Biochemical estimation of beta carotene in Indian Durum varieties

Biochemical estimation of β-carotene in Indian durum varieties

Meenakshi Santra, V. S. Rao and S. A. Tamhankar

Genetics and Plant Breeding Group, Plant Sciences Division, Agharkar Research Institute, Pune 411004, India.

Corresponding author: Meenakshi Santra

Present address: Department of Crop and Soil Sciences, Washington State University, Pullman 99163, WA, USA

E-mail: msantra@wsu.edu

Durum wheat is highly valued for pasta making in many parts of the world due to its attractive bright appearance which is caused by two kinds of pigments, namely xanthophylls and carotenoids. Durum wheat normally has higher pigment content than other wheat because of the presence of higher amount of xanthophylls and carotenoids (Dick and Matsuo 1988). Among carotenoids, β-carotene is important for pasta quality and nutrition. It acts as preservative and is a precursor of vitamin-A.

There are few reports on biochemical estimation of β-carotene in wheat. Goulden et al. (1934) studied carotenoid in a series of hybrid wheat and a group of standard varieties of hexaploid wheat using AACC methods. Markley (1937) estimated carotenoid pigment in individual plants of Triticum vulgare and Triticum durum to study the variability using standardized Evelyn photometric colorimeter and the range of carotenoid content was found to be 2.09 ppm to 3.74 ppm. Zechmeister and Cholnoky (1940) reported carotenoids in Hungarian wheat flour to detect the nature of pigment. Lacroix and Lier (1975) documented carotenoids in durum wheat variety Hercules at different developmental stages during two growing seasons. Lepage and Sims (1968) evaluated carotenoids in two varieties of wheat, Mindum (durum) and Thatcher (hard red spring) to study the chemical nature of the pigments. Taha and Sagi (1987) used seven durum wheat varieties in Hungary to reveal the effects of some intrinsic factors of semolina on macaroni colour. There is no report of biochemical analysis of pigments on Indian durum wheat varieties. Prior knowledge of β-carotene content in commonly used wheat breeding lines help breeders to design their breeding program. There is no such comprehensive report on evaluation of common Indian durum wheat varieties for β-carotene. The present work was undertaken to evaluate commonly used Indian durum wheat varieties with respect to β-carotene content following approved AACC method (14-50, 1995) and to compare its content in rainfed and irrigated condition.

Fifty Indian durum varieties (Table 1) have been selected to evaluate β-carotene content. The varieties were grown in two successive growing seasons of 1994-95 and 1995-96 both under rainfed and irrigated conditions at Hol, Pune using randomized block design with three replications.



β-carotene was estimated following approved AACC (14-50, 1995) method as described below. Eight gram flour was taken in 150 ml glass stoppered Erlenmeyer flask and 40 ml water saturated butanol (WSB) was added. The contents of the flasks were mixed vigorously for 1 minute and kept overnight (16-18 hrs) at room temperature under dark for complete extraction of β-carotene. Next day, the contents were shaken again and filtered completely through the Whatman no.1 filter paper into a 100 ml volumetric flask. The optical density of the clear filtrate was measured at 440 nm using HITACHI U-3210 spectrophotometer. Pure WSB was used as blank. The β-carotene content was calculated from calibration curve from known amount of β-carotene as discussed below and expressed as parts per million (ppm). Standard solution of β-carotene (Sigma) was prepared in water saturated butanol (WSB) at the concentration of 5 μg/ml. WSB is prepared by mixing n-butanol with distilled water in 8:2 ratios. Calibration curve is made from known amounts of pure β-carotene from 0.25 μg/ml to 1.5 μg/ml which are prepared after suitable dilutions of original stock with WSB in calibrated 10 ml volumetric flasks (from 0.5 ml to 3 ml of standard solution in 10 ml). Absorbance of each dilution is measured and a calibration curve is established. β-carotene content of unknown samples is calculated from standard curve.

The β-carotene content (mean of three replications) in Indian durum varieties is presented in Table 2. Enough seeds were not available for all 50 varieties in both the growing seasons. Therefore, data were available for 29 varieties during 1994-95 and 48 varieties during 1995-96 both for rainfed (RF) and irrigated (IR) conditions. A wide range of variation in β-carotene was observed in Indian durum wheat varieties grown both in rainfed (RF) and irrigated (RI) conditions in both the years. The β-carotene content in the varieties grown during 1994-95 ranged from 2.92 to 5.82 ppm (RF) and 2.73 to 5.89 ppm (IR) whereas the range was 2.97 to 5.95 ppm (RF) and 2.25 to 7.93 ppm (IR) among the varieties grown in 1995-96. Bhalegaon-4 and Kathia-25 showed the lowest (2.92 ppm) and highest (5.82 ppm) β-carotene varieties, respectively, among the varieties grown in 1994-95 under RF condition, where as Dasharkhed and MACS 9 showed the lowest (2.73 ppm) and highest (5.89 ppm) β-carotene, respectively grown under IR condition. Similarly in 1995-96 growing season, Datala-6 and MACS-9 showed the lowest (2.97 ppm) and highest (5.95 ppm) β-carotene, respectively grown under RF condition whereas A-1 and PDW-233 showed the lowest (2.25 ppm) and highest (7.93 ppm) β-carotene, respectively grown under IR condition. It is evident from Table 2 that some varieties differ in β-carotene content under irrigated and rainfed conditions. Some varieties are stable with respect to β-carotene in all the conditions, eg. Bhalegaon–4 shows low β-carotene content in both RF and IR conditions in two years. The β-carotene content is higher when a variety is grown in rainfed condition and it decreases when grown under irrigated condition for most of the varieties except few exceptions. Decrease in β-carotene content from RF to IR condition ranged from 0.8% in variety Bansi-288-18 to 45% in Kathia-25 during 1994-95 seasons whereas in 1995-96, it ranged from 1% in Kathia-21 to 48% in A-1-8-1.

Estimation of β-carotene content in Indian durum varieties has obviously great importance since durum varieties are becoming more popular day by day among Indian farmers because of their high resistance to disease, yield potentiality etc. Buyers always prefer attractive colour of semolina and pasta products. So varieties having high β-carotene content like PDW-233, Raj-6496, MACS-9 etc will be more popular and those can be used in breeding program. Development of variety with good agronomic value is always based on efficient selection method. The varieties having more than 5 ppm β-carotene content are JNK-4W-184, MACS-9, Bijapur 370-4, LR Dharwad, Raj-6496 and PDW-233. Most of the Indian durum varieties have lower β-carotene with a range of 3-6 ppm with an average of 4.5 ppm except two varieties Raj-6496 and PDW-233. These two varieties have been developed by Punjab Agricultural University, Ludhiana crossing with high β-carotene Mexican varieties. This study will enable wheat breeders in selection of parental lines for crossing in breeding program to develop high β-carotene cultivar, to study inheritance of this trait, tagging and mapping of the genes involved. Based on the result presented here three varieties with high β-carotene content viz. PDW-233. Raj-6496, MACS-9 and two varieties with low β-carotene content viz, Bhalegaon-4 and Baxi 6-1-1 were selected for crossing to study the inheritance of this trait.

References

American Association of Cereal Chemists method (1995) Approved Methods of the AACC, Method pp14-50. The association: St. Paul, MN.

Dick JW, Matsuo RR (1988) Durum wheat and pasta products. In: Wheat Chemistry and Technology, Vol. II. 3rd Ed. Y. Pomeranz, ed. Am. Assoc. Cereal Chem.: St Paul, MN.

Goulden CH, Geddes WF, Whiteside AGO (1934) A study of carotene content and other quality characters in a series of hybrid wheats. Cereal Chem 11: 557-566.

Lacroix LJ, Lier JB (1975) Carotenoids in durum wheat: developmental patterns during two growing seasons. Can J Plant Sci 55: 679-684.

Lepage M, Sims RPA (1968) Carotenoids of wheat flour: their identification and composition. Cereal Chem 45: 600-604.

Markley MC (1937) Variability in carotenoid pigment of individual plants of Triticum vulgare and Triticum durum. Cereal Chem 14: 400-409.

Taha SA, Sagi F (1987) Relationships between chemical composition of durum wheat semolina and macaroni quality. II. Ash, carotenoid pigments, and oxidative enzymes. Cereal Res Commun 15: 402-407.

Zechmeister L, Cholnoky L (1940) Carotenoids of Hungarian wheat flour. J Biol Chem 135: 31-36.