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
1
Department of Biochemistry, Faculty of Science, Khon Kaen University,
Khon Kaen 40002
2
Department of Horticulture, Faculty of Agriculture, Khon Kaen University,
Khon Kaen 40002
E-mail: kholom@kku.ac.th*
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
carotenoid extraction
maize,
carotenoids,
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 -carotene, and
monohydroxy carotenoids such as -cryptoxanthin
and -cryptoxanthin, have provitamin A activity
because of the presence of a -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 L, 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 (-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, βcryptoxanthin and β- carotene (data not shown).
Table 2. Total carotenoids in each maize line
Sample
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
5. Karw Naew
Pitsanulok (black),
6. Karw Naew
Pitsanulok (orange)
93.56±3.39
7. Lang Nong Bour
47.33±1.71
8. Krabngoo
9. Tien No.7
Chaingmai
20.45±1.38
10. Karw Nong Bour
5.89±0.88
11. Kolee Doum
12. Pan Ban
Surathanee
6.22±0.75
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
Table 1. Extraction of carotenoid from Pan Su Won
maize line (reference maize) for comparison of
extraction efficiency of each carotenoid extraction
method.
Method of
Total
% Recovery
extraction
carotenoid
of sample
content
mixed with
standard carotene
Harvest
Plus Method
Ben-Amotz
and Fishler Method
Kurilich and
Juvik Method
Panfili et al. Method
Kurilich and
Juvik Method with
modification
Total carotenoid(g/g
dry weight)
91.30±1.11
65.96±1.65
20.00±1.06
Maize sample
10.18 ±2.06
50±1.02
Fig. 3 Comparison of total carotenoid content in
maize lines.
17.17±1.21
76±3.86
4. Conclusions
47.70±3.07
86.68±5.34
59±0.91
10±2.04
12.60±1.31
60±1.06
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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