicals (2-4),
physically quench singlet oxygen (5), and generate vitamin A
(retinol) (6).
â-Carotene is present in nature in many isomeric forms. The
all-trans 1 is the main form encountered, but some organisms
contain large amounts of cis-isomers as for instance the
halotolerant alga Dunaliella bardawil, which can have twice
as much 9-cis 15 as all-trans (7). To date, most of the works
investigating the characteristics of â-carotene have dealt with
all-trans-â-carotene, but the 9-cis-isomer, mainly extracted from
this algae, has also been studied. The behavior of this latter
form in metabolism is rather different from that of the all-trans
one. It is, for instance, not absorbed in the same way (6), but it
seems to stimulate â-carotene absorption (8). Due probably to
the higher reactivity of the cis-bond, it has a higher in vitro
antioxidant activity (9) and in vivo antiperoxidative effect (10)
than the all-trans-isomer. However, this latter form is a better
Figure 1. Pathways for the synthesis of â-ionone 4, 5,6-epoxy-â-ionone
5, and DHA 6 from â-carotene 1 and through 5,6-epoxy- 2 and 5,8-
epoxy-â-carotene 3: (A) pathway proposed in the literature (16−18); (B)
pathway observed by Bosser et al. (17).
inhibitor of low-density lipoprotein oxidation (11). The two
forms seem also to have different roles in vitamin A synthesis
(12), although 9-cis-â-carotene can also generate 9-cis-retinal
16, -retinol, or -retinoic acids (9, 13-15).
However, nothing is known about the effect of cis/trans
isomerism on the generation of aroma compounds from carot-
enoids. â-Carotene, when cleaved, gives rise to various interest-
ing compounds (16). Among them, â-ionone 4, 5,6-epoxy-â-
ionone 5, and dihydroactinidiolide (DHA) 6 are often associated
with fruity, floral, and woody notes to varying degrees. Two
metabolic pathways originating the epoxy ionone and DHA have
been proposed (Figure 1). In the first one suggested by the
literature (16-18) (Figure 1A), â-carotene is first oxidized to
5,6-epoxy-â-carotene 2 and 5,8-epoxy-â-carotene 3, and these
compounds are cleaved to 5,6-epoxy-â-ionone and DHA,
respectively. In the second one observed by Bosser et al. (17)
* Author to whom correspondence should be addressed (e-mail ywache@
u-bourgogne.fr; fax ++33 3 80 39 66 41).
† Laboratoire de Microbiologie UMR UB/INRA 1082.
§ Present address: Diana Ingre´dients, 56000 Vannes, France.
# Laboratoire de Toxicologie alimentaire UMR UB/INRA 0938.
10.1021/jf021000g CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/22/2003

Degradation of â-Carotene Isomers to Volatile Compounds
J. Agric. Food Chem., Vol. 51, No. 7, 2003 1985
described previously (29). Qualitative identification of all volatiles was
realized by GC-MS with the ion-trap system Finnigan Mat ITS 40 by
comparing mass spectra from samples with those of pure products.
Molecular Modeling of the Oxidation Cleavage of â-Carotene.
Molecular modeling, based on the method of free energy perturbation,
was carried out using the software Molecular Advanced Design (MAD,
Oxford Molecular) in an IBM Risc 6000 H-355 workstation equipped
with a 24-plan graphic card. MAD is a molecular dynamic modeling
software that uses the fields of Allinger force as the initial data. We
calculated minimal free energies of the direct oxidation products of
all-trans-â-carotene and 9-cis-â-carotene, taking as a reference the
reaction of scission of one molecule of carotene to two retinals as
follows
(Figure 1B), â-carotene is cleaved to â-ionone, which is
oxidized to its 5,6-epoxy form and then lactonized to DHA.
This cleavage can be caused by enzymatic activities or physi-
cochemical conditions. This degradation has been and still is
the subject of many studies (19, 20) using enzymes (21-23)
and physical (24) or chemical media (25).
Technology and particularly biotechnologies have tried to take
advantage of this reaction to produce â-ionone and related
volatiles using physicochemical processes (26, 27) or enzyme-
generated free radicals (28-30). This latter reaction is called
co-oxidation because the enzyme (often lipoxygenase and
xanthine oxidase in our case) catalyzes a reaction generating
oxygen-derived radical species which, in turn, degrade carot-
enoids.
Unfortunately, yields are too low for industrial application
partly because the cleavage site is not mastered and also because
the production is divided into three interesting compounds. The
influence of the structure of the carotenoid on the cleavage site
and possible rearrangement has not been completely studied.
Some workers (31, 32) have investigated the relationship
between structure and reactivity in the free radical mediated
oxidation of carotenoids. Electron density along the polyene
chain as well as the presence of functional groups appears to
modify reactivity. Redox potentials of various carotenoids and
their related cations have also been studied (33), but this work
has not included the effect of cis-bonds in â-carotene on the
degradation products.
with ∆G1 + ∆G2 ) ∆G3 + ∆G4 and ∆G3 ) 0. The reaction resulting
in X + Y is then compared to the reference by calculating ∆∆G )
∆G4 - ∆G1 ) ∆G2. The more negative ∆∆G is, the more the reaction
resulting in X + Y is favored.
RESULTS AND DISCUSSION
Prediction of the Oxidation Cleavage Site of all-trans- and
9-cis-â-Carotene. To predict the cleavage site of all-trans-â-
carotene and that of its 9-cis-isomer, we calculated minimal free
energies of the oxidation products and compared them to the
formation of two molecules of retinal corresponding to a central
cleavage. Results are given in Figure 2. ∆∆G ) ∆G2 (free
energy variation of the reactions producing combination of
compounds other than retinal) - ∆G1 (free energy variation of
the reaction producing 2 retinals) shows the free energy
difference between the two reactions; the more negative ∆∆G
is, the more favored the noncentral scission reaction is compared
to central scission. The results showed that, for both isomers,
the lateral cleavage was favored. For the all-trans-isomer 1,
the favored cleavage was giving â-cyclocitral 14 and â-apo-
8′-carotenal 13, confirming the calculations using the method
of molecular orbitals to characterize electronically all-trans-â-
carotene (36). For the 9-cis-isomer 15, the favored cleavage bond
was the cis-bond (9-10) giving rise to â-ionone 4 and â-apo-
10-carotenal 19. This isomer was thus of potential interest for
aroma compound production.
These calculations have been carried out for molecular oxygen
oxidation without taking into account interference with the
medium such as the solvent and the primary reactants. In the
co-oxidation system we use, the degradation occurs through the
action of radicals and â-carotenyl is formed before scission (19,
20, 29). Several reactions can thus be involved. The impact of
high ratios of 9-cis-â-carotene on the degradation products was
then investigated.
Degradation of â-Carotene Isomer Mixtures. From the
different free energies of carotene isomers, we could expect
different cleavage sites for cis/trans-isomers and, thus, differ-
ences in the ratios of degradation products. We compared the
degradation of a mixture of isomers from photo-iodo-isomerized
all-trans-â-carotene containing 54% of all-trans-isomer and
25% of 9-cis-isomer to all-trans-â-carotene (96% all-trans and
0.8% 9-cis). In both cases, a high degradation happened in the
first 3 h (not shown) without any significant difference in the
rates of degradation of the various isomers (Table 1). In Figure
3 is presented the accumulation of â-ionone 4, epoxy-â-ionone
We investigated in this work the effect on the cleavage site
of â-carotene of cis/trans isomerism. The cis/trans isomerism
was investigated first with a predictive approach, then, using
isomer mixtures in the system used previously with all-trans-
â-carotene (29).
EXPERIMENTAL PROCEDURES
Degradation of â-Carotene in the Xanthine Oxidase/Acetalde-
hyde System. Reactions were carried out as previously described (29)
at 37 °C in a 2-L enzymatic reactor with 250 rpm stirring. The reaction
volume was 300 mL. The aqueous phase was composed of 27 × 10-3
units/mL xanthine oxidase (grade III from buttermilk), 48 mM
acetaldehyde, and 50 mM phosphate buffer, pH 8.0.
â-Carotene (Fluka, Sigma-Aldrich, St Quentin Fallavier, France) was
prepared according to the method of Ben Aziz et al. (34) as previously
described (29). It was dissolved in chloroform with Tween 80 and
evaporated to dryness under vacuum. The residue was then solubilized
in a 0.25% EDTA solution and filtered through filter paper. Thereafter,
sodium acetate buffer, pH 4.6, was added. The procedure was modified
for iodine-catalyzed photoisomerization by adding ethanol-solubilized
I2 (20 mg per 500 mg of â-carotene) and exposing it for 7 min to a
fluorescent lamp (20 W) according to the method of Tsukida (35). To
avoid interaction with the enzyme, I2 excess was reduced to I- prior to
the experiment by adding Na+HSO3-. Initial â-carotene concentration
was 90 mg/L in the reactor volume.
Analyses. â-Carotene isomers were quantified throughout the
reaction with a Merck HPLC system with a Merck L-6200 pump, a
10-µL loop injector, a Merck L-3000 photodiode detector in the
absorbance mode (λ ) 450 nm), and a Merck Lichrocart 250-4 column,
Purospher RP-18. The solvents used were methanol (95%) and
tetrahydrofuran (5%) with a 1.2 mL/min flow. The reference â-carotene
isomers were obtained from Roche (Basel, Switzerland), and each form
was checked according to its spectrometric spectrum.
For volatile analysis, samples (18 mL) were extracted with CH2Cl2.
After the addition of methyl isoeugenol as internal standard, they were
concentrated under N2 to a final volume of 3 mL. Analyses were carried
out in a Varian 3400 gas chromatograph equipped with a CP Wax-
58CB column (i.d., 0.25 mm; film thickness, 2 µm; length, 50 m) with
a 4 °C/min temperature increase from 90 to 220 °C. Both injector and
FID temperature were set to 250 °C. Details for analyses have been

1986 J. Agric. Food Chem., Vol. 51, No. 7, 2003
Wache´ et al.
Figure 3. Yields of bioconversion of â-carotene to â-ionone ([), 5,6-
epoxy-â-ionone (0), and dihydroactinidiolide (2) during the co-oxidation
of 90 mg/L of commercial (96% all-trans and 0.8% 9-cis) (A) or iodine-
catalyzed-photoisomerized (55% of all-trans and 24% of 9-cis) (B)
â-carotene. Results are the mean of three independent experiments, and
standard deviations are <10%.
Figure 2. Oxidation reactions of all-trans-â-carotene (A) and 9-cis-â-
carotene (B). Compounds [minimal energy] are 1, all-trans-â-carotene
[71.68]; 7, retinal [39.06]; 8, â-apo-14′-carotenal [37.60]; 9, â-apo-13-
carotenone [32.08]; 10, â-apo-12′-carotenal [42.93]; 11, â-ionylidene
acetaldehyde [30.71]; 12, â-apo-10′-carotenal [43.85]; 4, â-ionone [25.00];
13, â-apo-8′-carotenal [49.87]; 14, â-cyclocitral [16.89]; 15, 9-cis-â-
carotene [76.82]; 16, 9-cis-retinal [42.89]; 17, 9-cis-â-ionylidene acetal-
dehyde [33.82]; 18, â-apo-12-carotenal [42.93]; 19, â-apo-10-carotenal
[43.85]; and 20, 9-cis-â-apo-8-carotenal [58.82]. ∆G1 is the free energy
variation of the reaction producing two retinals (central scission), and ∆G2
is the free energy variation of the reactions producing other combinations
of compounds. ∆∆G ) ∆G2 − ∆G1 gives the free energy difference
between the two reactions; the more negative ∆∆G is, the more favored
the reaction is compared to central scission.
â-ionone was not higher, and only the concentration of DHA
was significantly increased. The rapid degradation of â-ionone
in the reaction medium is possible as the radical species might
also attack the products of degradation. Such a phenomenon
has been already observed by Mordi et al. (20): they expected
that the autoxidation of â-carotene would result in the major
formation of â-cyclocitral 14 and â-apo-8′-carotenal 13, but the
â-cyclocitral appeared only after a few hours and they did not
detect any â-apo-8′-carotenal at all. They explained these results
by the possible rapid isomerization and oxidation of the
degradation products in this highly reactive environment (20).
In our case, the “direct” expected oxidation product of all-
trans-â-carotene 1, â-ionone 4, does not accumulate more with
higher amounts of the 9-cis-isomer, whereas DHA accumulates
more from the beginning of the experiment. In the pathway
proposed by Bosser et al. (17), this product is more expected at
the end of the reaction, after rearrangement of â-ionone. Our
results suggest that 9-cis-â-carotene directly forms DHA without
first forming â-ionone. The chemical pathway may thus involve
the formation of 5,8-epoxy-â-carotene from 9-cis-â-carotene and
the further cleavage into DHA (Figure 1).
In conclusion, higher ratios of 9-cis-â-carotene increase the
formation of aroma compounds. However, we are not sure
whether the 9-10 cleavage site is favored, as proposed by our
prediction, or if the 9-cis-isomer is first transformed to its
epoxide and then cleaved at the 8-9 level. Further work is
necessary to determine the details of the mechanism.
This study shows once again that this cooxidation system
developed by Bosser and Belin (29) is able to generate different
carotenoid-derived degradation products from different sub-
strates. After â-ionone was obtained from all-trans-â-carotene
in the original study (29), the grasshopper ketone, a dama-
scenone precursor, identified later from neoxanthin solutions
(37), this study shows the rapid formation of dihydroactinidiolide
from 9-cis-â-carotene-rich mixtures, a compound that appears
Table 1. Relative Percentage of the Various Isomers of â-Carotene
from Isomerized â-Carotene during the Co-oxidation Experiment (±1%)
time (h)
all-trans
9-cis
13-cis
15-cis
0
1.5
4
55
54
53
53
24
23
24
23
19
18
19
17
3
4
4
5
6
5, and dihydroactinidiolide 6 during the degradation of â-
carotene in the xanthine oxidase/acetaldehyde system from the
different substrates: commercial â-carotene (96% all-trans) and
photoisomerized â-carotene (24% 9-cis). The presence of
â-cyclocitral 14 at trace level is not shown. With the all-trans
system, â-ionone accumulated in the first 4 h to almost 3% and
the concentrations decreased afterward to <2%. The profile of
accumulation of its epoxide was similar but reached lower
concentrations (maximum 2%). The lactone accumulated until
6 h, reaching a 3% concentration, and the concentration was
stable afterward. With the isomer mixture, â-ionone accumulated
in the first 2 h to only 2%, and its concentration then decreased.
Its epoxide’s concentration reached also a maximum after 2 h
but at a higher concentration than with the all-trans preparation
(2.8%). DHA accumulated very rapidly, reaching 6% after only
2 h and 8.5% after 24 h. By increasing the ratio of 9-cis-â-
carotene, it was thus possible to increase the total amount of
aroma compounds produced. However, the concentration of

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