6 THERMAL DEGRADATION OF CAROTENES AND INFLUENCE ON THEIR PHYSIOLOGICAL FUNCTIONS
Lena Jonsson SIK, The Swedish institute for food research P.O. Box 5401, S-402 29 G8teborg, Sweden
ABSTRACT Raw carrot juice contains a considerable amount of a -and f3-carotene, which makes carrot an excellent source of vitamin A. Heat treatment of the juice at temperatures comparable to those at pasteurization and boiling does not change the carotenes, while heating at temperatures used during sterillzation results In rearrangement of the carotene molecules and a decrease- in total carotenes. The all-trans £1'and f3-carotenes appear partly as cis-isomers, especially the 13-cls-isomer. Isomerization of the carotenes leads to a decrease In their vitamin A activity. Carotenes also seem to be anticarcinogens but the extent to which this property is influenced by isomerization is still unknown. INTRODUCTION Plenty of carotenolds occur in nature, both in vegetable and animal tissue. They are of varying color, such as yellow, orange, red or pink, and they also seem to have antioxidatlve properties. Nutritionally some carotenolds are known to exert vitamin A activity. The most potent vitamin A precursor Is f3-carotene. Recently it has also been shown In epidemiological studies that diets rich in carotenoid-containing green-yellow vegetables protect humans against certain forms of cancer, particularly lung cancer. Similarly, a high level of f3-carotene in serum reduces the risk of developing lung cancer. Long-term intervention trials with f3-carotene supplement are in progress to confirm this findings. The mechanisms behind the protective action of carotenoids and the molecular shape of carotenoids most effective 88 antJcarcinogens are still unknown.
M. Friedman (ed.), Nutritional and Toxicological Consequences of Food Processing © Springer Science+Business Media New York 1991
75
In vegetables, fj -carotene primarily occurs as the all-trans isomer. This is also the most vitamin A active isomer, as shown by Bauernfeind (1972). Carotenolds are, however, reactive substances. They can oxidize and isomerize. When all-trans fj-carotene Is exposed to intense light or heat many cis-isomers are found. During processing foods are often exposed to elevated temperatures, high or low pH levels, light, etc. - parameters which can accelerate the isomerization of carotenoids and decrease their vitamin A activity. The knowledge about the reaction rate of isomerization during common food preparation is Insufficient, due to the lack of proper analytical methods. At SIK we have developed a rapid and reliable method to determine different carotenoids and their isomers. This investigation was conducted to determine the effect of heat treatment on isomerization of a - and fj-carotene in carrot juice and to calculate the influence on the pro-vitamin A activity of the juice. EXPERIMENT AL SECTION Materials and reagents. Mature carrots of the Duke variety were harvested in July and stored at _4 0 C for one month before juice preparation. Thea- and fj-carotene standards were obtained from Sigma Chemical Company (St. Louis, MO). All chemicals used were either of analytical grade or of HPLC grade. Preparation of carrot juice. The carrots were washed and processed In a raw juice centrifuge. The carrot juice was bottled in 1.S ml brown, glass vials. The vials were stoppered with polyethylene caps and stored at _400 C up to six months before treatment. Treatments. The vials with frozen carrot juice were placed at +4 0 C for 16 hours before heating. Heat treatment was performed in a thermostatically controlled oil bath. The samples were heated to 80, 100, 121, 128 and 13SoC. For each temperature three vials were removed at specific time intervals during 240 minutes. Immediately upon reaching the specified heating duration the vials were cooled in an ice bath for one minute. Extraction of carotenes was performed immediately. Carotene extraction. The carotenes were extracted using equal amount of acetone and ethanol until the extraction solvent was colorless. The carotenes were then transferred to petroleum ether and evaporated to dryness using a rotary evaporator under vacuum and a water bath temperature of 3SoC. The carotenes were resuspended in the HPLC mobile phase (hexane:acetone, 99.5:0.5, v:v) and then analyzed using adsorption HPLC. To minimize exposure to light the extraction was performed in dimmed (red) light with aluminum foil around all glasswares.
76
- isomers of a - and JJ -carotene. The mobile phase consisted of hexane: acetone, 99.5:0.5 (v:v). Tentative identification was made by measuring the retention time and UV/vlsible adsorption spectra obtained with a diode array detector. The adsorption was recorded simultaneously at 286, 347, 400, 442 and 450 nm. The carotenes were quantified by comparing peak areas of standards of known concentrations with those of the samples. The cis-isomers were presumed to have the same adsorption Intensity as the corresponding trans-isomers. Detailed information of the HPLC procedure has been published elsewhere (Pettersson and Jonsson, 1990). Calculations of pro-vitamin A activity of Evaluation of data. different carotenes were made using biopotency values for the major carotene stereo-isomers reported by Zeichmeister et ale (1962), see Table 1. Table 1. Biopotency values of carotene stereo-isomers1) Isomer
Biopotency
All-trans- JJ-carotene 9-cis- ,8-carotene 13-cis- JJ-carotene
100 38 53
All- trans - a- carotene 9-cis-a- carotene 13-cis-a-carotene
53 13 16
1) Values reported by Zeichmesiter (1962) based on all-trans-Il-carotene
as 100.
Small amounts of other carotenoids or stereo-isomers of undetermined vitamin A activity were also present. As these compounds make up less than 5% of the total carotene content in the carrot juices studied, they have not been Included in the calculations. RESUL TS AN:) DISCUSSION Carotenes in carrot luice The raw carrot juice contained large amounts of a - and f3 -carotene, 21 and 88 ,ug/ml JUice, respectively. Trace amounts of other carotenoids, such tis phytoene and phytofluene were identified. The only stereo-isomer found in the raw juice was theall-trans form. Storage of the samples at _40 0 C before heat treatment did not change the carotene content. Influence of heat treatment Representative chromatograms of thermally treated and untreated juice samples are presented in Figure 1. 77
a..,I ...... 1
•
3!5
Til • •
2'"
H,..tIlBBIIBM
a
Figure 1. Representative chromatograms and spectra of carrot Juice samples. a) untreated samgle b) treated at 121 C for 60 minutes. (1) phytofluene; (2) phytoene; (3) all-trans- a-carotene; (4) 13-cls- JJ-carotene; (5) all-trans- JJ - carotene
78
As shown in Figure Ib, a considerable amount of the all-trans-carotenes are converted into cis-isomers during heating. Figure 2 shows the effect of heating carrot juice at different times and temperatures.
8)
120
110
to c
;;
c
!
8O'C
l00~~~;;;==============
l00'C 121'C
~ '~~:::::::::.::~~~;~~;;;;;;
128·0 135·0
&0 40 30
20
10 O~~---~~---~~---~~---~-'~
o
&0
100
150
200
250
tlme(mln)
120 ttO
b)
l00~~=---
80
,
80
, ...........
.
___________________________
8D'C l00'C
~~
~ t::==::=::=~:~~:~~~~~:~~:~;;~
121·0
128'C 135'C
20
10
o~~--~----r---~~---'----~ 150 200 250 100
o
50
tlme(mln)
Figure 2. Effect of heat treatment on all-trans-a- and fj-carotene in carrot Juice. a) all-trans-a-carotene b) all-trans-fj-carotene
79
During the first few minutes of treating the carrot Juice at BOoC, a small increase in the content of carotenes was found. Similar results have been reported earlier by Hojilla et al. (19B5), who thought that this Increase was due either to a transformation of cis-Isomers to the trans-form (they could not separate the different Isomers) or perhaps to water-soluble compounds draining from the sample, thus increasing the concentration of water-insoluble compounds, such as carotenes. As we are able to follow the changes taking place in the Isomers and because we extract the whole sample, we can reject the assumptions made by Hojilla and co-workers. We can not, however, explain the Increase In carotene during mild heat treatment. Further heat treatment of the carrot Juice at BOoC resulted only in trace amounts of cis-isomers. Samples heated at 1000 C during one hour lost about 10% of the initial amount of a - and J3-all-trans-carotene. Some change Into cis-Isomers was found. The predominant isomer was the 13-cis-isomer. When calculated as having the same adsorption intensity as the all-trans-isomer, the 13-cls- J3 -carotene amounted to between 3 and B% of all the J3 -carotene found. Even a short heating time at higher temperatures caused important loss of carotene. Ten minutes of heating at 121, 12B, and 1350 C resulted in 17, 32 and 42% loss of J3 -carotene, respectively. The a-carotene was much more stable during heating. Only about 10% of the a -carotene was destroyed during 10 minutes of heating at the temperatures studied. The rate of all-trans carotene destruction was highest during the first few minutes of heating. As shown in Figure 1, many cis-Isomers were found after heating at 1210 C for 60 minutes. About 15% of J3 -carotene existed as the 13-cis-isomer. Changes in vitamin A activity according to heat treatment The vitamin A activity In the carrot Juice was calculated using values presented by Bauernfeind (1972) and Zeichmelster (1949). According to these authors, one sixth of the all-trans- J3 -carotene is converted to active vitamin A. Seventy-five per cent of the activity exerted by the all-trans-form Is ascribed to the 13-cls-isomer. All-trans-a-carotene has about half as much vitamin A-activity as J3 -carotene. As the 9-cis-isomer of J3-carotene and ail the cis-isomers of a -carotene exert very little vitamin activity and, furthermore, occur In only smail amounts they will each contribute with less than 0.1 ,ug retinolequivalents per milliliter heated Juice. Therefore, we have decided to include only the all-trans-isomer of a -and J3 -carotene and the 13-cls-Isomer of J3 -carotene in the calculation of vitamin A activity In the juice after different heat treatments. The calculated vitamin A activity in the carrot Juice is reported in Table 2. Mild heat treatment resulted in a decrease In vitamin A activity by a few percentage units only, due to loss of all-trans- J3 -carotene. More intense heat treatment increased the transformation of all-trans- J3 -carotene into Its 13-cls isomer. This gave a rather smail decrease in vitamin A activity. 80
Table 2. Effect of heat treatment on the content of carotenes and vitamin A activity of carrot juice Heat treatment time min. 0
tBmp. C
all-trans a-carotene
all-trans
f} -carotene
13-cis
f} -carotene
/ug/ml Juice 2l!01)
Vitamin A activity retinol eqv·/ug/ml
ca 0 1!0 3!0
16.4
20!0 20!0
88!2 84!3 83!2
10 240
0 80 80
10 60 240
100 100 100
20!0 18!0 17!1
84!0 77!1 76!0
3!0 5!0 7!Z
15.9 14.9 14.9
10 60 Z40
121 1Zl lZI
17!1 16!0 15!1
73!1 64!0 55!1
8!0 10!0 13!0
14.6 13.2 1Z.1
10 30
lZ8 lZ8
15!Z 14!1
60!1 58!1
14!0 13!1
13.0 1Z.4
10 30
135 135
13!Z 1Z!Z
51!Z 45!4
16!1 17!1
11.6 10.6
15.7 15.7
1) Each value represents the mean! SO of 3 experiments.
Very high temperatures, however, resulted in a continuing increase in the amount of 13-cia-isomer, but the increase was nos comparable to the decrease in all-trans- fJ -carotene. Heating at 135 C for 10 minutes caused a 30% decrease in vitamin A activity. The 13-cls-lsomer accounted for almost one fifth of the vitamin activity. If calculation of vitamin A activity ia baaed merely on all-trans- fJ -carotene, then the activity was only corresponded to 58% of the activity in raw carrot Juice. CONCLUSIONS This study shows that low temperature heat treatment of relatively neutral foods, such as carrot, causes only insignificant changes in the carotenes and vitamin A activity. Examples of such heat treatment are cooking in water and pasteurization. However, when using high temperatures, as Is done during heat sterAlizatlon, the loss Is considerable. Conventional sterilization of cans at 121 C will result in a 20% reduction of the vitamin A activity. Very high temperatures, as those used during HTST treatment, enhance the need for proper control of the food process. The results also show the Importance of analyzing all occurring carotenes and their Isomers to get a more reliable vitamin A value fat' foods exposed to heat treatment. 81
There is no information in the literature about the possible anticarcinogen effect of carotenes when they are changed into cis-isomers. ACKNOWLEDGEMENTS This work was supported in part by grants from the Volvo Research Foundation, Goteborg, Sweden. The author thanks Anders Pettersson for excellent technical assistance. REFERENCES Bauernfeind, J.C. (1972). Carotenoid vitamin A precursors and analogs in foods and feeds. J. Agr. Food Chem., 20 (3), 456-473. Hojilla, M.P., Garcia, V.V. and Raymundo, L.C. (1985). Thermal degradation of [j -carotene in carrot juice. ASEAN Food Journal, 1 (4), 157-161. Pettersson, A. and Jonsson, L. (1990). Separation of cis-trans isomers of a-and [j-carotene by adsorption HPLC and identification with diode array detection. Accepted for publ. in Journal of Micronutrient Analysis. Zeichmeister, L. (1962). Cis-trans isomeric carotenoids. Press, London, pp.251.
82
Academic
Lena Jonsson SIK, The Swedish institute for food research P.O. Box 5401, S-402 29 G8teborg, Sweden
ABSTRACT Raw carrot juice contains a considerable amount of a -and f3-carotene, which makes carrot an excellent source of vitamin A. Heat treatment of the juice at temperatures comparable to those at pasteurization and boiling does not change the carotenes, while heating at temperatures used during sterillzation results In rearrangement of the carotene molecules and a decrease- in total carotenes. The all-trans £1'and f3-carotenes appear partly as cis-isomers, especially the 13-cls-isomer. Isomerization of the carotenes leads to a decrease In their vitamin A activity. Carotenes also seem to be anticarcinogens but the extent to which this property is influenced by isomerization is still unknown. INTRODUCTION Plenty of carotenolds occur in nature, both in vegetable and animal tissue. They are of varying color, such as yellow, orange, red or pink, and they also seem to have antioxidatlve properties. Nutritionally some carotenolds are known to exert vitamin A activity. The most potent vitamin A precursor Is f3-carotene. Recently it has also been shown In epidemiological studies that diets rich in carotenoid-containing green-yellow vegetables protect humans against certain forms of cancer, particularly lung cancer. Similarly, a high level of f3-carotene in serum reduces the risk of developing lung cancer. Long-term intervention trials with f3-carotene supplement are in progress to confirm this findings. The mechanisms behind the protective action of carotenoids and the molecular shape of carotenoids most effective 88 antJcarcinogens are still unknown.
M. Friedman (ed.), Nutritional and Toxicological Consequences of Food Processing © Springer Science+Business Media New York 1991
75
In vegetables, fj -carotene primarily occurs as the all-trans isomer. This is also the most vitamin A active isomer, as shown by Bauernfeind (1972). Carotenolds are, however, reactive substances. They can oxidize and isomerize. When all-trans fj-carotene Is exposed to intense light or heat many cis-isomers are found. During processing foods are often exposed to elevated temperatures, high or low pH levels, light, etc. - parameters which can accelerate the isomerization of carotenoids and decrease their vitamin A activity. The knowledge about the reaction rate of isomerization during common food preparation is Insufficient, due to the lack of proper analytical methods. At SIK we have developed a rapid and reliable method to determine different carotenoids and their isomers. This investigation was conducted to determine the effect of heat treatment on isomerization of a - and fj-carotene in carrot juice and to calculate the influence on the pro-vitamin A activity of the juice. EXPERIMENT AL SECTION Materials and reagents. Mature carrots of the Duke variety were harvested in July and stored at _4 0 C for one month before juice preparation. Thea- and fj-carotene standards were obtained from Sigma Chemical Company (St. Louis, MO). All chemicals used were either of analytical grade or of HPLC grade. Preparation of carrot juice. The carrots were washed and processed In a raw juice centrifuge. The carrot juice was bottled in 1.S ml brown, glass vials. The vials were stoppered with polyethylene caps and stored at _400 C up to six months before treatment. Treatments. The vials with frozen carrot juice were placed at +4 0 C for 16 hours before heating. Heat treatment was performed in a thermostatically controlled oil bath. The samples were heated to 80, 100, 121, 128 and 13SoC. For each temperature three vials were removed at specific time intervals during 240 minutes. Immediately upon reaching the specified heating duration the vials were cooled in an ice bath for one minute. Extraction of carotenes was performed immediately. Carotene extraction. The carotenes were extracted using equal amount of acetone and ethanol until the extraction solvent was colorless. The carotenes were then transferred to petroleum ether and evaporated to dryness using a rotary evaporator under vacuum and a water bath temperature of 3SoC. The carotenes were resuspended in the HPLC mobile phase (hexane:acetone, 99.5:0.5, v:v) and then analyzed using adsorption HPLC. To minimize exposure to light the extraction was performed in dimmed (red) light with aluminum foil around all glasswares.
76
- isomers of a - and JJ -carotene. The mobile phase consisted of hexane: acetone, 99.5:0.5 (v:v). Tentative identification was made by measuring the retention time and UV/vlsible adsorption spectra obtained with a diode array detector. The adsorption was recorded simultaneously at 286, 347, 400, 442 and 450 nm. The carotenes were quantified by comparing peak areas of standards of known concentrations with those of the samples. The cis-isomers were presumed to have the same adsorption Intensity as the corresponding trans-isomers. Detailed information of the HPLC procedure has been published elsewhere (Pettersson and Jonsson, 1990). Calculations of pro-vitamin A activity of Evaluation of data. different carotenes were made using biopotency values for the major carotene stereo-isomers reported by Zeichmeister et ale (1962), see Table 1. Table 1. Biopotency values of carotene stereo-isomers1) Isomer
Biopotency
All-trans- JJ-carotene 9-cis- ,8-carotene 13-cis- JJ-carotene
100 38 53
All- trans - a- carotene 9-cis-a- carotene 13-cis-a-carotene
53 13 16
1) Values reported by Zeichmesiter (1962) based on all-trans-Il-carotene
as 100.
Small amounts of other carotenoids or stereo-isomers of undetermined vitamin A activity were also present. As these compounds make up less than 5% of the total carotene content in the carrot juices studied, they have not been Included in the calculations. RESUL TS AN:) DISCUSSION Carotenes in carrot luice The raw carrot juice contained large amounts of a - and f3 -carotene, 21 and 88 ,ug/ml JUice, respectively. Trace amounts of other carotenoids, such tis phytoene and phytofluene were identified. The only stereo-isomer found in the raw juice was theall-trans form. Storage of the samples at _40 0 C before heat treatment did not change the carotene content. Influence of heat treatment Representative chromatograms of thermally treated and untreated juice samples are presented in Figure 1. 77
a..,I ...... 1
•
3!5
Til • •
2'"
H,..tIlBBIIBM
a
Figure 1. Representative chromatograms and spectra of carrot Juice samples. a) untreated samgle b) treated at 121 C for 60 minutes. (1) phytofluene; (2) phytoene; (3) all-trans- a-carotene; (4) 13-cls- JJ-carotene; (5) all-trans- JJ - carotene
78
As shown in Figure Ib, a considerable amount of the all-trans-carotenes are converted into cis-isomers during heating. Figure 2 shows the effect of heating carrot juice at different times and temperatures.
8)
120
110
to c
;;
c
!
8O'C
l00~~~;;;==============
l00'C 121'C
~ '~~:::::::::.::~~~;~~;;;;;;
128·0 135·0
&0 40 30
20
10 O~~---~~---~~---~~---~-'~
o
&0
100
150
200
250
tlme(mln)
120 ttO
b)
l00~~=---
80
,
80
, ...........
.
___________________________
8D'C l00'C
~~
~ t::==::=::=~:~~:~~~~~:~~:~;;~
121·0
128'C 135'C
20
10
o~~--~----r---~~---'----~ 150 200 250 100
o
50
tlme(mln)
Figure 2. Effect of heat treatment on all-trans-a- and fj-carotene in carrot Juice. a) all-trans-a-carotene b) all-trans-fj-carotene
79
During the first few minutes of treating the carrot Juice at BOoC, a small increase in the content of carotenes was found. Similar results have been reported earlier by Hojilla et al. (19B5), who thought that this Increase was due either to a transformation of cis-Isomers to the trans-form (they could not separate the different Isomers) or perhaps to water-soluble compounds draining from the sample, thus increasing the concentration of water-insoluble compounds, such as carotenes. As we are able to follow the changes taking place in the Isomers and because we extract the whole sample, we can reject the assumptions made by Hojilla and co-workers. We can not, however, explain the Increase In carotene during mild heat treatment. Further heat treatment of the carrot Juice at BOoC resulted only in trace amounts of cis-isomers. Samples heated at 1000 C during one hour lost about 10% of the initial amount of a - and J3-all-trans-carotene. Some change Into cis-Isomers was found. The predominant isomer was the 13-cis-isomer. When calculated as having the same adsorption intensity as the all-trans-isomer, the 13-cls- J3 -carotene amounted to between 3 and B% of all the J3 -carotene found. Even a short heating time at higher temperatures caused important loss of carotene. Ten minutes of heating at 121, 12B, and 1350 C resulted in 17, 32 and 42% loss of J3 -carotene, respectively. The a-carotene was much more stable during heating. Only about 10% of the a -carotene was destroyed during 10 minutes of heating at the temperatures studied. The rate of all-trans carotene destruction was highest during the first few minutes of heating. As shown in Figure 1, many cis-Isomers were found after heating at 1210 C for 60 minutes. About 15% of J3 -carotene existed as the 13-cis-isomer. Changes in vitamin A activity according to heat treatment The vitamin A activity In the carrot Juice was calculated using values presented by Bauernfeind (1972) and Zeichmelster (1949). According to these authors, one sixth of the all-trans- J3 -carotene is converted to active vitamin A. Seventy-five per cent of the activity exerted by the all-trans-form Is ascribed to the 13-cls-isomer. All-trans-a-carotene has about half as much vitamin A-activity as J3 -carotene. As the 9-cis-isomer of J3-carotene and ail the cis-isomers of a -carotene exert very little vitamin activity and, furthermore, occur In only smail amounts they will each contribute with less than 0.1 ,ug retinolequivalents per milliliter heated Juice. Therefore, we have decided to include only the all-trans-isomer of a -and J3 -carotene and the 13-cls-Isomer of J3 -carotene in the calculation of vitamin A activity In the juice after different heat treatments. The calculated vitamin A activity in the carrot Juice is reported in Table 2. Mild heat treatment resulted in a decrease In vitamin A activity by a few percentage units only, due to loss of all-trans- J3 -carotene. More intense heat treatment increased the transformation of all-trans- J3 -carotene into Its 13-cls isomer. This gave a rather smail decrease in vitamin A activity. 80
Table 2. Effect of heat treatment on the content of carotenes and vitamin A activity of carrot juice Heat treatment time min. 0
tBmp. C
all-trans a-carotene
all-trans
f} -carotene
13-cis
f} -carotene
/ug/ml Juice 2l!01)
Vitamin A activity retinol eqv·/ug/ml
ca 0 1!0 3!0
16.4
20!0 20!0
88!2 84!3 83!2
10 240
0 80 80
10 60 240
100 100 100
20!0 18!0 17!1
84!0 77!1 76!0
3!0 5!0 7!Z
15.9 14.9 14.9
10 60 Z40
121 1Zl lZI
17!1 16!0 15!1
73!1 64!0 55!1
8!0 10!0 13!0
14.6 13.2 1Z.1
10 30
lZ8 lZ8
15!Z 14!1
60!1 58!1
14!0 13!1
13.0 1Z.4
10 30
135 135
13!Z 1Z!Z
51!Z 45!4
16!1 17!1
11.6 10.6
15.7 15.7
1) Each value represents the mean! SO of 3 experiments.
Very high temperatures, however, resulted in a continuing increase in the amount of 13-cia-isomer, but the increase was nos comparable to the decrease in all-trans- fJ -carotene. Heating at 135 C for 10 minutes caused a 30% decrease in vitamin A activity. The 13-cls-lsomer accounted for almost one fifth of the vitamin activity. If calculation of vitamin A activity ia baaed merely on all-trans- fJ -carotene, then the activity was only corresponded to 58% of the activity in raw carrot Juice. CONCLUSIONS This study shows that low temperature heat treatment of relatively neutral foods, such as carrot, causes only insignificant changes in the carotenes and vitamin A activity. Examples of such heat treatment are cooking in water and pasteurization. However, when using high temperatures, as Is done during heat sterAlizatlon, the loss Is considerable. Conventional sterilization of cans at 121 C will result in a 20% reduction of the vitamin A activity. Very high temperatures, as those used during HTST treatment, enhance the need for proper control of the food process. The results also show the Importance of analyzing all occurring carotenes and their Isomers to get a more reliable vitamin A value fat' foods exposed to heat treatment. 81
There is no information in the literature about the possible anticarcinogen effect of carotenes when they are changed into cis-isomers. ACKNOWLEDGEMENTS This work was supported in part by grants from the Volvo Research Foundation, Goteborg, Sweden. The author thanks Anders Pettersson for excellent technical assistance. REFERENCES Bauernfeind, J.C. (1972). Carotenoid vitamin A precursors and analogs in foods and feeds. J. Agr. Food Chem., 20 (3), 456-473. Hojilla, M.P., Garcia, V.V. and Raymundo, L.C. (1985). Thermal degradation of [j -carotene in carrot juice. ASEAN Food Journal, 1 (4), 157-161. Pettersson, A. and Jonsson, L. (1990). Separation of cis-trans isomers of a-and [j-carotene by adsorption HPLC and identification with diode array detection. Accepted for publ. in Journal of Micronutrient Analysis. Zeichmeister, L. (1962). Cis-trans isomeric carotenoids. Press, London, pp.251.
82
Academic
