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Food Science and Technology

Print version ISSN 0101-2061On-line version ISSN 1678-457X

Ciênc. Tecnol. Aliment. vol.18 no.1 Campinas Jan./Apr. 1998 



Helena Maria PINHEIRO-SANT’ANA2, Paulo César STRINGHETA3, Sebastião César Cardoso BRANDÃO3, Héctor Hernando PÁEZ4, Valéria Maria Vitarelli de QUEIRÓZ5




This study aims to analyze the influence of dehydration and different preparation methods during home processing related to a-carotene, b-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 a- and b-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 a- and b-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 a- and b-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.


AVALIAÇÃO DE CAROTENÓIDES TOTAIS, a E b-CAROTENO EM CENOURA (Daucus carota L.) DURANTE PROCESSAMENTO A NÍVEL DOMÉSTICO. O presente estudo teve como objetivo analisar a influência da desidratação e de diferentes métodos de preparo a nível doméstico sobre a estabilidade de a-caroteno, b-caroteno e carotenóides totais em cenouras. Os valores de vitamina A foram avaliados após os diferentes tratamentos. Para tanto, amostras de cenoura foram submetidas à cocção a vapor, cocção em água com e sem pressão, cocção úmida/seca e à desidratação convencional. Para a determinação de a e b-caroteno utilizou-se Cromatografia Líquida de Alta Eficiência (CLAE). As condições utilizadas (desenvolvidas por nós) constaram de dectecção espectrofotométrica UV-visível a 470 nm; coluna RP-18 e metanol: acetonitrila: acetato de etila (80: 10: 10) como fase móvel. Os carotenóides totais foram quantificados por espectrofotometria a 449 nm. A retenção dos carotenóides analisados após os tratamentos variou de 60,13 a 85,64%. A cocção em água sem pressão permitiu os maiores níveis de retenção de a- e b-caroteno e também dos valores de vitamina A. Os carotenóides totais foram melhor preservados quando a cocção em água com pressão foi utilizada. O processo de desidratação provocou as maiores perdas nos três tipos de carotenóides analisados. Os resultados mostraram que, entre os métodos rotineiramente utilizados a nível doméstico, a cocção em água sem pressão, se realizada em condições controladas de tempo e tempertura, é o melhor método para reduzir perdas no conteúdo de a- e b-caroteno, principais carotenóides provitamínicos A da cenoura. Apesar das perdas significativas de carotenóides, cenouras preparadas a nível doméstico permanecem como uma rica fonte de provitamina A.




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 (a-carotene, b-carotene, g-carotene, b-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 a- and b-carotene, carrots are therefore high in provitamin A (6). The main carotenoids in carrots of the "orange" variety are a- and b-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 b-carotene (51,3%), a-carotene (29,5%) and d-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 a- and b-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 b-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 a-carotene and 14% for b-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 a-carotene and b-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.



2.1 - 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 Viçosa (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 25-26°C 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 300°C 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 64-65°C 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 -54°C 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 -18°C, 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 a- and b-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 Büchner 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 b-carotene) and the absorbance of the pigment solution was used. An absorptivity coefficient of 2592 was use for b-carotene standard quantification (19).

The a and b-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 standard’s 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 µm membrane and injected in the liquid chromatographic column. The following apparatus were used: liquid chromatograph, CG-480 (Instrumentos CG, São Paulo, Brazil) equipped with Rheodyne 7125 injector, 100 µL 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 µm, 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 (0.45µm).

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 a and b-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, a- and b-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 µg of b-carotene and 1.2 µg of a-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 study’s 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 a and b-carotene were quantified, since the standards used were trans- a and b-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).



3.1 - Qualitative Separation of a and b-Carotene by HPLC

A typical chromatographic separation of a and b-carotene from carrot samples is shown in Figure 1.



3.2 - Recovery of Added a and b-Carotenes

The results on recovery of added a and b-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 a-carotene, b-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 a-carotene and b-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 a and b-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 a-carotene, b-carotene and total carotenoids in carrots prepared in small quantities is presented in Table 7.



The results showed that a-carotene presented lower retention rates than b-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 b-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 b-carotene retention, possibly due to oxidation and isomerization. Besides variations in the stability of a and b-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, a- and b-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 a-carotene and 16% for b-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 a and b-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 b-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 a and b-carotene were quantified in both samples of raw carrots and carrots subject to preparation methods. This occurred since the standards used were trans a and b-carotene.



- The preparation methods utilized for small quantities of carrots promoted 14.4 to 39.9% carotenoid losses. Dehydration promoted the highest degradation level for a-carotene, b-carotene and total carotenoids. Provitamin A losses were expressive and statistically different among the analyzed methods, following the b-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 a- and b-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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We gratefully thank Prof. Marcelo José Vilela for review of this manuscript and Prof. Alice Jham for its translation into English.



1 Recebido para publicação em 01/07/97. Aceito para publicação em 14/04/98.

2 Professora do Departamento de Nutrição e Saúde, Universidade Federal de Viçosa, MG-Brazil - 36 571-000. Tel.: (031) 899 2545; Fax 55 (031) 899 2541; E-mail:

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

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

5 Departamento de Nutrição e Saúde, Universidade Federal de Viçosa, MG-Brazil - 36 571-000.

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