CONTENT OF CAROTENOIDS AND CHLOROPHYLL PIGMENTS IN KALE (BRASSICA OLERACEA L. VAR. ACEPHALA) DEPENDING ON THE CULTIVAR AND THE HARVEST DATE

Anna Korus¹, Waldemar Kmiecik^2
1 Department of Fruit, Vegetable and Mushroom Processing, University of Agriculture in Cracow, Poland
² Department of Raw Materials and Processing of Fruit and Vegetables, Agricultural University of Cracow, Poland

ABSTRACT

The levels of total carotenoids, beta-carotene, chlorophyll a and b and total chlorophylls were determined in kale leaves of the cultivars Winterbor F₁, Redbor F₁ and Srednio Wysoki Zielony Kędzierzawy. The investigation was carried out in two successive years; in each year the raw material was obtained in three harvests carried out 10, 14 and 18 weeks after planting young kale plants in the field. During the whole period of the investigation the average content of carotenoids in 100 g fresh matter of kale was 23.1-26.0 mg; that of beta-carotene 3.80-4.53 mg; and that of total chlorophylls 118-145 mg. The ratio of chlorophyll a to b was 1:0.41-1:0.42. The greatest content of all the analysed constituents was found in the cultivar Winterbor F₁. During the entire period of the investigation there was practically no difference in carotenoids content between the first and second harvests, the content of beta-carotene increasing by 27% and that of chlorophylls by 4%. In the third harvest an 18% increase was found in the content of carotenoids and a 48% increase in beta-carotene, the content of chlorophylls decreasing by 11%. The proportion of beta-carotene in the level of carotenoids varied from 15% to 19% depending on the cultivar and the time of harvest.

Key words: beta-carotene, carotenoids, chlorophylls, kale, time of harvest .

INTRODUCTION

Awareness of the significance of vegetable consumption can play an important role in maintaining health and reducing the risk of illness [1]. In particular, increased vegetable consumption helps reduce the risk of cancer [2]. That is why vegetables as well as fruits are widely recommended as healthy food [3]. Vegetables are also a valuable part of the diet owing to their nutritive values [4]. They are a low-energy food containing small amounts of carbohydrates and fats and high proportion of dietary fibre, minerals and vitamins. In the selection of products for consumption, however, consumers pay attention above all to sensory traits. According to Lisiewska et al. [5], it is chiefly the colours, including the level of chlorophyll pigments and their ratios, that have a decisive bearing on the attractiveness of the raw material. Tijskens et al. [6] also stress that colour is the most important trait used in consumer evaluation of product quality, playing a decisive role in the acceptability of the product.

Chlorophyll pigments are accompanied by carotenoid ones, whose colour, from yellow to orange, is frequently masked by green chlorophylls [7]. Carotenoids are important nutritive and biological constituents common in the plant world. Important sources of carotenoids are green vegetables, with kale as a predominant species [1]. Of the carotenoids, beta-carotene is the most valuable, showing the activity of pro-vitamin A [8]. This compound is classed among a group of constituents with antioxidative properties, which play a protective role in the human body.

The aim of the presented investigation was to determine the level of chlorophyll and carotenoid pigments in kale leaves depending on the cultivar and the harvest date.

MATERIAL AND METHODS

Material
The investigated materials were fresh leaves of kale obtained in three harvests:

• first (I) – 10 weeks after planting seedlings in the field,

• second (II) – 14 weeks after planting seedlings in the field,

• third ( III) – 18 weeks after planting seedlings in the field.

Two hybrid cultivars produced by Dutch Bejo Zaden b.d. breeders: Winterbor F₁ and Redbor F₁ and the cultivar Srednio Wysoki Zielony Kędzierzawy produced by Polish breeders were used in the experiment.

Kale was grown in 2003 and 2004 in an experimental field lying in the western outskirts of Krakow on soil of good horticultural quality characterized by the following traits during the first and second years of the investigation: pH in H₂O 7.08 and 7.09; humus 1.96 and 1.55%; nitrogen 25 and 21 mg/dm³; phosphorus 81 and 46 mg/dm³; potassium 200 and 90 mg/dm³ and calcium 1200 and 960 mg/dm³. The kale was grown in the third year after manure fertilization, with leguminous vegetables preceding kale and a mixture of green manure plants used as a forecrop in the year of the experiment. Mineral fertilizers were applied, and the fertility of the soil and the nutritional requirements of the crop being taken into account, the following doses of fertilizer were used in the years of the investigation:

• Nitrogen before planting seedlings 40 kg N/ha, and 50 kg N/ha as a side dressing in the form of 34% ammonium nitrate;

• Phosphorus before planting seedlings 80 and 100 kg P₂O₅ in the form of 46% triple superphosphate (50% of the dose during ploughing and 50% during harrowing);

• Potassium before planting seedlings 150 and 200 kg K₂O in the form of 50% potassium sulfate (50% of the dose during ploughing and 50% during harrowing).

Kale seeds were sown in boxes on June 3. The seedlings were planted in the field at the stage of 3-4 leaves, i.e. on June 25 in 2003 and on June 28 in 2004 at a spacing of 50 cm x 50 cm. The cultivation measures during plant growth included mechanical weed control, side dressing with nitrogen and protection against diseases and pests as necessary.

The first harvest was carried out when the number of leaves of commercial value per plant was 25-30; this was observed during the first five days of September in both years of the experiment. The second harvest was carried out after four weeks and the third after a further four weeks. During harvest whole plants were cut, non-marketable yellowing leaves, usually growing at the base of the plant, and leaves less than 10 cm in length from the apical bud of the plant being discarded.

Methods
From marketable leaves, i.e. leaves of good colour, undamaged by diseases or pests, the main rib was removed and the material for analyses of chemical composition was sampled in four replications of 1000 g each.

Total carotenoids and chlorophylls was determined according to the methods of Lichtenthaler and Buschmann [9, 10]. The method consisted of repeated acetone extraction, until obtained colourless residue, with a pestle and mortar and filtered over cotton pad. The extracts were made up to 50 ml with acetone. The concentration of carotenoids was measured at 470 nm, chlorophyll a at 662 nm, chlorophyll b at 645 nm in a Shimadzu UV 160A spectrophotometer.

Acetone extracts prepared for carotenoid determinations were used in the extraction of beta-carotene. Beta-carotene analysis consisted of the extraction procedures of pigment, followed by liquid/liquid partitioning with hexane, concentration and column chromatography [11]. Hexane extracts were filtered over anhydrous sodium sulphate on filter paper (Whatman No.1 equivalent) and was made up to a known volume. The extract was 10 fold concentrated by evaporation and loaded onto the column. Columns (150x10 mm) were packed with aluminium oxide to a length of 100 mm and covered with a 10 mm of anhydrous sodium sulphite then were washed with hexane contained 1% of acetone. The orange coloured eluent contained beta-carotene was collected to a volumetric flask. The concentration of beta-carotene was measured at 450 nm in a described above Shimadzu UV 160A spectrophotometer and compared with beta-carotene reference standard. All the analyses were conducted in four replications, each in two parallel samples.

The results were statistically verified on the basis of analysis of variance, using the Snedecor F test and the Student T test, the least significant difference (LSD) being calculated for the probability level of p=0.01. The computer program was Statistica ver.6.1.

RESULTS AND DISCUSSION

Carotenoids are compounds characterized by strong antioxidative properties [7]. Leaf vegetables are a rich source of carotenoids [12]. In kale leaves the average content of carotenoids in 100 g fresh matter was 26.0 mg in the cultivar Winterbor F₁, 23.4 mg in the cultivar Srednio Wysoki Zielony Kędzierzawy and 23.1 mg in the cultivar Redbor F₁ in the two years of the investigation (Table 1). Of the cultivars compared, a significantly greater content of carotenoids was found in the cultivar Winterbor F₁ in the first year of the experiment, while in the following year differences between the cultivars in the content of these compounds were non-significant.

The recorded quantities of carotenoids exceeded the level of 12.7-18.7 mg/100 g reported by Mercadante and Rodriguez-Anaya [13], but were much lower than the content of 77 mg/100g given by Khachik et al. [14]. In comparison with other cabbage and leaf vegetables the content of carotenoids in kale exceeded that in broccoli [15], Brussels sprouts, green lettuce or spinach [16] but was similar to that in parsley [17] and in dill [5]. Saric et al. [18] found the greatest content of carotenoids in the oldest leaf blades of cabbage. In the presented experiment the authors also observed a tendency for the carotenoids content to increase with the growth of the plant, particularly in the first year of the investigation. A mean 40% increase in the content of pigments was recorded in the period between the first and third harvest date. In the second year of the experiment, differences in the level of carotenoids between the harvest dates were observed in the cultivar Redbor F₁; the level remained stable in the cultivar Œrednio Wysoki Zielony Kędzierzawy, while in the cultivar Winterbor F₁ a 13% increase was noted between the first and the third harvest.

Bhaskarachary et al. [19] investigated the content of carotenoids in 17 species of leaf vegetables and found that beta-carotene was the dominant carotenoid in the analysed vegetables. In 100 g fresh matter of the investigated cultivars of kale the mean content of beta-carotene was 3.80-4.53 mg for the two years, constituting 16-19% of the carotenoids, which is similar to the data given by Mercadante and Rodriguez-Amaya [13], Azevedo and Rodriguez-Amaya [3] and Kurlich et al. [20] (Table 2).

The content of this pigment in 100 g fresh matter of leaf vegetables varied from 3.5 to 10.6 mg in parsley [21]; 4.43-5.23 mg in dill [22] and 3.00 mg and 4.62 mg in New Zealand and common spinach respectively [23]. A smaller content was found in broccoli, cauliflower and Brussels sprouts [20]. During the entire experiment the cultivars Winterbor F₁ and Redbor F₁ did not significantly differ in the level of beta-carotene, while in the cultivar Srednio Wysoki Zielony Kędzierzawy the content of this constituent was smaller. In 2003 the content of beta-carotene in 100 g fresh matter was 4.74 in the cultivar Winterbor F₁, being comparable with the amount found in the cultivar Redbor F₁, but 33% greater than in the cultivar Œrednio Wysoki Zielony Kędzierzawy. In the following year the highest level of this constituent was also found in the cultivar Winterbor F₁, exceeding the cultivars Redbor F₁ and Srednio Wysoki Zielony Kędzierzawy by 5% and 7% respectively. Kopsell et al. [24] noted 8.7-12.3 mg beta-carotene in 100 g fresh matter depending on the amount of fertilizer applied. According to the above authors, the cultivar Redbor F₁ contained 10% more beta-carotene than the cultivar Winterbor F₁.

In all the cultivars the content of beta-carotene increased with the growth of the plant. In comparison with the first harvest, the average increase in the level of beta-carotene in the second harvest was 39% in 2003 and 16% in 2004; in the third harvest the increases were 55% and 42% respectively. Azevedo and Rodriguez-Amaya [3] also found more beta-carotene in more mature leaves of kale. However, Drews et al. [25, 26] noted lower beta-carotene content in the course of maturation of butter head and iceberg lettuce.

Chlorophyll pigments are classed among the least stable natural pigments, the yellowing of green plants being associated with their degradation [27]. Kale is one of the vegetables with a relatively high content of chlorophylls, similar to that in dill [28] and parsley leaves [29] and exceeding that in spinach [23]. The total content of chlorophyll pigments varied in the different years: the highest was found in the cultivar Winterbor F₁ – 132-158 mg/100 g; it was statistically lower in the cultivar Redbor F₁ – 127-146 mg; and the lowest levels were seen in the cultivar Srednio Wysoki Zielony Kędzierzawy – 117-118 mg; in both years differences between the cultivars did not exceed 20% (Table 3).

In 2003 the greatest statistically significant content of chlorophylls was found in leaves from the second harvest: 151 mg/100 g on average; in the following year the highest content of pigments was recorded in the first and second harvests, being roughly equal at 133 mg/100 g on average. Analyses of the chlorophyll content according to the time of harvest showed an 11% increase in the level of these pigments in the second harvest compared with the first in 2003, and a 2% increase in 2004 in the cultivar Winterbor F₁; in the cultivar Srednio Wysoki Zielony Kędzierzawy a 24% increase was recorded in 2003 and one of 2% in 2004, followed by decreases of 2-4% and 37-40% respectively. In the leaves of the cultivar Redbor F₁ the level of chlorophylls fell by 7% on average in the second harvest of the first year and did not change in the third; in the second year the amounts of pigments fell by 17% in the third harvest compared with the first. Kopsell et al. [24] found 270-488 mg chlorophylls in 100 g and Khachik et al. [14] 186 mg/100 g. Leja et al. [30] investigated three stages of maturity of broccoli florets and observed that chlorophyll content decreased during ripening.

The quantitative ratio of chlorophylls a and b shows a considerable preponderance of chlorophyll a (Table 4). This phenomenon should be regarded as favourable since chlorophyll a is characterized by a more intense green colour significantly affecting the coloration of the raw material.

In both years of the present experiment, the average ratio of chlorophyll a to chlorophyll b was 1: 0.42 in the cultivar Winterbor F₁, 1:0.44 in Redbor F₁ and 1:0.41 in Srednio Wysoki Zielony Kędzierzawy, this exceeding the figures given by Kopsell et al. [24], who found an average ratio of 1:0.29 for the cultivar Winterbor F₁ and 1:0.39 for the cultivar Redbor F₁. However, Lisiewska et al. [28] quoted a ratio of 1:0.34 for dill and Lisiewska and Kmiecik [31] 1:0.27 for chive. As the period of growth progressed in the first year of the investigation, the content of chlorophyll b decreased from the first to the third harvest, while the content of chlorophyll a was virtually unchanged, bringing about a decrease in the chlorophyll a:b ratio from 1:0.52 to 1:0.39, on average. In the second year the ratio for all harvest dates remained more or less constant at 1:0.40, on average.

CONCLUSIONS

Kale leaves were characterized by a high content of carotenoids, beta-carotene and chlorophylls. The compared cultivars differed in the level of the analysed pigments. Compared with Redbor F₁ and Srednio Wysoki Zielony Kędzierzawy over the two years, the leaves of Winterbor F₁ contained, on average, 13% and 11% more carotenoids; 3% and 19% more beta-carotene; and 6% and 23% more total chlorophylls respectively. In most samples the level of carotenoids and beta-carotene increased right through the period of growth, while that of chlorophylls increased in the second harvest but later fell. The most abundant source of carotenoids and beta-carotene was found in leaves harvested after 18 weeks of growth. Compared with 10-week-old plants, they contained 18% more carotenoids and 48% more beta-carotene, while compared with 14-week-old plants, the respective increases were 19% and 16%. The highest level of chlorophylls was noted in leaves from the second harvest: the content of these pigments increased by an average of 4% in both years compared with leaves from the first harvest date, and by 17% compared with the third harvest.

REFERENCES

1. Kalt W., 2005. Effects of production and processing factors on major fruit and vegetable antioxidants. J. Food Sci. 70, 11-19.

2. Steinmetz K. A., Potter J. D., 1991. Vegetables, fruit and cancer. I. Epidemiology. Cancer Cause Control. 2, 325-357.

3. Azevado, C. H.-de, Rodriguez-Amaya, D. B., 2005. Carotenoid composition of kale as influenced by maturity, season and minimal processing. J. Sci. Food Agric. 85,591-597.

4. Ramesh M. N., 2000. The performance evaluation of a continuous vegetable cooker. Int. J. Food Sci. Technol. 35, 377-384.

5. Lisiewska Z., Kmiecik W., Słupski J., 2004. Contents of chlorophylls and carotenoids in frozen dill: effect of usable part and pre-treatment on the content of chlorophylls and carotenoids in frozen dill (Anethum graveolens L.), depending on the time and temperature of storage. Food Chem. 84, 511-518.

6. Tijskens L. M. M., Barringer S. A., Biekman E. S. A., 2001. Modelling the effect of pH on the colour degradation of blanched broccoli. Innov. Food Sci. Emerg. Technol. 2, 315-322.

7. Kidmose U., Knuthsen P., Edelenbos M., Justesen U., Hegelund E., 2001. Carotenoids and flavonoids in organically grown spinach (Spinacia oleracea L.) genotypes after deep frozen storage. J. Sci. Food Agric. 81, 918-923.

8. Nishino H., Tokuda H., Satomi Y., Masuda M., Bu P., Onozuka M., Yamaguchi S., Okuda Y., Takayasu J., Tsuruta J., Okuda M., Ichiishi E., Murakoshi M., Kato T., Misawa N., Narisawa T., Takasuka N., Yano M., 1999. Cancer prevention by carotenoids. Pure Appl. Chem. 7, 2273-2278.

9. Lichtenthaler H. K., Buschmann C., 2001. Extraction of photosynthetic tissues: chlorophylls and carotenoids. Curr. Prot. Food Anal. Chem. 1, F4.2.1-F4.2.6.

10. Lichtenthaler H.K., Buschmann C., 2001. Chlorophylls and carotenoids: measurement and characterization by UV-VIS spectroscopy. Curr. Prot. Food Anal. Chem. F4.3.1 – F 4.3.8.

11. ISO 6558-2, 1992. Fruits, vegetables and derived products – Determination of carotene content. Part 2: Routine methods. International Organization for Standardization, Case Postale 56, CH-1211, Geneve 20, Switzerland.

12. Prakash D., Nadh P., Pal M., 1995. Composition and variation in vitamin C, carotenoids, protein, nitrate and oxalate contents in Celesia leaves. Plant Foods Hum. Nutr. 47, 221-226.

13. Mercadante A. Z., Rodriguez-Amaya D. B., 1991. Carotenoid composition of a leafy vegetable in relation to some agricultural variables. J. Agric. Food Chem. 39, 1094-1097.

14. Khachik F., Beecher G. R., Whittaker N. F., 1986. Separation, identification, and quantification of the major carotenoid and chlorophyll constituents in extracts of several green vegetables by liquid chromatography. J. Agric. Food Chem. 34, 603-616.

15. Kmiecik W., Budnik A., 1997. Wpływ dwóch sposobów gotowania brokuła na poziom wybranych wskazników fizykochemicznych. Bromatologia Chem. Toksykol. 30, 303-309.

16. Müller H., 1997. Determination of the carotenoid content in selected vegetables and fruit by HPLC and photodiode array detection. Z. Lebens. Unters. F. A., 204, 88-94.

17. Kmiecik W., Lisiewska Z., 1999. Comparison of leaf yields and chemical composition of the Hamburg and leafy types of parsley. II. Chemical composition. Folia Hort. 11, 65-74.

18. Saric M., Zatezalo S., Krstic B., 1990. The amount of pigments and basic mineral elements in the leaf blades of cabbage of different ages. Fiziol. Biokhim. Kult., 22, 32-37.

19. Bhaskarachary K., Sankar Rao D.S., Deosthale Y.G., Reddy V., 1995. Carotene content of some common and less familiar foods of plant origin. Food Chem. 54, 189-193.

20. Kurilich A. C., Tsau G. J., Brown A., Howard L., Klein B. P., Jeffery E. H., Kushad M., Wallig M. A., Juvik J.A., 1999. Carotene, tocopherol, and ascorbate contents in subspecies of Brassica oleracea. J. Agric. Food Chem. 47, 1576-1581.

21. Hart D. J., Scott J., 1995. Development and evaluation of an HPLC method for the analysis of carotenoids in foods, and the measurement of the carotenoid content of vegetables and fruits commonly consumed in the UK. Food Chem. 54, 101-111.

22. Kmiecik W., Lisiewska Z., Jaworska G., 2001. Effect of storage conditions on the technological value of dill (Anethum graveolens L.). Folia Hort. 13, 33-43.

23. Jaworska G., Kmiecik W., 1999. Effect of the date of harvest on the selected traits of the chemical composition of spinach (Spinacia oleracea L.) and New Zealand spinach (Tetragonia expansa Murr.). Acta Agr. et Silv. s. Agr. 37,15-26.

24. Kopsell D. E., Kopsell D. A., Randle W. M., Coolong T. W., Sams C. E., Curran-Celentano J., 2003. Kale carotenoids remain stable while flavor compounds respond to changes in sulfur fertility. J. Agric. Food Chem., 51, 5319-5325.

25. Drews M., Schonhof I., Krumbein A., 1995. Gehalt und Verteilung von Inhaltsstoffen in Kopfsalat. Gartenbauwissenschaft, 60, 287-293.

26. Drews M., Schonhof I., Krumbein A., 1997. Gehalt an Inhaltsstoffen in Eissalat (Lactuca sativa var. capitata L.) in Abhaengigkeit von Sorte und Stadium der Kopfentwicklung beim Anbau im Gewaechshaus. Gartenbauwissenschaft, 62, 65-72.

27. Yamauchi N., Watada A.E., 1991. Related chlorophyll degradation in spinach leaves during storage. J. Am. Soci. Hort. Sci., 116, 58-62.

28. Lisiewska Z., Słupski J., Korus A., 2001. Influence of cultivation period, cultivar and usable part on content of chlorophylls and volatile oils in dill (Anethum graveolens L.). Elec. J. Pol. Agric. Univ., Food Sci. Technol. 4, 2, http://www.ejpau.media.pl/series/volume4/issue2/food/art-18.html.

29. Kmiecik W., Lisiewska Z., 1999. Content of selected pigments in parsley leaves depending on biological factors and the conditions of growth. Acta Agr. et Silv. s. Agr., 37, 3-13.

30. Leja M., Mareczek A., Starzyńska A., 2002. Some antioxidant and senescence parameters of broccoli as related to its developmental stages. Acta Physiol. Plant. 24, 237-241.

31. Lisiewska Z., Kmiecik W., 1998. Dependence of dried chive (Allium schoenoprasum) quality upon the drying method and storage period.). Elec. J. Pol. Agric. Univ., Food Sci. Technol. 1, 1, http://www.ejpau.media.pl/series/volume1/food/art- 06.html.

Accepted for print: 15.02.2007

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