Technology and Innovation for Sustainable Development Conference (TISD2008) Faculty of Engineering, Khon Kaen University, Thailand
28-29 January 2008
Screening of biofortified maize with high carotenoid content in Northeast of Thailand
Khomsorn Lomthaiong1*, Ubonrat Keawrang1, Kamol Lertrat2
1Department of Biochemistry, Faculty of Science, Khon Kaen University,
Khon Kaen 40002
2Department of Horticulture, Faculty of Agriculture, Khon Kaen University,
Khon Kaen 40002
E-mail: *
Abstract
In Thailand, biofortification of maize to increase carotenoid content has the potential to improve vitamin A status in vitamin A deficient populations. The objective of this study was to screen biofortified maize with high carotenoid content in 15 lines of maize. Kurilich and Juvik extraction method was employed to extract total carotenoids in all maize line samples. Yellow-orange maize line No. 14 KK2 has found to have highest total carotenoid level among all the lines tested. However line Luse xanfeng, line PI919C2M1, and line Karw Neaw Pitsanulok (black) have high amount of carotenoids, and line No. 14 KK3, line Karw Neaw Pitsanulok (orange) contain moderate level of total carotenoids respectively. Thin Layer Chromatography (TLC) was also used to analyze types of carotenoids with mobile phase using were petroleum ether: diethyl ether: acetone (4: 1: 1, v/v). The major carotenoids found in yellow maize are lutein/zeaxanthin, β-cryptoxanthin and β- carotene. In conclusion, we have successfully screened biofortified maize on the level of total carotenoids that can then be used for further line selection and breeding.
Keywords: biofortified maize, carotenoids, carotenoid extraction
1. Introduction
Biofortification has been proved to be valuable method to increase micronutrient in particular crops [1, 2]. In Thailand, many plant breeding programs are underway to boost up some micronutrient in plant foods [3]. Maize is the basic crops grown throughout the country that can be easy right to consume for people and many farm animals. It is important as basic staple food for sub-rural region in Thailand as maize provides a rich provitamin-A source. Increasing the supply of provitamin A rich maize cultivars may contribute to better vitamin A status in children and vitamin A deficiency people in rural region in this country. Compared to most carotenoid-containing foods, maize has high protein and oil content, i.e., 8-11 and 3-18% dry weight, respectively [4]. It has also been shown that the carotenoids profile of maize varies among the different maize cultivars [5, 6, 7]. Fig. 1 shows the major carotenoids that have been detected in maize [8]. Carotenes such as a carotene and b-carotene, and monohydroxy carotenoids such as a-cryptoxanthin and b-cryptoxanthin, have provitamin A activity because of the presence of a b-ionone ring structure in the molecule. However, lutein, zeaxanthin and zeinoxanthin lack pro-vitamin A activity but has been shown to have antioxidant activity [9]
Here, we screened the maize with high level of carotenoid content using the most reproducible extraction method selected from five different extraction methods and the one that give best extraction efficiency was employed to determine carotenoids in all maize lines.
2. Materials and methods
2.1 Maize samples
All biofortified maize lines were generously provided by Plant Breeding Research Centre for Sustainable Agriculture, faculty of Agriculture, Khon Kaen University. The maize lines were called as code name composed of 1. Yanhejin, 2. Lusa xanfeng, 3. PI 919 C2S1, 4. PI 919 C2M1, 5.Karw Naew Pitsanulok (black), 6. Karw Naew Pitsanulok (orange), 7. Lang Nong Bour, 8. Krabngoo, 9.Tien No.7 Chaingmai, 10. Karw Nong Bour, 11. Kolee Doum, 12. Pan Ban Surathanee, 13. No.13 KK1, 14. No14. KK2, 15. No14. KK3 (shown in fig. 2). Upon receipt, maize was stored at -80 °C. Samples of maize were analyzed at least in triplicate by different methods to determine the optimum protocol for carotenoid analysis. All sample preparations, extractions, and analyses were performed under gold or UV-filtered white fluorescent lighting.
Fig.1 Major carotenoids detected in maize [8]
2.1 Extraction methods
HarvestPlus Method [10]. Brifly, carotenoids were extracted by grinding the maize using a mortar and pestle with 50 mL acetone. The residue was filtered with Whatman #2 filter paper. One-third of the filtrate was transferred to a separatory funnel containing 20 mL of petroleum ether, to which 300 mL of distilled water was added. After the aqueous and organic layers separated, the aqueous layer was discarded. The organic phase was washed 3 times with 200 mL of distilled water and passed through anhydrous sodium sulfate (~15 g) into a round-bottom flask. The sample was concentrated with a rotary evaporator and dried under nitrogen gas and kept in -20 C freezer. Extracted samples were measured spectrophotometrically for total carotenoids at 450 nm, using absorption coefficients (E1% 1 cm) of 2500 [11].
Ben-Amotz and Fishler Method [12]. The maize powder (0.6 g) was extracted twice with tetrahydrofuran/methanol (5 mL, 50:50 v/v) by mixing and centrifugation. Hexanes (10 mL) and sodium chloride (2 mL, 10% w/v) were added to the combined organic layers. A large glass test tube (50 mL) were used to separate the layer. The organic layer was transferred to a new tube and dried under nitrogengas.
Kurilich and Juvik Method [9]. Carotenoids were released from dried maize (0.6 g) by adding ethanol (6 mL) containing 0.1% BHT (w/v), mixing by vortex for 20 s, and placing in an 85 °C water bath for 5 min. Potassium hydroxide (120 mL, 80% w/v) was added to the heated ethanol-maize mixture, to saponify potentially interfering oils. Samples were mixed by vortex and returned to the 85 °C water bath for 10 min with an additional mixing at 5 min. After saponification, samples were immediately placed in ice, and cold deionized water (~3 mL) was added. Carotenoids were extracted 3 times with hexanes (~3 mL) using centrifugation (1200g) to separate the layers. Combined organic layers were washed with deionized water (~3 mL), and the organic layer was removed to a new test tube and dry under nitrogen gas. Several modifications were performed to improve chromatography and verify importance of saponification and heating steps.
Panfili et al. Method [13]. The maize sample (2 g) was placed in a screw-capped vial. Ethanolic pyrogallol (5 mL, 60 g/L), ethanol (2 mL, 95% v/v), sodium chloride (2 mL, 10 g/L), and potassium hydroxide (2 mL, 600 g/L) were added. Samples were heated in a 70 °C water bath for 45 min with mixing every 5-10 min. Samples were transferred to an ice bath immediately following saponification. Sodium chloride (15 mL, 10 g/L) was added. The carotenoids were extracted twice with hexanes/ethyl acetate (15 mL, 9:1 v/v), and the combined organic layers were dried using rotary evaporation.
Kurilich and Juvik method with modification. Carotenoids were released from dried ground maize (0.6 g) by heating at 50 °C for 10 min in ethanol with BHT (0.1% w/v). The carotenoids were extracted from the maize twice using petroleum ether/diethyl ether (6 mL, 4 mL, 2:1 v/v) and combined in a new test tube. The carotenoid extract was saponified with ethanolic potassium hydroxide (1 mL, 40% w/v) on ice for 2 min and at room temperature for 3 min. Then, distilled water (3 mL) was added, and the organic layer was removed to a new tube. The remaining aqueous layer was further extracted twice more with petroleum ether/diethyl ether (5 mL, 3 mL, 2:1 v/v), and the combined organic layers were dried under argon.
3. Result and discussion
Comparison of carotenoid extraction efficiency of five different extraction method was shown that the Kurilich and Juvik method was the most reproducible method for carotenoid extraction. Although the Panfili et al. method was shown to be the best extraction method giving highest amount of carotenoid but it was not preferred because this method has caused an error with artificial carotenoids resulting higher spectrophotometrically absorption of A450 nm [11].
We have also found that vigorously extraction condition by heating of potassium and extraction solvent in the Panfili et al. method has made an inconsistency of carotenoid extraction as the percent of standard (b-carotene) mixed sample recovery of this method was very low comparing to all other methods. Kurilich and Juvik method was preferred as it is easy, rapid and need little amount of maize sample for extraction.
We found that yellow-orange maize line No. 14 KK2 has the highest total carotenoid content among all the lines tested. However line Luse xanfeng, line PI919C2M1, line Karw Neaw Pitsanulok (black) have high amount of total carotenoids, and line No. 14 KK3, line Karw Neaw Pitsanulok (orange) contain moderate level of carotenoids respectively.
Fig.2. Maize lines that were used for carotenoids extraction.
Thin Layer Chromatography (TLC) was also used to analyze types of carotenoids with mobile phase using were petroleum ether: diethyl ether : acetone (4 : 1 : 1 , v/v). The major carotenoids found in yellow maizes are lutein/zeaxanthin, β-cryptoxanthin and β- carotene (data not shown).
Table 1. Extraction of carotenoid from Pan Su Won maize line (reference maize) for comparison of extraction efficiency of each carotenoid extraction method.
Method ofextraction / Total carotenoid content / % Recovery of sample mixed with standard b-carotene
Harvest
Plus Method / 10.18 ±2.06 / 50±1.02
Ben-Amotz
and Fishler Method / 17.17±1.21 / 76±3.86
Kurilich and
Juvik Method / 47.70±3.07 / 59±0.91
Panfili et al. Method / 86.68±5.34 / 10±2.04
Kurilich and
Juvik Method with modification / 12.60±1.31 / 60±1.06
Table 2. Total carotenoids in each maize line
Sample / Total carotenoid(mg/g dry weight)1. Yanhejin, / 3.73±0.38
2. Lusa xanfeng / 91.84±2.83
3. PI 919 C2S1 / 28.96±1.66
4. PI 919 C2M1 / 93.56±3.39
5. Karw Naew Pitsanulok (black), / 91.30±1.11
6. Karw Naew Pitsanulok (orange) / 65.96±1.65
7. Lang Nong Bour / 47.33±1.71
8. Krabngoo / 20.45±1.38
9. Tien No.7 Chaingmai / 20.00±1.06
10. Karw Nong Bour / 5.89±0.88
11. Kolee Doum / 6.22±0.75
12. Pan Ban Surathanee / 11.59±0.20
13. No.13 KK1 / 44.08±1.34
14. No.14 KK2 / 106.29±2.33
15. No.14 KK3 / 72.49±4.40
Maize sample
Fig. 3 Comparison of total carotenoid content in maize lines.
4. Conclusions
In this study, we examined the carotenoid content in biofortified maize to select the line with high carotenoid level. Kurilich and Juvik method was the most reliable method for the extraction of carotenoids from maize samples. This method was used to extract all the maize samples in this study. We found that maize line No. 14 KK2 has the highest carotenoid level, and line Lusa xanfeng and line PI 919 C2M1 contain high amount of carotenoids.
In conclusion, the selected maize lines based on high carotenoid can be breed into different maize cultivars that have preferred characteristic. Therefore these maize lines can be distributed and cultivated in Northeastern of Thailand. Biofortification of maize to increase provitamin A level would help children and vitamin A deficiency population in Thailand benefit from this program.
Acknowledgments
This research was financially supported by the Department of Biochemistry, Faculty of Science, Khon Kaen University.
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