Carotenoid conversion kinetics depend on the carotenoid

type and on the food system

Ines Colle, Lien Lemmens, Griet Knockaert, Sandy Van Buggenhout, Ann Van Loey, Marc Hendrickx

Laboratory of Food Technology and Leuven Food Science and Nutrition Research Centre (LFoRCe), Department of Microbial
and Molecular Systems (M2S), KU Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium

ABSTRACT

The kinetics of thermally induced degradation and isomerization of -carotene and lycopene in different
food systems were investigated in detail. To study the degradation reaction, a single response modelling
approach was used, while multi-response modelling was applied to describe isomerization reactions. A
significant different set of kinetic parameters was obtained for -carotene degradation in olive oil/carrot
emulsion than for lycopene degradation in olive oil/tomato emulsion. In addition, lycopene isomerization
reactions in the olive oil/tomato emulsion as a whole occurred more slowly than in olive oil. Furthermore,
different sets of kinetic parameters describing -carotene and lycopene isomerization in olive oil were
obtained. The results obtained clearly show that carotenoid conversion kinetics depend on the carotenoid
type as well as on the food system.

1 Introduction

Recent interest in carotenoids arises from their purported health benefits. Some carotenoids (e.g. -
carotene) can provide vitamin A activity within the body. Moreover, carotenoids are potent antioxidants and can
play a role in preventing/delaying chronic diseases. The increasing awareness of potential health benefits of
certain carotenoids has triggered the food industry to create functional food products with carotenoid containing
ingredients. The same carotenoid properties that may lead to their functional properties, create challenges with
regard to carotenoid stability during processing. Carotenoids are expected to undergo two types of changes:
degradation and isomerization. Obviously, carotenoid degradation should be prevented to maintain biological
activity while the latter reaction might be of interest to either prevent or promote depending on the carotenoid
type. Quantitative kinetic models describing carotenoid changes as a function of process parameters are valuable
tools for process optimization. The objective of this research was to investigate -carotene and lycopene
degradation and isomerization during thermal processing using a kinetic approach. Different food systems
(enriched olive oil, olive oil/carrot and olive oil/tomato emulsions) containing either lycopene or -carotene are
considered.

2 Material and Methods

2.1 Sample preparation

Fresh carrots of the cultivar Nerac were peeled, cut into pieces, and blended with water (1:1). Mature
red-ripe tomatoes of the cultivar Heinz 9997 were processed into a tomato puree free from skin and seeds.
Stable olive oil/vegetable emulsions were prepared by adding 5% of olive oil to carrot or tomato puree and
applying high pressure homogenization at 100 bar (Panda 2K, Gea Niro Soavi, Mechelen, Belgium). To obtain
an oil phase enriched with carrot -carotene or enriched with tomato lycopene, the carotenoids were transferred
from the vegetable matrix to the oil phase. Hereto 16% (w/w) of olive oil was added to the vegetable puree and
incubated during 5 h while rotating end-over-end. The enriched oil phase was collected following
centrifugation.

2.2 Thermal treatments

Stainless steal reactor tubes were filled with enriched olive oil or vegetable emulsions. The closed reactor
tubes were equilibrated at 40 C and immediately after equilibration immersed into an oil bath at a preset
temperature during preset time intervals. Treatment temperatures ranged from 70 C to 140 C and treatment
time ranged from 0 min to 120 min. During the thermal treatment the temperature profile in one tube was

InsideFood Symposium, 9-12 April 2013, Leuven, Belgium 1|P a g e

registered every 2 seconds. The actual time/temperature profiles were included in the kinetic parameter
estimation procedures.

2.3 Carotenoid isomer analysis

-carotene and lycopene were extracted from the samples according to the method of Sadler et al. (1990)
with some modifications (Colle et al., 2013; Knockaert et al., 2012; Lemmens et al., 2010). The carotenoid
extract was analyzed using RP-HPLC-DAD (Agilent Technologies 1200 Series, Diegem, Belgium). Carotenoid
isomers were separated on a C30 column and eluted using gradient elution with methanol, methyl-t-butyl ether
and water (Colle et al., 2013; Lemmens et al., 2010).

2.4 Data analysis

Carotenoid degradation was analyzed independently of carotenoid isomerization.

To describe -carotene and lycopene degradation the drop in total (all-E + Z) -carotene and lycopene
content was considered. For this purpose, a single response kinetic approach was followed and possible
differences between isomers were not taken into account. Experimental data are presented in the dimensionless
form X = C/C0 with C the total -carotene or lycopene content after a specific heating interval, and C 0 the initial
total -carotene or lycopene content. The change in the dimensionless carotenoid content is described using the
fractional conversion model:

Eq 1

with Xfinal the dimensionless total carotenoid content after prolonged heating, k the reaction rate constant (min -1)
and t the reaction time (min). The temperature dependency of the rate constant k can be expressed using the
activation energy (Ea) (J/mol) according to the Arrhenius equation:

Eq 2

where R represents the universal gas constant (8.314 J/mol K), T the temperature concerned, and k ref the
reaction rate constant at a reference temperature Tref. Equation 2 was substituted in equation 1 and kref and Ea
were estimated simultaneously by minimizing the sum of squares of errors by one-step nonlinear regression
taking into account the registered time/temperature profiles. Joint confidence regions (JCR) (90%) were
constructed using equation 3 to evaluate the statistical confidence of the estimated parameters.

Eq 3

where SSQ () represents the error sum of squares associated with the least square estimate , p the number of
simultaneously estimated parameters, m the number of observations and F the upper 1- quantile for a F-
distribution with p and m-p degrees of freedom. The JCR takes into account the correlation between
simultaneously estimated parameters.

To study exclusively the carotenoid isomerization, the contribution of each isomer to the total content
was calculated as follows:

Eq 4

The kinetic model was derived from the simplified reaction network proposed in Fig. 1. For each step a
differential equation was set up (Colle et al., 2013). The temperature dependence of the rate constants k was
expressed using the activation energy (Ea) (J/mol) according to the Arrhenius equation (Equation 2). The
mathematical model was obtained by substitution of the Arrhenius equation into the individual differential
equations. The model was fitted simultaneously on the four responses taking into account the initial isomer
contributions measured. The kinetic parameters were estimated by non-linear regression using the Bayesian
approach. Conform to statistical demands for multi-response modelling, the commonly used least-squares
minimization was replaced by the determinant criterion (Colle et al., 2013).

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 For -carotene: For lycopene:
X = -carotene contribution X = lycopene contribution
Z1 = 9-Z Z1 = x-Z (unidentified Z-isomers)
Z2 = 13-Z Z2 = 9-Z
Z3 = 15-Z Z3 = 13-Z

Fig. 1 Reaction network used to derive the kinetic model for carotenoid isomerization.

3 Results

3.1 Carotenoid degradation

The evolution in total -carotene content and total lycopene content in heat treated olive oil is shown in
Fig. 2A and Fig. 2B. The evolution in total -carotene content and total lycopene content in heat treated olive
oil/vegetable emulsion is shown in Fig. 2C and Fig. 2D. The data points represent the experimental values. Both
carotenoids in both reaction media initially degraded quickly during heating. When heating was continued,
degradation ceased and a plateau value was reached. Generally degradation increased with increasing treatment
temperature and a temperature dependent plateau value was reached after prolonged heating. The kinetic
parameters, kref and Ea for carotenoid degradation (except for -carotene in olive oil) were estimated
simultaneously and are shown in Table 1. The predicted values are shown in Fig. 2 as full lines.

A B

C D
70 C 85 C 90 C 100 C 110 C 115 C 120 C 130 C 140 C

Fig. 2 Time course of the relative total -carotene content (A) and time course of the relative total lycopene content (B) after
thermal treatment of an enriched olive oil. Time course of the relative total -carotene content (C) and time course of the
relative total lycopene content (D) after thermal treatment of an olive oil/vegetable emulsion. The single data points
represent the experimental values, whereas the full lines represent the values predicted by the fractional conversion model.

Table 1 Estimated kinetic parameters ( standard error) for the degradation of total -carotene and total lycopene estimated
by one-step regression analysis using a fractional conversion model.

Tref = 110 C k (min-1) Ea (kJ/mol)

-carotene in enriched olive oil nd nd
Lycopene in enriched olive oil 0.10 0.02 25.4 6.6
-carotene in olive oil/carrot emulsion 0.10 0.01 45.0 8.6
Lycopene in olive oil/tomato emulsion 0.15 0.02 28.1 8.8
nd: not determined

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 To correctly compare the simultaneously estimated parameters obtained for -carotene degradation in an
olive oil/carrot emulsion with the simultaneously estimated parameters obtained for lycopene degradation in an
olive oil/tomato emulsion 90% JCR were constructed (Fig. 3).

Fig. 3 Joint confidence region (90%) for total -carotene

degradation kinetics in olive oil/carrot emulsion () and for
total lycopene degradation kinetics in olive oil/tomato
emulsion (). The symbols represent the least-squares
estimates; the lines represent the JCRs.

3.2 Carotenoid isomerization

To gain insight into thermally induced -carotene and lycopene isomerization reactions, changes in -
carotene and lycopene isomer contributions during thermal treatment of enriched olive oil and olive
oil/vegetable emulsions were investigated. The results for enriched olive oil are shown in Fig. 4. Similar trends
were obtained for -carotene and lycopene isomerization in the olive oil/vegetable emulsions (data not shown).
The all-E-carotenoid contributions decreased with increasing treatment time and temperature which was
accompanied by an increase in Z-carotenoid contributions. Some Z-isomers showed a maximal contribution
after a certain treatment time. This treatment time shortened as treatment temperature increased. Isomerization
reactions were described using the reaction mechanism in Fig. 1 and the corresponding differential equations.
Four response models were constructed and are illustrated with full lines in Fig. 4. The estimated kinetic
parameters are listed in Table 2.
120 18 90 35
All-E--carotene contribution (%)

All-E-lycopene contribution (%)

9-Z--carotene contribution (%)

16 80

x-Z-lycopene contribution (%)

100 30
14 70
25
80 12 60
10 50 20
60
8 40 15
40 6 30
10
4 20
20 5
2 10
0 0 0 0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140
Treatment time (min) Treatment time (min) Treatment time (min) Treatment time (min)

16 9 30 14
15-Z--carotene contribution (%)
13-Z--carotene contribution (%)

13-Z-lycopene contribution (%)

8
9-Z-lycopene contribution (%)

14 12
25
12 7
10
6 20
10
5 8
8 15
4 6
6
3 10
4
4 2
5 2
2 1
0 0 0 0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140
Treatment time (min) Treatment time (min) Treatment time (min) Treatment time (min)

A B
70 C 85 C 90 C 100 C 110 C 115 C 120 C 130 C 140 C

Fig. 4 Time course of -carotene isomer contribution in heated olive oil (A) and time course of lycopene isomer contribution
in heated olive oil. The solid lines represent the fit of the Fig. 1 model, while (mean) experimental data are represented by
single data points.

Table 2 Estimated kinetic parameters ( 95% HPD interval) based on Fig. 1 and multi response data analysis describing -
carotene and lycopene isomerization in olive oil and olive oil/vegetable emulsion heated at temperatures between 70 C and
140 C.

Olive oil Olive oil/ carrot emulsion Olive oil Olive oil/ tomato emulsion
-carotene Temperature range: 85-130 C Temperature range: 85-130 C Lycopene Temperature range: 70-130 C Temperature range: 80-140 C
k 100 C (min-1) Ea (kJ/mol) k100 C (min-1) Ea (kJ/mol) k 100 C (min-1) Ea (kJ/mol) k100 C (min-1) Ea (kJ/mol)
all-E to 9-Z 0.0010 0.0003 89 8 0.0007 0.0002 87 14 all-E to x-Z 0.0165 0.0041 60 10 0.0073 0.0030 85 19
all-E to 13-Z 0.0265 0.0020 129 8 0.0337 0.0048 120 15 all-E to 9-Z 0.0049 0.0004 127 5 0.0019 0.0003 143 8
all-E to 15-Z 0.0112 0.0021 131 22 0.0092 0.0019 76 17 all-E to 13-Z 0.1435 0.0541 101 17 0.0482 0.0122 114 25
9-Z to all-E 0.0173 0.0062 22 6 0.0012 0.0036 47 45 x-Z to all-E 0.0379 0.0101 43 13 0.0349 0.0166 57 22
13-Z to all-E 0.1489 0.0130 124 8 0.1357 0.0229 108 16 9-Z to all-E 0.0150 0.0026 105 8 0.0073 0.0017 125 10
15-Z to all-E 0.1777 0.0379 106 23 0.0691 0.0167 59 19 13-Z to all-E 0.8111 0.3109 100 18 0.3761 0.1025 92 25

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4 Discussion

4.1 Carotenoid degradation

One-step regression using the fractional conversion model and the Arrhenius equation resulted in suitable
models which correlated the experimental values well with the predicted values. The obtained activation
energies are rather low compared to the typical activation energy (about 100 kJ/mol) of a chemical reaction
(Van Boekel, 2008). This might be explained by the fact that radical intermediates play an important role during
carotenoid degradation (Boon et al., 2010) and that radical reactions are only slightly temperature dependent
(Van Boekel, 2008). At first sight Table 1 shows that the Ea-value for lycopene degradation is slightly lower
than the Ea-value for -carotene degradation. In addition, the JCRs do not overlay (Fig. 3) which indicates that
the correlated estimated parameters are significantly different from one another. This difference can be
attributed to the carotenoid type studied (lycopene versus -carotene). However, it can not be excluded that the
difference arises from the difference in carotenoid source and the structural organization of the carotenoids in
the different matrices (tomato versus carrot).

4.2 Carotenoid isomerization

Multi-response modelling proved to be a valuable tool to describe carotenoid isomerization reactions.
The rate constants of Z-isomer formation were generally lower than the rate constants of back isomerization
suggesting that after a certain treatment time the Z-contributions will attain a maximum. Higher rate constants
for isomerization of lycopene dissolved in olive oil than in olive oil/tomato emulsion were obtained. Since
lycopene isomerization hardly occurs in plain tomato pulp (Colle et al., 2010) isomerization in the olive
oil/tomato emulsion should have occurred primarily in the oil phase. Smaller rate constants for lycopene
isomerization in the olive oil/tomato emulsion than in the olive oil are consequently comprehensible since the
rate constants were expressed with regard to the system as a whole. It is important to realize that the model
parameters, in particular the rate constants, are apparent. This was not as straightforward in the case of -
carotene. Furthermore, the sets of kinetic parameters describing lycopene and -carotene isomerization in olive
oil were different. This was also the case when carotenoid isomerization was compared in the olive oil/vegetable
emulsions.

5 Conclusions

In this study, the thermal degradation and isomerization of -carotene and lycopene in different food
systems were compared. To study the degradation reaction, a single response modelling approach was used,
while multi-response modelling was applied to describe isomerization reactions. The latter approach has the
advantage that information on multiple responses is used resulting in more precise parameter estimates and more
realistic/mechanistic models. The models (fractional conversion model and isomerization reaction scheme)
derived were applicable to describe the changes in both -carotene and lycopene for all the different food
systems studied. A significantly different set of kinetic parameters was obtained for -carotene and lycopene
degradation in respectively olive oil/carrot emulsion and olive oil/tomato emulsion. In addition, lycopene
isomerization reactions in the olive oil/tomato emulsion as a whole occurred more slowly than in olive oil.
Furthermore, different sets of kinetic parameters describing -carotene and lycopene isomerization in olive oil
were obtained. The results obtained clearly show that carotenoid conversion kinetics depend on the carotenoid
type as well as on the food system. Therefore process optimization demands product specific kinetic data.

Acknowledgements

This work is financially supported by the Institute for the Promotion of Innovation through Science and
Technology in Flanders, by the Research Foundation-Flanders and by the Research Fund KU Leuven.

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