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β-carotene losses during different methods of cooking green leafy vegetables in Bangladesh
JOURNAL
OF FOOD COMPOSITION
3,47-53 (1990)
AND ANALYSIS
P-Carotene Losses during Different Methods of Cooking Green Leafy Vegetables in Bangladesh M. MUJIBUR International
Centrefor
RAHMAN,’ Diarrhoeal
M. A. WAHED,
Disease
Research,
GPO
AND M. AKBAR ALI Box 128. Dhaka
1000,
Bangladesh
Received May 19, 1989, and in revised form January 30, 1990 Green leafy vegetables containing p-carotene (provitamin A) may serve as an alternative inexpensive source ofvitamin A for the majority ofthe poor people in developing countries. Previous studies showed conflicting results as to whether considerable amounts of P-carotene in vegetables are destroyed by cooking. The present study was undertaken to assessthe extent of P-carotene loss in vegetables subjected to three traditional methods ofcooking practiced in Bangladesh: Method I: boiling for 7-9 min, followed by frying in oil for 4-6 min in an open pot with stirring and allowing the water portion to evaporate or to be thrown away; Method II: simple boiling for 8-10 min with the lid of the pot on for most of the cooking time; and Method III: placing vegetables on the surface of partially cooked rice until cooking is done and then mashing it into a paste with condiments. Housewife volunteers (8-10) participated in the cooking experiments using each of 6 types of commonly eaten vegetables; only one type was used in Method III. A modified Holden’s method which was used to measure &carotene was statistically comparable to the conventional method of the Association of Official Analytical Chemists. The percentage loss of p-carotene for Method I was 3 1 to 43%; Method II, 11 to 14%; and Method III, 2.3 to 11%. The b-carotene contents of 14 types of commonly eaten green leafy vegetables in Bangladesh ranged between 5400 and 16000 hg/ 100 g. The cause and implication of the high loss of b-carotene by cooking Method I are discussed. Health planners of developing countries should educate people to use cooking methods similar to Methods II and III. o 1990 Academic PKW, IX.
INTRODUCTION
Vitamin A deficiency in Bangladesh is of public health significance (Cohen et al., 1986). Foods such as dairy and meat products containing preformed vitamin A are often too expensive for the majority of people in developing countries. Green leafy vegetables are an alternative, less expensive source of provitamin A or P-carotene (Vinod and Reddy, 1970). It has been shown in developing countries that 82% of the vitamin A in the diet is present in the form of P-carotene (Simpson, 1983; Krause and Mahan, 1984). People in developing countries usually cook green leafy vegetables2 before eating them. The loss of vitamin A after cooking in both Indian and Western food has been shown to be insignificant (Gopalan et al., 1982; NIH, 1972; Ahmed, 1980), but the methods of cooking in these studies were not clearly described. It has also been reported that both retinol and P-carotene remain stable during conventional cooking, although some losses may occur at temperatures above 100°C when butter or palm oil is used for frying (Davidson et al., 1979). The loss of vitamin A can be minimized by reducing the cooking time and lowering the temperature of cooking (Sood and ’ To whom correspondence should be addressed at Biochemistry and Nutrition, 128, Dhaka 1000, Bangladesh. z Hereafter green leafy vegetables will simply be termed vegetables. 41
ICDDR,B, GPO Box
0889-l 575190 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
48
RAHMAN,
WAHED,
AND
AL1
Bhat, 1974). Poor retention of P-carotene has been shown in vegetables after cooking using traditional methods in open vessels which allow the evaporation of water (Rao and Reddy, 1979). In the studies referred to above, the details of the cooking methods were not given. The present study was undertaken to assessthe extent of ,&carotene losses in vegetables cooked by three different methods commonly used in Bangladesh. In addition, the P-carotene contents of 14 commonly eaten vegetables were analyzed. MATERIALS
AND
METHODS
Fourteen types of fresh vegetables (Table 1) were collected on 3 to 14 occasions between October 1983 and October 1984 during the growing season of each vegetable from different markets in Dhaka city. Cooking Method The following methods of cooking green leafy vegetables traditionally practiced in Bangladesh were used in the study. Method I. Tender green leaves of vegetables are chopped into small pieces and washed. They are heated for about 7-9 min in the water adhering to the leaves (extra water is not added), and the fluid which collects at the bottom of the cooking vessel is either thrown away or allowed to evaporate. The vegetables are then transferred to another pot in which salt, onion, garlic, and fresh or dried chilies have been fried in a small quantity of oil, and the mixture is fried for 4-6 min. Open topped earthen, aluminum, or iron vessels are used in this method of cooking, and the vegetables are stirred from time to time. This method is most commonly used in rural Bangladesh. Method II. The chopped and washed pieces of vegetables with quantities of salt, oil, and condiments similar to those used in Method I are cooked by simple boiling for 8- 10 min with the lid of the pot on. The lid is opened two to four times to allow evaporation. This method is practiced by both rural and urban people. Method III. When rice is partially cooked, vegetables such as lau sak (Lugenaria vulgaris) and mishti kumra sak (Cucurbita maxima) are placed with a few fresh chilies on the surface of boiling rice. When the rice is cooked, the vegetables are removed and mashed with raw onion, salt, and mustard oil to make a paste which is eaten with rice. This method is used infrequently by both rural and urban people in only two districts of Bangladesh, Tangail, and Mymensingh. Study Area, Participating
Housewives, and Collection of Samples
The study was performed in Zinjira, a suburb of Dhaka city. Twenty-eight housewives volunteered to cook vegetables by each of the three methods: 10 used Method I, 10 used Method II, and 8 used Method III. The 20 housewives using Methods I and II were accustomed to cook by the respective methods, but 8 were selected and trained to cook lau sak using Method III. Six varieties of most commonly used vegetables were used in this part of the study, namely, la1 sak, mula sak, motor sak, palang sak, pui sak, and lau sak. Only one type was used for each day of investigation in Methods I and II. Only lau sak was used with Method III. The vegetables were purchased by one of the investigators from a local market in the morning of each day of the investigation. After cleaning and wash-
P-CAROTENE
LOSSES
DURING
COOKING
49
ing, the vegetable was left for lo- 15 min on a perforated tray made of bamboo strips to drain off water. A portion of the fresh vegetable was kept in a covered-glass container for duplicate measurements of P-carotene before cooking. The remainder was distributed among the housewives, each receiving a quantity according to the needs of the family. The exact weights of the vegetable before and after cooking were determined in the office of Terre des Hommes, Netherlands, a voluntary organization which is centrally located in the community. The vegetable was then cooked in the home of the participant using one of the three methods of cooking described above. The duration of cooking was noted and afterward about 5 g of the cooked vegetable was collected in a covered-glass container to measure its P-carotene content after cooking. Specimens of each kind of vegetable were processed and analyzed individually without making any pool. Method of P-Carotene Analysis The method of Holden ( 198 1) was modified for measurement of P-carotene. One gram cooked or raw vegetable was chopped and ground rapidly using a mortar and pestle with l-2 g of sand, 5 ml of acetone [Analar grade, British Drug House (BDH)], and 5 ml of petroleum ether (Analar grade; bp, 40-60; BDH). The liquid was poured into a 25-ml volumetric flask (Pyrex) and the extraction process was repeated three times by addition of 5 ml of petroleum ether on each occasion. The extract was made up to the volume (25 ml) with petroleum ether, and the flask was stoppered and stored in the dark until analysis. P-Carotene was extracted from the vegetable extract using a glass column (2.5 X 40 cm) with a sintered glass disk at the bottom holding 8 cm of an adsorbent mixture of alumina (activated aluminium oxide, Brockman, activity II, BDH) and anhydrous sodium sulfate, in equal proportions by weight. Ten milliliters of the extract was placed on the top of the column bed. A deep yellow band containing P-carotene traveled down the column leaving other pigments adsorbed onto the bed. Petroleum ether was added until the eluate turned colorless. The eluate was made up to a final volume of 20 ml with petroleum ether. The absorbance of the eluate was read on a Pye Unicam Sp-500 spectrophotometer, (Pye-Unicam, Cambridge, UK) at 450 nm using a l-cm cell. A 1 pg/ml solution of the P-carotene (Sigma Chemical Co.) was prepared in petroleum ether as a standard which, under the test conditions used, showed an absorbance of 0.25. Standardization of the Present Method in Reference to That ofAOAC The results of five measurements of P-carotene in 1 g of fresh pat sak using the modified Holden (198 1) method were compared with AOAC method, sections 43.014, 43.015, and 43.016 (AOAC, 1984). No significant difference was found between the results of the two methods (present method, mean + SD = 138 + 6.7, coefficient of variation = 4.86%; vs AOAC method, mean + SD = 13 1 ? 4.0, coefficient of variation = 3.08%; P = ~0.069). Similar agreement was found with a low P-carotene vegetable pui sak. Five measurements showed no significant difference (mean + SD = 44 + 1.9, CV% = 4.36 vs mean f SD = 42 * 1.4, CV% = 3.1%; P = ~0.12). Reproducibility The reproducibility of the present test was determined by five estimations on different days of the same quantity of dried powder of a high ,&carotene-containing
50
RAHMAN,
WAHED.
AND
AL1
1
TABLE
ANALYSISOF~CAROTENECONTENTOF~~COMMONLYEATEN GREENLEAFYVEGETABLESINBANGLADESH
Types leafy
of green vegetables
1.
Helencha (Enhydra
sak fluctuans)
2.
Kachu sak (Colocasia
3.
Kalmi sak (Ipomoea reptans)
4.
La1 sak (Amaranthus
Beta-carotene Range mg/lQw
content Mean&SD mg/lOOg
5
7.95-8.66
8.2kO.33
6
7.04-9.70
821.2
6
6.70-9.69
8.3kO.35
11
6.64-9.26
8.OkO.92
5
6.20-6.94
6.6kO.30
9
7.80-9.92
8.9LO.88
14
5.68-7.64
6.3+0.60
5
5.05-5.76
5.4kO.30
Number of samples
antiquorum)
gangeticus)
5.
Lau sak (Lagenaria
6.
Motor
7.
Mula sak (Raphanus
8.
Nunia sak (Portulaca
9.
Palang sak (Spinacia oleracea)
14
5.04-7.69
6.420.97
10.
Pat sak (Chorchorus
12
8.29-11.80
1Okl.l
11.
Pui sak (Basella
alba)
12
4.16-6.74
5.650.69
12.
Sharisha (Brassica
sak campestris)
3
8.44-9.66
8.8kO.74
13.
Sharisha (Brassica
phool (flower) campestris)
6
16.06-17.04
16 + 1.0
14.
Thankuni (Centella
sak asiatica)
3
6.02-6.27
6.lt0.13
vulgaris)
sak
(Pisum
sativus) sativus) oleracea)
capsularis)
kalmi sak (mean k SD = 107 + 4.3, CV% = 4.05%). Thus the present method was statistically comparable to the currently used AOAC method and was simple, quick, economical, and fairly reproducible. The percentage loss of P-carotene after each type of cooking was determined by a comparison of the mean value of P-carotene contents in 10 cooked samples (each measurement was in duplicate) with the mean value of two measurements of the corresponding amounts of fresh vegetable performed on the same day (only 8 cooked samples for Method III).
B-CAROTENE
LOSSES
DURING
TABLE CHANGE
of green vegetables'
Method
Beta-carotene value mg,100g
After cooking Mean+SD
7.85
5.4+0.49
7.19
Palang sak (spinacia oleracea) Pui sak (Basella
Mula sak (Raphanus
(Pisum
sak sativus)
'N=lO
for
Motor
all
Method
Beta-carotene value mg,100g
II Change ($ loss) of Beta-carotene
After
3126.2 (22.8-41.0)
8.17
7.2kO.31
12+3.9 (6.0-17.6)
4.x+1.1
40214.4 (21.8-63.0)
6.89
5.9+0.49
14k4.4 (7.0-21.0)
7.34
4.2kO.41
43+5.5 (34.3-53.0)
5.91
5.320.24
llk4.4 (3.8-17.0)
5.47
3.6+0.7X
33+13.4 (14.6-51.X) 8.97
7.96+0.47
11+5.2 (3.3-19.0)
sativus)
Mean+SD (R="g=)
Cooking
Before cooking
gangeticus)
alba)
I
Change (% loss) Of Beta-carotene
Before cooking
La1 sak (Amaranthus
2
IN P-CAROTENE CONCENTRATION OFGREEN LEAFY VEGETABLES BEFORE AND AFTER DIFFERENT METHODS OF COOKING PRACTICED IN BANGLADESH Cooking
Types leafy
51
COOKING
cooking Mean+SD
Me.3"+SD (R="ge)
vegetables
RESULTS
Table 1 shows the P-carotene contents of 14 types of commonly eaten vegetables in Bangladesh. Values ranged between 5400 and 16,000 pg/ 100 g. Table 2 shows the losses of P-carotene due to cooking by the two main methods used. P-Carotene loss in Method I ranged from 3 1 to 43%; in Method II, from 11 to 14%. In Method III, using only lau sak, the loss of P-carotene was very low, between 2 and 11% and the results are as follows: before cooking, 8230 wg/ 100 g; after cooking, mean + SD = 7721 + 245 pg/lOO g; mean percentage less of P-carotene with mean +- SD = 6.2 + 2.9 (range, 2.3 to 11.2%). DISCUSSION
The wide range in P-carotene contents of the 14 types of vegetables (Table 1) is most likely a function of different methods of cultivation with their different handling conditions (Bureau and Bushway, 1986) since the observed variation is larger than that of our analytical method. Whether a-carotene was concurrently eluted from the leafy vegetables by the modified Holden method of extraction was not checked. However, if any a-carotene was present, it must have been at a negligible level since it did not disturb the determination of P-carotene by spectrophotometry. Bureau and Bushway (1986) showed-by the very highly sensitive method of HPLC-that the a-carotene content of spinach is below the level of detection. &Carotene from vegetable sources is available to human systems as (vitamin A) retinol. Six micrograms of P-carotene is equivalent to 1 pg of retinol (Simpson, 1983). Accordingly, 30-50 g of a vegetable of high p-carotene content is sufficient to meet the daily allowance (a minimum of 400 pg of retinol equivalent) for a preschool child
52
RAHMAN,
WAHED,
AND AL1
(Begum and Pereira, 1977; Krause and Mahan, 1984). To our knowledge there is no dependable data on how much leafy vegetable a Bangladeshi preschool child consumes on the average. If we estimate that the quantity is 40 g and calculate in terms of la1 sak, the p-carotene content in 40 g fresh la1 sak would be 32 18 pg, which is equivalent to 536 pg of retinol-sufficient to meet the minimum daily requirement of vitamin A for a preschool child. It has been shown in the present study that the mean loss in P-carotene in a vegetable after cooking is about 37% for Method I, 12% for Method II, and 6% for Method III. We believe that the destruction of vitamin A by Method II was less because the cooking pot remained covered, there was no separate frying in oil, there was less stirring, and the cooking time was shorter. This explanation is in keeping with the findings of Proudfit ( 1965) Davidson et al. ( 1979) Sood and Bhat (1974) that some loss of vitamin A may occur at temperatures above 100°C when butter or palm oil is used for frying and the vegetable is stirred in an open pot while cooking. On the basis of the results of our study we recommend that health planners of developing countries educate their people to consume daily requisite quantities of green leafy vegetables (sak) using cooking methods similar to Methods II and III. ACKNOWLEDGMENTS This research was supported by the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B). ICDDR,B is supported by countries and agencies which share its concern about the impact of diarrhoeal diseases on the developing world. Current major donors giving assistance to ICDDR,B are: The Aga Khan Foundation, Arab Gulf Fund, Australia, Bangladesh, Belgium, Canadian International Development Agency (CIDA), Danish International Development Agency (DANIDA), France, the Ford Foundation, Japan, Netherlands, Norwegian Agency for International Development (NORAD), Saudi Arabia, Switzerland (SDC), United Kingdom, United Nations Development Programme (UNDP), United Nations Children’s Fund (UNICEF), World Bank, World Health Organization (WHO), World University Services of Canada (WUSC), United Nations Capital Development Fund (UNCDF), and United States Agency for International Development (USAID). We are grateful to Dr. Zafar Ullah Sikder, Medical Officer, Terre Des Hommes Netherlands, Zinjira, Dhaka for allowing the use of facilities in carrying out the investigation in the community, to Dr. Ayesha Mollah, Dr. M. Mujibur Rahman, Dr. David A. Sack and Professor Roger Eeckels for their keen interest and encouragement during this work. We also acknowledge the assistance of Dr. Andrew Hall and Dr. A. Ahmed for manuscript review, and Mr. Shafiqul Islam Khan for his assistance in the laboratory.
REFERENCES AHMED, K. (1980). Nutritive Value ofBangladeshi Foods. Institute of Nutrition and Food Science. University of Dhaka, Bangladesh. AOAC (1984). Ojicial Methods ofAnalysis (S. Williams, Ed.), 14th ed., p. 834. Association of Official Analytical Chemists, Washington DC. BEGUM, A., AND PEREIRA, S. M. (1977). The beta-carotene of edible green leaves. Trap. Geogr. Med. 29, 47-50. BUREAU, J. L., AND BUSHWAY, R. J. (1986). HPLC determination of carotenoids in fruits and vegetables in the United States. J. Food Sci. 51, 128-l 30. COHEN, C., JALIL, M. A., RAHMAN, H., LEEMHUIS, DE REGT, E., SPRAGUE, J., AND MITRA, M. (1986). Blinding malnutrition in rural Bangladesh. J. Trap. Pediatr. 32,73-78. DAVIDSON, S., PASSMORE,R., BROCK, J. F., AND TRUSWELL, A. S. (1979). Human Nutrition and Dietics, 7th ed., pp. 117-12 I. Churchill Livingstone, Edinburg, London. GOPALAN, C., RAMASASTRI, B. V., AND BALASUBRAMANIAN, S. C. (1982). Nutrition Value oj‘ Indian Foods, pp. 37-42. National Institute of Nutrition, Indian Council of Research, Hyderabad, India.
P-CAROTENE
LOSSES DURING
COOKING
53
HOLDEN, M. (198 1). A Rapid and Simple Methodfor Determining Carotene in Plant Materials, Bulletin No. 22. Xerophthalmia Club, UK. KRAUSE, M. V., AND MAHAN, L. K. (1984). Food, Nutrition and Diet Therapy, 7th ed., pp. 102-105. Saunders, Philadelphia/London. NIH (1972). Food Composition Tablefor Use in East Asia. Department of Health Education and Welfare, Maryland. PROUDF~T,F. T. (1965). Normal and Therapeutic Nutrition, 12th ed., pp. 115-l 19. Oxford & IBH Publ. Co., Calcutta, India. RAO, S., AND REDDY, U. M. (1979). Proceedings, 1st. Indian Conv. Food Sci. Technol. 11, 12, A871. ROBINSON, C. H., AND LAWER, M. R. (1982). Normal and Therapeutic Nutrition, 16th ed., pp. 187-193. Macmillan, New York. SIMPSON, K. L. (1983). Relative value of carotenoids as precursors of vitamin A. Proc. Nutr. Sot. 42, 815. SOOD, S., AND BHAT, C. M. ( 1974). Food Sci. Technol. 11, 13 1. VINOD, R. L., AND VINODINY, R. (1970). Absorption of p-carotene from green leafy vegetables in undernourished children. Amer. J. Clin. Nutr. 23. 110-l 13.
OF FOOD COMPOSITION
3,47-53 (1990)
AND ANALYSIS
P-Carotene Losses during Different Methods of Cooking Green Leafy Vegetables in Bangladesh M. MUJIBUR International
Centrefor
RAHMAN,’ Diarrhoeal
M. A. WAHED,
Disease
Research,
GPO
AND M. AKBAR ALI Box 128. Dhaka
1000,
Bangladesh
Received May 19, 1989, and in revised form January 30, 1990 Green leafy vegetables containing p-carotene (provitamin A) may serve as an alternative inexpensive source ofvitamin A for the majority ofthe poor people in developing countries. Previous studies showed conflicting results as to whether considerable amounts of P-carotene in vegetables are destroyed by cooking. The present study was undertaken to assessthe extent of P-carotene loss in vegetables subjected to three traditional methods ofcooking practiced in Bangladesh: Method I: boiling for 7-9 min, followed by frying in oil for 4-6 min in an open pot with stirring and allowing the water portion to evaporate or to be thrown away; Method II: simple boiling for 8-10 min with the lid of the pot on for most of the cooking time; and Method III: placing vegetables on the surface of partially cooked rice until cooking is done and then mashing it into a paste with condiments. Housewife volunteers (8-10) participated in the cooking experiments using each of 6 types of commonly eaten vegetables; only one type was used in Method III. A modified Holden’s method which was used to measure &carotene was statistically comparable to the conventional method of the Association of Official Analytical Chemists. The percentage loss of p-carotene for Method I was 3 1 to 43%; Method II, 11 to 14%; and Method III, 2.3 to 11%. The b-carotene contents of 14 types of commonly eaten green leafy vegetables in Bangladesh ranged between 5400 and 16000 hg/ 100 g. The cause and implication of the high loss of b-carotene by cooking Method I are discussed. Health planners of developing countries should educate people to use cooking methods similar to Methods II and III. o 1990 Academic PKW, IX.
INTRODUCTION
Vitamin A deficiency in Bangladesh is of public health significance (Cohen et al., 1986). Foods such as dairy and meat products containing preformed vitamin A are often too expensive for the majority of people in developing countries. Green leafy vegetables are an alternative, less expensive source of provitamin A or P-carotene (Vinod and Reddy, 1970). It has been shown in developing countries that 82% of the vitamin A in the diet is present in the form of P-carotene (Simpson, 1983; Krause and Mahan, 1984). People in developing countries usually cook green leafy vegetables2 before eating them. The loss of vitamin A after cooking in both Indian and Western food has been shown to be insignificant (Gopalan et al., 1982; NIH, 1972; Ahmed, 1980), but the methods of cooking in these studies were not clearly described. It has also been reported that both retinol and P-carotene remain stable during conventional cooking, although some losses may occur at temperatures above 100°C when butter or palm oil is used for frying (Davidson et al., 1979). The loss of vitamin A can be minimized by reducing the cooking time and lowering the temperature of cooking (Sood and ’ To whom correspondence should be addressed at Biochemistry and Nutrition, 128, Dhaka 1000, Bangladesh. z Hereafter green leafy vegetables will simply be termed vegetables. 41
ICDDR,B, GPO Box
0889-l 575190 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
48
RAHMAN,
WAHED,
AND
AL1
Bhat, 1974). Poor retention of P-carotene has been shown in vegetables after cooking using traditional methods in open vessels which allow the evaporation of water (Rao and Reddy, 1979). In the studies referred to above, the details of the cooking methods were not given. The present study was undertaken to assessthe extent of ,&carotene losses in vegetables cooked by three different methods commonly used in Bangladesh. In addition, the P-carotene contents of 14 commonly eaten vegetables were analyzed. MATERIALS
AND
METHODS
Fourteen types of fresh vegetables (Table 1) were collected on 3 to 14 occasions between October 1983 and October 1984 during the growing season of each vegetable from different markets in Dhaka city. Cooking Method The following methods of cooking green leafy vegetables traditionally practiced in Bangladesh were used in the study. Method I. Tender green leaves of vegetables are chopped into small pieces and washed. They are heated for about 7-9 min in the water adhering to the leaves (extra water is not added), and the fluid which collects at the bottom of the cooking vessel is either thrown away or allowed to evaporate. The vegetables are then transferred to another pot in which salt, onion, garlic, and fresh or dried chilies have been fried in a small quantity of oil, and the mixture is fried for 4-6 min. Open topped earthen, aluminum, or iron vessels are used in this method of cooking, and the vegetables are stirred from time to time. This method is most commonly used in rural Bangladesh. Method II. The chopped and washed pieces of vegetables with quantities of salt, oil, and condiments similar to those used in Method I are cooked by simple boiling for 8- 10 min with the lid of the pot on. The lid is opened two to four times to allow evaporation. This method is practiced by both rural and urban people. Method III. When rice is partially cooked, vegetables such as lau sak (Lugenaria vulgaris) and mishti kumra sak (Cucurbita maxima) are placed with a few fresh chilies on the surface of boiling rice. When the rice is cooked, the vegetables are removed and mashed with raw onion, salt, and mustard oil to make a paste which is eaten with rice. This method is used infrequently by both rural and urban people in only two districts of Bangladesh, Tangail, and Mymensingh. Study Area, Participating
Housewives, and Collection of Samples
The study was performed in Zinjira, a suburb of Dhaka city. Twenty-eight housewives volunteered to cook vegetables by each of the three methods: 10 used Method I, 10 used Method II, and 8 used Method III. The 20 housewives using Methods I and II were accustomed to cook by the respective methods, but 8 were selected and trained to cook lau sak using Method III. Six varieties of most commonly used vegetables were used in this part of the study, namely, la1 sak, mula sak, motor sak, palang sak, pui sak, and lau sak. Only one type was used for each day of investigation in Methods I and II. Only lau sak was used with Method III. The vegetables were purchased by one of the investigators from a local market in the morning of each day of the investigation. After cleaning and wash-
P-CAROTENE
LOSSES
DURING
COOKING
49
ing, the vegetable was left for lo- 15 min on a perforated tray made of bamboo strips to drain off water. A portion of the fresh vegetable was kept in a covered-glass container for duplicate measurements of P-carotene before cooking. The remainder was distributed among the housewives, each receiving a quantity according to the needs of the family. The exact weights of the vegetable before and after cooking were determined in the office of Terre des Hommes, Netherlands, a voluntary organization which is centrally located in the community. The vegetable was then cooked in the home of the participant using one of the three methods of cooking described above. The duration of cooking was noted and afterward about 5 g of the cooked vegetable was collected in a covered-glass container to measure its P-carotene content after cooking. Specimens of each kind of vegetable were processed and analyzed individually without making any pool. Method of P-Carotene Analysis The method of Holden ( 198 1) was modified for measurement of P-carotene. One gram cooked or raw vegetable was chopped and ground rapidly using a mortar and pestle with l-2 g of sand, 5 ml of acetone [Analar grade, British Drug House (BDH)], and 5 ml of petroleum ether (Analar grade; bp, 40-60; BDH). The liquid was poured into a 25-ml volumetric flask (Pyrex) and the extraction process was repeated three times by addition of 5 ml of petroleum ether on each occasion. The extract was made up to the volume (25 ml) with petroleum ether, and the flask was stoppered and stored in the dark until analysis. P-Carotene was extracted from the vegetable extract using a glass column (2.5 X 40 cm) with a sintered glass disk at the bottom holding 8 cm of an adsorbent mixture of alumina (activated aluminium oxide, Brockman, activity II, BDH) and anhydrous sodium sulfate, in equal proportions by weight. Ten milliliters of the extract was placed on the top of the column bed. A deep yellow band containing P-carotene traveled down the column leaving other pigments adsorbed onto the bed. Petroleum ether was added until the eluate turned colorless. The eluate was made up to a final volume of 20 ml with petroleum ether. The absorbance of the eluate was read on a Pye Unicam Sp-500 spectrophotometer, (Pye-Unicam, Cambridge, UK) at 450 nm using a l-cm cell. A 1 pg/ml solution of the P-carotene (Sigma Chemical Co.) was prepared in petroleum ether as a standard which, under the test conditions used, showed an absorbance of 0.25. Standardization of the Present Method in Reference to That ofAOAC The results of five measurements of P-carotene in 1 g of fresh pat sak using the modified Holden (198 1) method were compared with AOAC method, sections 43.014, 43.015, and 43.016 (AOAC, 1984). No significant difference was found between the results of the two methods (present method, mean + SD = 138 + 6.7, coefficient of variation = 4.86%; vs AOAC method, mean + SD = 13 1 ? 4.0, coefficient of variation = 3.08%; P = ~0.069). Similar agreement was found with a low P-carotene vegetable pui sak. Five measurements showed no significant difference (mean + SD = 44 + 1.9, CV% = 4.36 vs mean f SD = 42 * 1.4, CV% = 3.1%; P = ~0.12). Reproducibility The reproducibility of the present test was determined by five estimations on different days of the same quantity of dried powder of a high ,&carotene-containing
50
RAHMAN,
WAHED.
AND
AL1
1
TABLE
ANALYSISOF~CAROTENECONTENTOF~~COMMONLYEATEN GREENLEAFYVEGETABLESINBANGLADESH
Types leafy
of green vegetables
1.
Helencha (Enhydra
sak fluctuans)
2.
Kachu sak (Colocasia
3.
Kalmi sak (Ipomoea reptans)
4.
La1 sak (Amaranthus
Beta-carotene Range mg/lQw
content Mean&SD mg/lOOg
5
7.95-8.66
8.2kO.33
6
7.04-9.70
821.2
6
6.70-9.69
8.3kO.35
11
6.64-9.26
8.OkO.92
5
6.20-6.94
6.6kO.30
9
7.80-9.92
8.9LO.88
14
5.68-7.64
6.3+0.60
5
5.05-5.76
5.4kO.30
Number of samples
antiquorum)
gangeticus)
5.
Lau sak (Lagenaria
6.
Motor
7.
Mula sak (Raphanus
8.
Nunia sak (Portulaca
9.
Palang sak (Spinacia oleracea)
14
5.04-7.69
6.420.97
10.
Pat sak (Chorchorus
12
8.29-11.80
1Okl.l
11.
Pui sak (Basella
alba)
12
4.16-6.74
5.650.69
12.
Sharisha (Brassica
sak campestris)
3
8.44-9.66
8.8kO.74
13.
Sharisha (Brassica
phool (flower) campestris)
6
16.06-17.04
16 + 1.0
14.
Thankuni (Centella
sak asiatica)
3
6.02-6.27
6.lt0.13
vulgaris)
sak
(Pisum
sativus) sativus) oleracea)
capsularis)
kalmi sak (mean k SD = 107 + 4.3, CV% = 4.05%). Thus the present method was statistically comparable to the currently used AOAC method and was simple, quick, economical, and fairly reproducible. The percentage loss of P-carotene after each type of cooking was determined by a comparison of the mean value of P-carotene contents in 10 cooked samples (each measurement was in duplicate) with the mean value of two measurements of the corresponding amounts of fresh vegetable performed on the same day (only 8 cooked samples for Method III).
B-CAROTENE
LOSSES
DURING
TABLE CHANGE
of green vegetables'
Method
Beta-carotene value mg,100g
After cooking Mean+SD
7.85
5.4+0.49
7.19
Palang sak (spinacia oleracea) Pui sak (Basella
Mula sak (Raphanus
(Pisum
sak sativus)
'N=lO
for
Motor
all
Method
Beta-carotene value mg,100g
II Change ($ loss) of Beta-carotene
After
3126.2 (22.8-41.0)
8.17
7.2kO.31
12+3.9 (6.0-17.6)
4.x+1.1
40214.4 (21.8-63.0)
6.89
5.9+0.49
14k4.4 (7.0-21.0)
7.34
4.2kO.41
43+5.5 (34.3-53.0)
5.91
5.320.24
llk4.4 (3.8-17.0)
5.47
3.6+0.7X
33+13.4 (14.6-51.X) 8.97
7.96+0.47
11+5.2 (3.3-19.0)
sativus)
Mean+SD (R="g=)
Cooking
Before cooking
gangeticus)
alba)
I
Change (% loss) Of Beta-carotene
Before cooking
La1 sak (Amaranthus
2
IN P-CAROTENE CONCENTRATION OFGREEN LEAFY VEGETABLES BEFORE AND AFTER DIFFERENT METHODS OF COOKING PRACTICED IN BANGLADESH Cooking
Types leafy
51
COOKING
cooking Mean+SD
Me.3"+SD (R="ge)
vegetables
RESULTS
Table 1 shows the P-carotene contents of 14 types of commonly eaten vegetables in Bangladesh. Values ranged between 5400 and 16,000 pg/ 100 g. Table 2 shows the losses of P-carotene due to cooking by the two main methods used. P-Carotene loss in Method I ranged from 3 1 to 43%; in Method II, from 11 to 14%. In Method III, using only lau sak, the loss of P-carotene was very low, between 2 and 11% and the results are as follows: before cooking, 8230 wg/ 100 g; after cooking, mean + SD = 7721 + 245 pg/lOO g; mean percentage less of P-carotene with mean +- SD = 6.2 + 2.9 (range, 2.3 to 11.2%). DISCUSSION
The wide range in P-carotene contents of the 14 types of vegetables (Table 1) is most likely a function of different methods of cultivation with their different handling conditions (Bureau and Bushway, 1986) since the observed variation is larger than that of our analytical method. Whether a-carotene was concurrently eluted from the leafy vegetables by the modified Holden method of extraction was not checked. However, if any a-carotene was present, it must have been at a negligible level since it did not disturb the determination of P-carotene by spectrophotometry. Bureau and Bushway (1986) showed-by the very highly sensitive method of HPLC-that the a-carotene content of spinach is below the level of detection. &Carotene from vegetable sources is available to human systems as (vitamin A) retinol. Six micrograms of P-carotene is equivalent to 1 pg of retinol (Simpson, 1983). Accordingly, 30-50 g of a vegetable of high p-carotene content is sufficient to meet the daily allowance (a minimum of 400 pg of retinol equivalent) for a preschool child
52
RAHMAN,
WAHED,
AND AL1
(Begum and Pereira, 1977; Krause and Mahan, 1984). To our knowledge there is no dependable data on how much leafy vegetable a Bangladeshi preschool child consumes on the average. If we estimate that the quantity is 40 g and calculate in terms of la1 sak, the p-carotene content in 40 g fresh la1 sak would be 32 18 pg, which is equivalent to 536 pg of retinol-sufficient to meet the minimum daily requirement of vitamin A for a preschool child. It has been shown in the present study that the mean loss in P-carotene in a vegetable after cooking is about 37% for Method I, 12% for Method II, and 6% for Method III. We believe that the destruction of vitamin A by Method II was less because the cooking pot remained covered, there was no separate frying in oil, there was less stirring, and the cooking time was shorter. This explanation is in keeping with the findings of Proudfit ( 1965) Davidson et al. ( 1979) Sood and Bhat (1974) that some loss of vitamin A may occur at temperatures above 100°C when butter or palm oil is used for frying and the vegetable is stirred in an open pot while cooking. On the basis of the results of our study we recommend that health planners of developing countries educate their people to consume daily requisite quantities of green leafy vegetables (sak) using cooking methods similar to Methods II and III. ACKNOWLEDGMENTS This research was supported by the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B). ICDDR,B is supported by countries and agencies which share its concern about the impact of diarrhoeal diseases on the developing world. Current major donors giving assistance to ICDDR,B are: The Aga Khan Foundation, Arab Gulf Fund, Australia, Bangladesh, Belgium, Canadian International Development Agency (CIDA), Danish International Development Agency (DANIDA), France, the Ford Foundation, Japan, Netherlands, Norwegian Agency for International Development (NORAD), Saudi Arabia, Switzerland (SDC), United Kingdom, United Nations Development Programme (UNDP), United Nations Children’s Fund (UNICEF), World Bank, World Health Organization (WHO), World University Services of Canada (WUSC), United Nations Capital Development Fund (UNCDF), and United States Agency for International Development (USAID). We are grateful to Dr. Zafar Ullah Sikder, Medical Officer, Terre Des Hommes Netherlands, Zinjira, Dhaka for allowing the use of facilities in carrying out the investigation in the community, to Dr. Ayesha Mollah, Dr. M. Mujibur Rahman, Dr. David A. Sack and Professor Roger Eeckels for their keen interest and encouragement during this work. We also acknowledge the assistance of Dr. Andrew Hall and Dr. A. Ahmed for manuscript review, and Mr. Shafiqul Islam Khan for his assistance in the laboratory.
REFERENCES AHMED, K. (1980). Nutritive Value ofBangladeshi Foods. Institute of Nutrition and Food Science. University of Dhaka, Bangladesh. AOAC (1984). Ojicial Methods ofAnalysis (S. Williams, Ed.), 14th ed., p. 834. Association of Official Analytical Chemists, Washington DC. BEGUM, A., AND PEREIRA, S. M. (1977). The beta-carotene of edible green leaves. Trap. Geogr. Med. 29, 47-50. BUREAU, J. L., AND BUSHWAY, R. J. (1986). HPLC determination of carotenoids in fruits and vegetables in the United States. J. Food Sci. 51, 128-l 30. COHEN, C., JALIL, M. A., RAHMAN, H., LEEMHUIS, DE REGT, E., SPRAGUE, J., AND MITRA, M. (1986). Blinding malnutrition in rural Bangladesh. J. Trap. Pediatr. 32,73-78. DAVIDSON, S., PASSMORE,R., BROCK, J. F., AND TRUSWELL, A. S. (1979). Human Nutrition and Dietics, 7th ed., pp. 117-12 I. Churchill Livingstone, Edinburg, London. GOPALAN, C., RAMASASTRI, B. V., AND BALASUBRAMANIAN, S. C. (1982). Nutrition Value oj‘ Indian Foods, pp. 37-42. National Institute of Nutrition, Indian Council of Research, Hyderabad, India.
P-CAROTENE
LOSSES DURING
COOKING
53
HOLDEN, M. (198 1). A Rapid and Simple Methodfor Determining Carotene in Plant Materials, Bulletin No. 22. Xerophthalmia Club, UK. KRAUSE, M. V., AND MAHAN, L. K. (1984). Food, Nutrition and Diet Therapy, 7th ed., pp. 102-105. Saunders, Philadelphia/London. NIH (1972). Food Composition Tablefor Use in East Asia. Department of Health Education and Welfare, Maryland. PROUDF~T,F. T. (1965). Normal and Therapeutic Nutrition, 12th ed., pp. 115-l 19. Oxford & IBH Publ. Co., Calcutta, India. RAO, S., AND REDDY, U. M. (1979). Proceedings, 1st. Indian Conv. Food Sci. Technol. 11, 12, A871. ROBINSON, C. H., AND LAWER, M. R. (1982). Normal and Therapeutic Nutrition, 16th ed., pp. 187-193. Macmillan, New York. SIMPSON, K. L. (1983). Relative value of carotenoids as precursors of vitamin A. Proc. Nutr. Sot. 42, 815. SOOD, S., AND BHAT, C. M. ( 1974). Food Sci. Technol. 11, 13 1. VINOD, R. L., AND VINODINY, R. (1970). Absorption of p-carotene from green leafy vegetables in undernourished children. Amer. J. Clin. Nutr. 23. 110-l 13.