Food Science and Technology (Campinas)

Print version ISSN 0101-2061

On-line version ISSN 1678-457X

Cinc.Tecnol.Aliment.vol.18no.1CampinasJan./Apr.1998

http://dx.doi.org/10.1590/S0101-20611998000100009

EVALUATION OF TOTAL CAROTENOIDS, - AND -

CAROTENE IN CARROTS (Daucus carota L.) DURING
HOME PROCESSING1

Helena Maria PINHEIRO-SANTANA2, Paulo Csar

STRINGHETA3, Sebastio Csar Cardoso BRANDO3, Hctor
Hernando PEZ4, Valria Maria Vitarelli de QUEIRZ5

SUMMARY

This study aims to analyze the influence of dehydration and different preparation
methods during home processing related to-carotene, -carotene and total
carotenoids stability in carrots. Vitamin A values were evaluated after different
treatments. Thus, carrots were submitted to steam cooking, water cooking with and
without pressure, moist/dry cooking and conventional dehydration. Determination
of - and -carotenes was made by High-Performance Liquid Chromatography
(HPLC) (conditions were developed by us) using spectrophotometric detection
visible-UV at 470 nm; a RP-18 column and methanol: acetonitrile: ethyl acetate
(80: 10: 10) as mobile phase. Total carotenoids quantification was made by 449 nm
spectrophotometer. The retention of the analyzed carotenoids ranged from 60.13 to
85.64%. Water cooking without pressure promoted higher retention levels of-
and -carotene and vitamin A values, while water cooking with pressure promoted
higher retention levels of total carotenoids. Dehydration promoted the highest
carotenoid losses. The results showed that, among the routinely utilized methods
under domestic condition, cooking without pressure, if performed under controlled
time and temperature, is the best method as it reduces losses in the amount of -
and -carotene, the main carotenoids present in the carrots. Despite the significant
carotenoid losses, carrots prepared through domestic methods, remain a rich
source of provitamin A.

Key words: Carrot, carotenoid, stability, HPLC.

RESUMO
AVALIAO DE CAROTENIDES TOTAIS, E -CAROTENO EM CENOURA (Daucus
carota L.) DURANTE PROCESSAMENTO A NVEL DOMSTICO. O presente estudo
teve como objetivo analisar a influncia da desidratao e de diferentes mtodos de
preparo a nvel domstico sobre a estabilidade de -caroteno, -caroteno e
carotenides totais em cenouras. Os valores de vitamina A foram avaliados aps os
diferentes tratamentos. Para tanto, amostras de cenoura foram submetidas
coco a vapor, coco em gua com e sem presso, coco mida/seca e
desidratao convencional. Para a determinao de e -caroteno utilizou-se
Cromatografia Lquida de Alta Eficincia (CLAE). As condies utilizadas
(desenvolvidas por ns) constaram de decteco espectrofotomtrica UV-visvel a
470 nm; coluna RP-18 e metanol: acetonitrila: acetato de etila (80: 10: 10) como
fase mvel. Os carotenides totais foram quantificados por espectrofotometria a
449 nm. A reteno dos carotenides analisados aps os tratamentos variou de
60,13 a 85,64%. A coco em gua sem presso permitiu os maiores nveis de
reteno de - e -caroteno e tambm dos valores de vitamina A. Os carotenides
totais foram melhor preservados quando a coco em gua com presso foi
utilizada. O processo de desidratao provocou as maiores perdas nos trs tipos de
carotenides analisados. Os resultados mostraram que, entre os mtodos
rotineiramente utilizados a nvel domstico, a coco em gua sem presso, se
realizada em condies controladas de tempo e tempertura, o melhor mtodo
para reduzir perdas no contedo de - e -caroteno, principais carotenides
provitamnicos A da cenoura. Apesar das perdas significativas de carotenides,
cenouras preparadas a nvel domstico permanecem como uma rica fonte de
provitamina A.

1 - INTRODUCTION
Carotenoids have been extensively studied due to their important biological
functions for humans and also as a natural pigment. In 1919, relations beetween
carotenoid and vitamin A were found, and in 1930 it was established that some of
them have provitamin A activity (-carotene, -carotene, -carotene, -zeacarotene
and others), which could be transformed in vitamin A inside the animal organism
(7, 20). Due to the functions and several actions attributed to carotenoids, their
studies have assembled researchers from several areas, such as chemistry,
agriculture, medicine, engineering and nutrition (21).

Known as one of the best sources of - and -carotene, carrots are therefore high
in provitamin A (6). The main carotenoids in carrots of the "orange" variety are -
and -carotene, ranking from 80 to 90% of total carotenoids. The remaining
consists of xanthophylls and non-corored polyenic pigments (3). According to
RAMOS, 1991 (17) the main carotenoids in raw carrots of the Nantes variety are -
carotene (51,3%), -carotene (29,5%) and -carotene (5,1%).

Carotenoids are present in nature in the trans configuration, which is more stable.
However, the cis-isomers may occur and increase during cooking methods as well
as during industrial processing (24). Nutritionally, the difference between trans- and
cis-isomers is very important, since cis-configuration exhibits lesser potency,
resulting in a drastic reduction of vitamin A activity (9, 19). Raw carrots show a
variation in the incidence of cis-isomers of - and -carotene, i. e., they may not be
detected or be present in small quantities (8). Almeida and Penteado, 1987 (1)
have found an increase of approximately 6% in cis-isomers, folloowed by a
reduction of trans-isomers in raw carrots.

Nowadays, the nutritional content of food after preparation has become an

important concern to health and food professionals. It is also a concern for
consumers and manufacturers who, without doubt, have contributed to better food
quality and, consequently, to consumers health.

In vegetables and intact fruits, the cellular structure and its complex combination
with proteins confer carotenoids some stability. However, during several processing
phases, the ultra-structure of carotenoids and their complexes can be broken,
exposing the pigments to adverse factors that can lead to their destruction.
Stability varies largely during the stages of processing and storage, depending upon
carotenoid structure, temperature, oxygen availability, light exposure, humidity
content, water activity and acid, metal, anti-oxidant and pro-oxidant presence (5,
6, 21).

SPEEK et al. (23) evaluated the effect of food processing on total carotenoids
and -carotene contents in vegetables, finding evident losses. Average loss of
vitamin A activity after cooking, frying, fermentation, sun drying and sun drying
followed by cooking, were 14, 24, 29, 44 and 60%, respectively. Also, the effect of
cooking was studied by ALMEIDA & PENTEADO (1). After ten minutes of cooking
had losses of 12% for -carotene and 14% for-carotene.

There is a general agreement that among several forms of processing food, drying
and dehydration involve the highest carotenoid losses. Decaying affects color,
nutritive value, texture and flavor of vegetables during dehydration and storage (2).

The main objective of this study was to evaluate the effect of conventional
dehydration and different home preparation methods on -carotene and -carotene
in carrots, since these are the main carotenoids in carrots as well as their main
source of provitamin A. Total carotenoids and vitamin A values also evaluated in
carrots.

2 - MATERIALS AND METHODS

2.1 - Raw Material

Carrots (Daucus carota L.) of the variety Nantes, grown under standardized
conditions (same type of soil, same planting techniques and same fertilizer
treatment), were used in this study at the Universidade Federal de Viosa (UFV),
MG, Brazil. The entire lot was obtained from the same planting date and harvested
approximately 4 months later. The samples were taken from field to laboratory
inside plastic containers at 25-26C inside a closed vehicle. Samples were prepared
the day after harvest.

2.2 - Sample Preparation

After washing and mechanical peeling, carrots were submitted to the preparation
methods described in Table 1. Dehydration of pre-prepared roots was also
undertaken. Three repetitions were performed for each treatment. The chemical
analyses were carried out in duplicate.
Carrot samples of about 1 x 1 x 1 cm were cut manually, before cooking. Aluminum
pans of one liter capacity were used for immersion of samples, cooked in water
without pressure. The moist / dry cooking method consisted of sample immersion in
water without pressure, followed by a second heat exposure in oven. In the steam
cooking method a 2-liter aluminum pan was used. Cooking under pressure was
done in an autoclave, with capacity for 2.5 liters. An adapted thermometer was
used for temperature control. A mercury thermometer with a scale ranging from 0
to 300C was used for other measurements.

Dehydration was conducted using about 1.5 kg of carrots, for each replication.
Shredded samples were blanched and the peroxidase test performed to verify the
efficiency of the process. Dryness was conducted for 7 and a half hours, at 64-65C
in an oven with forced air. After this, the samples were inserted into glasses,
wrapped in aluminum paper and stored in a freezer for further analysis.

Except for the dehydrated samples, all the others were codified and frozen by the
fast frozen method (a method which uses liquid nitrogen aspersion over the
samples and freezers them instantaneously) at -54C in a cryogenic chamber.
Following this, the samples were placed into transparent plastic bags, with excess
of air being removed, wrapped in aluminum paper and stored in a freezer at -18C,
for further analysis.

2.3 - Solids Analysis

The moisture content of the samples was initially determined and the total solid
content obtained by subtraction (22). The insoluble solids were determined
according to the analytical procedures of Instituto Adolfo Lutz (11). The soluble
solids were also determined by a subtraction between the total and insoluble solids.

2.4 - Preparation of Standards

Stock solutions of trans - and -carotenes (obtained from Basf do Brasil) were
prepared by weighing 50 mg of each in stock solutions of 100 mL of the mobile
phase used for HPLC analysis. Standard purity was checked in pet. ether using
Lambert-Beer law. Increasing concentrations of stock solutions of carotenoids were
prepared to build standard curves, which were used for determination of
carotenoids in the samples. Sudan {1-(phenylazo) 2-naphthalenol} was used as
internal standard according to Quackembush & Smallidge (16). For its preparation,
0.1 g of this standard was weighed and diluted in a solution of 500 ml of methanol:
chloroform (9: 1)and further diluted for sample analysis.
2.5 - Carotenoid Extraction

Carotenoid extraction was based on the procedure described by Rodriguez et al.

(18). Samples (5 g) were ground with the help of chilled acetone and a
microgrinder model TE 102, Tecnal and vacuum filtered using a Bchner funnel.
This procedure was repeated until the residue became colorless and pigments were
transferred to pet. ether, each fraction being washed with distilled water for
complete acetone removal.

2.6 - Carotenoid Analysis

After extraction, the total carotenoid content of the pigments extracted was
determined by spectrophotometer at 449 nm, as proposed by Ramos (17). A
standard curve correlating total carotenoid concentration (expressed in-carotene)
and the absorbance of the pigment solution was used. An absorptivity coefficient of
2592 was use for -carotene standard quantification (19).

The and -carotene analysis was carried out by HPLC. Pigments were clarified in a
MgO: hyflosupercel (1:1) mini column, according to Carvalho (7). This procedure
was adopted to avoid overlapping of the internal standards retention time with
that of other pigments in the carrot. Clarification allows the elimination of
carotenoids other than the provitaminic A ones. Samples thus obtained were
evaporated under nitrogen, re-dissolved in known acetone volume, a constant
volume of internal standard added, filtered using 0.45 m membrane and injected
in the liquid chromatographic column. The following apparatus were used: liquid
chromatograph, CG-480 (Instrumentos CG, So Paulo, Brazil) equipped with
Rheodyne 7125 injector, 100 L loop; UV-VIS detector at 470 nm and absorbance
range at 0.5; an integrator (CG 200). The column used was a 25 cm x 4 mm RP-18
(Lichrospher, 5 m, Merck). The mobile phase used was methanol: acetonitrile:
ethyl acetate (80:10:10) with a flow rate of 2mL/min.

Solvents for HPLC (Lichrosolv-Merck) were filtered immediately before use utilizing
the Millipore vacuum filtration system, degassed under ultrasonic bath. Samples
and standards were filtered through a FH 1300 Millipore membrane (0.45m).

2.7 - Recovery Experiments

Since carotenoids are rather unstable compounds, their stability during sampling
preparation was determined through addition and recovery of known quantities
of and -carotene standards to the carrot samples, followed by HPLC analysis, as
previously described.

2.8 - Vitamin A Value Calculation

Calculation was performed based on A vitamin activity of the carotenoid

precursors, - and -carotenes, according to Bauernfeind (4) and to the conversion
factors provided by the National Academy of Sciences - National Council Research
(NAS-NCR) (13). Vitamin A value was expressed in RE (retinol equivalents)/100 g
of sample. It is known that 0.6 g of -carotene and 1.2 g of -carotene and other
carotenoids are equivalent to 1 IU (International Unit), with 1 RE being equivalent
to 10 IU.

Although the differentiation between trans- and cis-isomers is very important in

determination vitamin A value, this studys objective was not to separate such
isomers. Besides, for both raw carrot samples and samples of carrots subject to
preparation methods, only trans-isomers of and -carotene were quantified, since
the standards used were trans- and -carotene.

2.9 - Experimental Design

The experiment was arranged in a completely randomized design. Statistical

analyses were performed based on the SAEG program, 5.0 version (System for
Statistical and Genetic Analyses developed by the Data Processing Center of the
UFV). Variance analysis was carried out in order to detect significant differences
among the treatments. The Duncan test was applied to analyze the differences
between treatment averages presenting significant differences (15).

3 - RESULTS AND DISCUSSION

3.1 - Qualitative Separation of and -Carotene by HPLC

A typical chromatographic separation of and -carotene from carrot samples is

shown in Figure 1.

3.2 - Recovery of Added and -Carotenes

The results on recovery of added and -carotenes to carrot samples are shown
in Table 2 and 3. As can be observed the % recovery was very good. Hence, it can
be concluded that the results obtained on the concentration of carotenoids are due
to the treatments conducted.
3.3 - Quantitative Analysis of Carotenoids

Tables 4, 5 and 6 show -carotene, -carotene and total concentrations expressed

on moist, dry and insoluble bases, respectively.
The values of carotenoids expressed on the moist basis (Table 4) are much higher
in the dehydrated samples. As the moist content in the dehydrated samples is much
lower than in the other samples, an enhanced level of carotenoids is observed when
the values are expressed on a moist basis.
The slightly higher sample concentration of carotenoids submitted to the moist / dry
cooking method can be attributed to a lower moist content in these samples.
Samples obtained from steam cooking and water cooking with and without pressure
presented small humidity level differences, probably due to the reduced and similar
cooking time (Table 1), although they were statistically different and also had a
difference in carotenoid concentration. In this case, the samples submitted to
drastic treatments presented higher carotenoid levels and statistically different
concentrations. Thus, moist base concentration does not account for the distinct
loss of the soluble solids, nor to the great difference between humidity and total
solids, making it difficult to evaluate stability levels of carotenoids on a moist basis.

As it can be seen in Tables 4, 5 and 6, the sum of -carotene and -carotene values
do not correspond to the total carotenoid values. This can be explained by the use
of two different techniques for analysis of the compounds, by the greater HPLC
method sensibility and by the presence of other carotenoids in the carrots,
besides and -carotenes.

The expression of the carotenoid values on the dry basis (Table 5) also shows high
levels in the samples submitted to dehydration. In this case, the highest solid total
level of dehydrated samples contributed to the apparent increase. On the other
hand, there are soluble solid losses by leaching during blanching and by
caramelization during the drying process. In practice, when the amount of nutrients
in the diet is calculated, the values are expressed on the moist basis, as the
majority of the Chemistry Composition Food Tables do. The presence of carotenoids
in the insoluble solid fraction makes the expression of the results on this basis more
adequate and reliable.

Table 6 shows that the lowest carotenoid values are those of samples which were
submitted to dehydration, as expected, confirming that the expression of the
results in the insoluble solid basis is indeed more adequate.

3.4 - Evaluation of Carotenoid Stability

The stability of -carotene, -carotene and total carotenoids in carrots prepared in

small quantities is presented in Table 7.
The results showed that -carotene presented lower retention rates than -
carotene. The uniformity among retention index contents of the samples seems to
be due to similar cooking time and temperature, as well as to the similarity of
water/food relationship and similar slicing processes before cooking (Table 1).

According to Lachance and Erdman, 1975 (12), when the vegetables are cooked in
water, on a domestic scale, the nutrient losses vary depending on the water
quantity, cooking time and type of equipment used. Besides this, sliced vegetables
are specially more sensitive. Many studies, using small and large food quantities,
indicated that vegetable nutrient loss during cooking is caused, in most cases, by
water extraction rather than by thermal destruction.

When the -carotene levels were analyzed, large losses (25 to 35%) were
observed. The treatments which involved higher heat exposure (moist/dry cooking
and dehydration) were, coincidentally, the ones which presented lower -carotene
retention, possibly due to oxidation and isomerization. Besides variations in the
stability of and -carotenes, the methods used during preparation did cause
visible and important provitamin A losses.

Total carotenoids losses found in this study in dehydrated carrot (38%) were close
to those (45%, 40% and 40%) found, respectively, by WEIER and STOCKING, 1946
(25), DELLA-MONICA and McDOWELL, 1965 (10) and PARK, 1987 (14).

In our study, - and -carotene losses in samples of carrot cooked in water without
pressure (30 and 22%, respectively) were higher than those found by ALMEIDA and
PENTEADO,1987 (1) (12 and 14%, respectively) in cooked carrots in similar
conditions. However, this study does not report size of carrot cuts nor water/food
ratio used.

RAMOS, 1991 (17), in the other hand, found losses of 4% for -carotene and 16%
for -carotene carrots subject to steam bleaching.

A higher variation between retention rates was noted in relation to the total
carotenoids. Water cooking without pressure promoted lower stability and water
cooking with pressure, the highest carotenoid retention. These results were not
expected, as they should have the profile shown by and -carotenes, since they
account for 80 to 90% of carotenoids in carrots.

3.5 - Provitamin A Loss Evaluation

Variations of the vitamin A values in the samples prepared on adomestic scale can
be seen in Table 8. Losses follow the same profile of those found in -carotene
(Table 7), since this is the main carotenoid with provitamin A activity in carrots.
The average loss of 35.20% in dehydrated samples was lower than that found by
Speek et al. (23), who submitted several vegetables to sun drying, with 44% loss.
It has been known that when using the sun drying technique, parameters cannot be
adequately controlled since sun radiation, associated with oxygen, affects vitamins
causing a higher carotenoid degradation.

Although it is known that cis-isomers of provitamin A carotenoids show a lower

activity than their trans forms and despite the fact that vitamin A activity had not
been analyzed and determined separately for each isomer, one must emphasize
that only the trans-isomers of and -carotene were quantified in both samples of
raw carrots and carrots subject to preparation methods. This occurred since the
standards used were trans and -carotene.

4 - CONCLUSIONS
- The preparation methods utilized for small quantities of carrots promoted 14.4 to
39.9% carotenoid losses. Dehydration promoted the highest degradation level
for -carotene, -carotene and total carotenoids. Provitamin A losses were
expressive and statistically different among the analyzed methods, following the -
carotene profile.

- The results obtained show that, for methods routinely used on a domestic level,
water cooking without pressure, if carried out under controlled time and
temperature, is the best method to reduce losses of - and -carotene present in
carrots. Hence, since cooking time and temperature are important factors for
carotenoid degradation, the least cooking time and lowest temperature are vital to
prevent greater losses. In spite of expressive carotenoid losses, carrots prepared by
home food preparation methods remain a rich source of provitamin A. The
methodology developed for this study is being presently tested for determination of
carotenoids by HPLC in other vegetables.
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ACKNOWLEDGMENTS
We gratefully thank Prof. Marcelo Jos Vilela for review of this manuscript and Prof.
Alice Jham for its translation into English.
1
Recebido para publicao em 01/07/97. Aceito para publicao em 14/04/98.

2
Professora do Departamento de Nutrio e Sade, Universidade Federal de
Viosa, MG-Brazil - 36 571-000. Tel.: (031) 899 2545; Fax 55 (031) 899 2541; E-
mail: hsantana@mail.ufv.br

3
Professores do Departamento de Tecnologia de Alimentos, Universidade Federal
de Viosa, MG-Brazil - 36 571-000.

4
Professor do Departamento de Engenharia de Alimentos, Universidade de La
Serena, Chile.

5
Departamento de Nutrio e Sade, Universidade Federal de Viosa, MG-Brazil -
36 571-000.