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Oxidation of Crude Corn Oil with and without Elevated Tocotrienols

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Kernels and oil from corn with increased concentrations of tocotrienols (T3) due to the transgenic expression of a homogentisic acid geranylgeranyl transferase (HGGT) gene from two growing years were characterized for tocol and fatty acid compositions. The crude extracted oil was analyzed for oxidative properties and compared to non-transformed material derived from the plants grown at the same location and during the same year. No significant difference was observed in composition of major fatty acids. Both the seed (kernel) and extracted crude oil from the HGGT expressing corn had a 18-fold increase in tocotrienol content from 300 to 5,400ppm in oil. There was a concurrent 18% decrease in tocopherol content, 1,150ppm total tocopherols in control oil declining to 940ppm in HGGT oil. Although tocopherols and tocotrienols are generally considered antioxidants, they may exhibit prooxidant effects at higher concentrations and they should be tested. Crude oil was extracted from control and transformed corn produced during 2005 and 2006 yielding four oil samples that were evaluated for their oxidative properties. The formation of lipid hydroperoxides, a primary oxidation product, was evaluated at 60°C over 9days by measuring the peroxide value (PV). Resistance to oxidation or induction period (IP) was measured using an Oxidative Stability Instrument. There was a slight decrease in hydroperoxide formation in the HGGT oil compared to the corresponding control but was less than the year to year differences. The induction period was the same for the 2005 oils, with or without the increased tocotrienol content, but the crude oil with enhanced tocotrienol had a longer IP than the control crude oil in the 2006 samples. KeywordsAntioxidants–Corn oil–Homogentisic acid geranylgeranyl transferase–Oxidative stability index–Peroxide value–Tocotrienol–Tocopherol
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ORIGINAL PAPER
Oxidation of Crude Corn Oil with and without Elevated
Tocotrienols
David Dolde
Tong Wang
Received: 17 October 2010 / Revised: 30 January 2011 / Accepted: 9 March 2011 / Published online: 26 March 2011
Ó AOCS 2011
Abstract Kernels and oil from corn with increased con-
centrations of tocotrienols (T3) due to the transgenic
expression of a homogentisic acid geranylgeranyl trans-
ferase (HGGT) gene from two growing years were char-
acterized for tocol and fatty acid compositions. The crude
extracted oil was analyzed for oxidative properties and
compared to non-transformed material derived from the
plants grown at the same location and during the same
year. No significant difference was observed in composi-
tion of major fatty acids. Both the seed (kernel) and
extracted crude oil from the HGGT expressing corn had a
18-fold increase in tocotrienol content from 300 to
5,400 ppm in oil. There was a concurrent 18% decrease in
tocopherol content, 1,150 ppm total tocopherols in control
oil declining to 940 ppm in HGGT oil. Although toc-
opherols and tocotrienols are gener ally considered antiox-
idants, they may exhibit prooxidant effects at higher
concentrations and they should be tested. Crude oil was
extracted from control and transformed corn produced
during 2005 and 2006 yielding four oil samples that were
evaluated for their oxidative properties. The formation of
lipid hydroperoxides, a primary oxidation product, was
evaluated at 60 °C over 9 days by measuring the peroxide
value (PV). Resistance to oxidation or induction period (IP)
was measured using an Oxidative Stability Instrument.
There was a slight decrease in hydroperoxide formation in
the HGGT oil compared to the corr esponding control but
was less than the year to year differences. The induction
period was the same for the 2005 oils, with or without the
increased tocotrienol content, but the crude oil with
enhanced tocotrienol had a longer IP than the control crude
oil in the 2006 samples.
Keywords Antioxidants Corn oil Homoge ntisic acid
geranylgeranyl transferase Oxidative stability index
Peroxide value Tocotrienol Tocopherol
Introduction
Tocotrienols and tocopherols are lipophilic, plant-produced
antioxidants exhibiting vitamin E activity. The composition
of the eight tocol homologues differs considerably in dif-
ferent plant species. Common sources of tocopherols, such
as nuts and oils of soy, corn, canola and sunflower, contain
little or no tocotrienols. Because of their similar phenolic
structure, all tocols exhibit antioxidative properties at parts
per million (ppm) concentrations in oil or lipid systems.
Recent research suggests indivi dual tocotrienols provide
benefits beyond these antioxidation properties [1, 2].
The reported benefits of tocotrienols include inhibiting
cholesterogenesis and the coronary heart disease [13]. By
lowering plasma triacylglycerides, tocotrienols may reduce
the effects of metabolic syndrome. Tocot rienols have
shown prom ise to control tumor growth and suppress
growth of specific melanoma cells in vitro and in animal
studies. There may also be synergistic advantages to using
tocotrienols with other chemotherapies [4].
Using molecular biology to manipulate metabolic
pathways in plants for increased tocol production and
varying their compositions were reviewed and accom-
plished by Cahoon and others in 2003 [57]. The
D. Dolde
Pioneer Hi-Bred International, Inc., Johnston, IA 50131, USA
T. Wang (&)
Department of Food Science and Human Nutrition,
Iowa State University, Ames, IA 50011-1061, USA
e-mail: tongwang@iastate.edu
123
J Am Oil Chem Soc (2011) 88:1367–1372
DOI 10.1007/s11746-011-1805-0
assumption is that genetic modification of the tocopherol
and tocotrienol biosynthetic pathways can reduce oxidative
stress in plants, increase crop productivity, increase storage
duration of seed and plant material, improve shelf life of
vegetable oils, and meet increasing demands for nutra-
ceutical or therapeutic markets [7]. As research continues
on the genetics and biochemistry of tocol biosynthesis,
further enhancements to tocol-fortified seeds are expected.
Tocols at higher concentrations, however, may act as
prooxidants [ 8 ]. As little as 250 ppm of a-tocopherol has
been reported as having prooxidative effects in the for-
mation of lipid hydroperoxides [9]. In tocotrienol- spiked
coconut fat at 60 °C, the addition of a- and b- tocotrienols
at 100, 500 and 1,000 ppm increased fat oxidation as
measured by PV compared to the control. This prooxidant
effect increased with increased tocotrienol concentration
[10]. Both c- and d-tocotrienols had the opposite effect and
they reduced lipid hydroperoxide formation, although the
effect was not correlated with c-tocotrienol concentration
[10]. The primary goal of this research was to determine
the oxidative effects of very high levels of tocotrienols
([5,000 ppm) in crude oil from corn genetically trans-
formed with a homogentisate geranylgeranyl transf erase
(HGGT) gene expressing increased tocotrienols.
Materials and Methods
Material
Four bulk samples of approximately 8 kg of corn each
were provided by Pioneer Hi-Bred International, Inc.
(Johnston, IA, USA ). Two samples from each of 2005 and
2006 were provided. One sample was genetically trans-
formed with the barley HG GT gene to express increased
amounts of tocotrienols, and the second was a sample
control with a similar pedigree but lack ing the transgene.
No field or yield data was provided. The samples were
stored in the laboratory as whole kernels in the dark at
ambient temperature. The moisture content of all samples
was below 10%.
a-, c- and d-Tocotrienol standards were obtained
from Davos Life Sciences, PTE Ltd (Singapore). a- and
d-Tocopherol were purchased from MP Biomedicals, LLC
(Solon, OH, USA). Acetic acid, chloroform, hexanes,
potassium iodide, sodium thiosulfate, potato starch, sali-
cylic acid were all purchased through VWR International,
LLC (West Chester, PA, USA).
Oil Extraction Sample Preparation
The corn was ground with a Thomas Wiley lab mill with a
1-mm screen. Sixty gram batches were extracted with
hexanes by using a Buchi B811 (New Castle, DE), an
automated Soxhlet extraction system. The extraction effi-
ciency was 97% by using a minimum of 10 cycles and 1-h
extraction. The mean percentages of oil recovered was
2.63, 2.66, 2.99 and 2.91 for the 2005 control, 2005 HGGT,
2006 control and 2006 HGGT mater ial, respectively. The
crude extracted oils were stored under argon in amber glass
at -23 °C until tested.
Three 1-mL aliquots of the crude oils were each placed
in 1.8-mL uncapped amber vials for each of the 9 days of
accelerated oxidation. Twelve vials were removed from a
60 °C oven each day at the same time over nine subsequent
days. The samples were blanketed with argon, capped and
stored at -23 °C until analyzed for peroxide value. Day
zero samples were also placed in amber vials and stored
under argon at -23 °C.
Tocopherol and Tocotrienol Content
The tocotrienols and tocopherols were determined by using
a modification of AOCS Offi cial Method Ce 8-89 [
11].
Tocols were separated by using a Waters HPLC Alliance
2695 (Milford, MA, USA) with a 3 l NH
2
100A,
150 mm 9 3.0 mm column and detected by fluorescence
(Waters 2475) with EXk = 292 nm and EMk = 335 nm.
An external calibration curve of 0.05, 0.1, 0.2, 0.5, 1.0, 2.5
and 5 ppm of each tocol was used for quantificatio n. For
single seed tocol analysis, individual kernels were ground
using a ball mill. The weight of the ground material was
recorded and then extracted with 2 mL of hexane under
reduced lighting. Total tocotrienols in single seed was
expressed as lg/g kerne l. An aliquot was reserved for
methylation and subsequent fatty acid analysis.
Fatty Acid Composition
The fatty acid compositions for the major fatty acids of the
crude corn oils were determined by GC by using a ZB-wax
column (Pheno menex, Torrance, CA, USA) at 220 °C.
About 50 lL of oil was diluted in 1 mL of hexane and
methylated with trimethylsulfonium hydroxide [12, 13].
Free Fatty Acid Content
The free fatty acid (FFA) percentages in the crude oils were
determined by using AOCS method Ca 5a-40 [11]. Per-
centage FFA was expressed as percentage of oleic acid.
Primary Lipid Oxidation Products Determined
by Peroxide Value
Three samples of each extracted crude oil from the four
bulk corn samples were oxidized at 60 °C for each of
1368 J Am Oil Chem Soc (2011) 88:1367–1372
123
9 days and analyzed by using the AOCS Cd 8-53 method
adapted by Crowe and White [14].
Resistance to Oxidation as Induction Period (IP)
Determined by Oil Stability Instrument (OSI)
Five samples of each extracted crude oil from the four bulk
corn samples were analyzed by using an ADM OSI unit
(Oxidative Stability Instruments, Omnion, Rockland, MA,
USA). The 5 g samples were oxidized at 100 °C with
airflow of 110 mL/min. Both the induction period and the
actual plot of conductivity by time were recorded.
Statistical Analysis
All experiments were conducted with repeated treatments
as noted. Data analyses were done by using Microsoft
Office Excel 2003 (Microsoft Corporation, Redmond, WA,
USA). One-way analysis of variance (ANOVA) was used
and least significant difference was calculated at P \ 0.05
(LSD
0.05
).
Results and Discussion
Fatty Acid Composition
The major fatty acids in the crude extracted corn oil as
molar percentages are given in Table 1. There were mini-
mal differences in the profile of the four crude oils, and
modification of the tocol pathway did not appreciably
affect fatty acid composition beyond the minor differences
that can normally be attri buted to environmental condi-
tions. Thus, fatty acid composition, one of the main
determinants of oxidative stability, was not expected to
influence the PV and OSI values obtained.
Free Fatty Acids
The FFA percentages in the crude oils expressed as oleic
acid, were 10.69 for the 2005 control oil, 10.97 for the
2005 HGGT oil, 10.42 for the 2006 control oil and 12.30
for the 2006 HGGT oil. All values were much higher than
expected for oils extracted from high quality, undamaged
grains. A commodity corn grain sample extracted using the
same equipment and process had a more reasonable FFA of
2.44%, indicating that the high FFA percentages observed
did not result from the extraction process or laboratory
storage conditions. No yield and other field performance
data were provided with the corn, therefore, the cause is
unknown. The initial hydroperoxide values for the crude
oils were 1.45, 1.32, 1.10 and 1.04 meq/kg for the 2005
control, 2005 HGGT, 2006 control and 2006 HGGT,
respectively, indicating no major damage or degradation of
the seed during storage. The high FFA is a concern as FFA
can be prooxidants [1517]. Thus, the oxidative stability of
the crude oils may not reflect the stability of the refined,
bleached and deodorized final product after the FFA are
removed. However, as both the control oils and the HGGT
oils had similar FFA content, their influence on oxidative
stability should also be similar. Idea lly, the crude oil should
have been fully refined and then evaluated for oxidative
stability. However, due to the limit ed amount of oil
obtained, refining was not possible.
Tocol Content
Both samples from each year were grown at the same
location. No significant differences were observed in the
single kernel analysis of the control corn compared with
HGGT corn for moisture, oil content, kerne l weight and
fatty acid composition. As expected, there were slight year-
to-year differences. There were, however, significant dif-
ferences in tocol amounts and tocol distribut ion between
the control and the HGGT corn. The 2005 HGGT kerne ls
segregated into two distinct populations, one with high T3
concentration (*75% total analyzed population) and one
with low T3 concentration, similar to that of the control
sample. This finding suggests that the dominant HGGT
transgene was segregating in the 2005 HGGT sample.
Thus, the genetics of this trait may not have been fully
fixed in the parent seed until the following year. This
phenomenon was not seen in the 2006 HGGT sample
(Fig. 1).
The concentrations of tocopherols and tocotrienols in
the oils studied are presented in Table 2. Modification of
the tocol biosynthetic pathway in HGGT corn resulted in
dramatic increases of all tocotrienols, especially c-tocotri-
enol. As there was no modification of the endogenous tocol
methyltransferase, excess tocols would be expected to
accumulate as the gamma homologue. There was a slight
decrease in a- and c-tocopherols and a slight increase in
b- and d-tocopherols in the modified HGGT corn oil
compared with the unmodified control oil. Major tocot-
rienols were less abundant in the 2005 HGGT oil than the
2006 HGGT oil. The reduction may have been a result of
the segregating population in 2005.
Table 1 Fatty acid composition (molar %) of crude corn oils
16:0 18:0 18:1 18:2 18:3
Control Corn Oil 2005 12.40 1.93 23.15 59.53 1.35
HGGT Corn Oil 2005 12.07 1.96 23.65 59.37 1.28
Control Corn Oil 2006 11.47 1.76 24.29 59.77 1.27
HGGT Corn Oil 2006 11.69 1.91 24.28 59.53 1.23
J Am Oil Chem Soc (2011) 88:1367–1372 1369
123
Accumulation of Primary Oxidation Products
The rates of hydroperoxide development in the crude corn
oils are given in Table 3. Due to the linearity of PV
development during the sampling period as shown in
Fig. 2, expressing the rate of PV change as D-PV/day was
reasonable and thus used. The R
2
for the linear relationship
ranged from a low value of 0.9826 for the 2006 control to a
high value of 0.9976 for the 2005 control (Fig. 2). The rate
of PV development of the HGGT crude corn oil was
significantly lower (P \ 0.05) than that in the control crude
oils of the same year. Thus, even at unusually high con-
centrations, the tocols appear to have an antioxidant effect
in the crude oil. Results from our model system [18] and
research by others suggest, however, that the optimum
levels of tocotrienols for antioxidation in bulk oils should
be lower [10, 19, 20]. The higher concentrations used in
these other experiments promoted the formation of the
primary lipid oxidation products in the purified fats and oils
but this was not observed with the crude extracted corn
oils. Other native antioxidants in the crude oils, such as
carotenoids, may provide synergies not found in highly
purified oils [17, 21]. This would allow the tocotrienols and
tocopherols to retain their effectiveness at very high levels.
These endogenous compounds provide an alternate path for
sequestering free radicals resulting from tocol oxidation
[21, 22].
2005
2006
0
5
10
15
20
25
30
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270
Total tocotrienol ug/g kernel
% of kernels
Fig. 1 Histogram of total
tocotrienols in corn kernels
modified with a homogentisate
geranylgeranyl transferase
(HGGT) gene
Table 2 Tocol composition of extracted crude corn oil in ppm and relative %
a- b- c- d- Total a- b- c- d- Total
Tocotrienols (ppm) Tocopherols (ppm)
Control 2005 192 ND 55 1 248 501 20 730 28 1,279
HGGT 2005 643 18 3,081 442 4,184 301 26 641 56 1,024
Control 2006 187 1 108 7 303 373 29 647 23 1,072
HGGT 2006 690 20 4,051 618 5,379 185 31 565 60 841
Tocotrienols (%) Tocopherols (%)
Control 2005 12.6 ND 3.6 0.1 16.2 32.8 1.3 47.8 1.8 83.8
HGGT 2005 12.3 0.3 59.2 8.5 80.3 5.8 0.5 12.3 1.1 19.7
Control 2006 13.6 0.1 7.9 0.5 22.0 27.1 2.1 47.1 1.7 78.0
HGGT 2006 11.1 0.3 65.1 9.9 86.5 3.0 0.5 9.1 1.0 13.5
ND not detectable
Table 3 Change in average daily peroxide value ± one standard
deviation over 9 days in crude corn oil at 60 °C in the dark
D-PV/day 2005 2006
Control 11.96 ± 1.06 9.19 ± 0.34
HGGT 9.83 ± 0.32 8.48 ± 0.27
1370 J Am Oil Chem Soc (2011) 88:1367–1372
123
Oxidative Stability of Crude Oils as Measured by OSI
The IP and conductivity values at the IP time are given in
Table 4. Although IP time is typically used to differentiate
oil stabilities, a better understandin g of the stability index
and accumulation of po lar degradation products is deduced
from the graph of the actual conductance of the five sam-
ples as seen in Fig. 3. Both the control and HGGT crude
oils from 2005 had the same IP value but exhibited dif-
ferent curve shapes and different conductivities at the IP
time. The control oil had a lower, much sharper and well-
defined inflection point than the HGGT crude oil contain-
ing increased tocotrienol levels. The 2006 samples also
exhibited the same pattern but this similarity is less obvious
as the IPs were significan tly different from each other
(Fig. 3). Similar findings of a smoothing of the curve and
less defined inflec tion point of high toco l oils have also
been reported when hexanal formation at lower tempera-
tures was used to track secondary oxidation [22]. Because
of the high temperature and artificial conditions used to
generate the IP time, the value of this type of testing has
caused considerable controversy, but it continues to be
widely used.
The actual slopes or curve shapes generated by OSI
suggest that the mechanism and antioxidative properties
may have shifted as the concentrations of tocols increase in
bulk oils. The initial rate of the evolution of degradation
products may have increased slightly more rapidly in high
tocol oils than in those oils containing less tocols. Yet the
high tocol oils continued to inhibit polar or acidic com-
pounds generation for a longer time. The classical model of
a long induction period of little polar compounds formation
followed by a very rapid increase in these compounds as
measured by OSI is less useful for oils having very high
tocol concentrations. Using IP time alone to compare crude
oils with high tocol levels is thus insufficient to determ ine
the actual oxidative effects.
Conclusion
Crude oil from corn expressing tocotrienols at
4,200–5,400 ppm exhibited no prooxidant effects. High
tocotrienol concentrations displayed slight antioxidative
properties in the reduction of the formation of lipid
hydroperoxides in crude oils. The induction period was
the same or extended for these oils but the curve of the
oxidative stability index shifted away from a sharp
inflection point indicating a possible shift in the oxidation
kinetics. Crude corn oil with increased tocol levels
remains as oxidatively viable as crude oil from non-
enhanced corn.
2005 Control
2005 HGGT
2006 Control
2006 HGGT
0
20
40
60
80
100
120
140
012345678910
Day
Peroxide Value, meq/kg
Fig. 2 Peroxide values of the
extracted crude corn oil from
control kernels and kernels
modified with homogentisate
geranylgeranyl transferase
(HGGT) from 2005 to 2006 at
60 °C and in the dark
Table 4 OSI induction period (IP, h) and conductivity (at IP) ± one
standard deviation of the extracted crude corn oil
OSI Induction
period (h)
OSI conductivity
at induction time
Control corn oil 2005 11.57 ± 0.22
b
6,650 ± 403
c
HGGT corn oil 2005 11.61 ± 0.51
b
10,548 ± 756
b
Control corn oil 2006 9.86 ± 0.57
c
6,869 ± 1,550
c
HGGT corn oil 2006 13.82 ± 1.04
a
13,015 ± 1,804
a
LSD
0.05
0.87 1,673
Values not sharing a common superscript are significantly different
OSI temperature is 100 °C
J Am Oil Chem Soc (2011) 88:1367–1372 1371
123
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Control 2005
HGGT 2005
0
5000
10000
15000
20000
25000
30000
35000
0 5 10 15 20 25
Hours
0 5 10 15
20
25
Hours
Conductivity
Control 2006
HGGT 2006
0
5000
10000
15000
20000
25000
30000
35000
Conductivity
Fig. 3 Oxidative stability index at 100 °C of five subsamples each of
extracted crude corn oil from control kernels and kernels modified
with homogentisate geranylgeranyl transferase
1372 J Am Oil Chem Soc (2011) 88:1367–1372
123
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... Besides redirection of tocopherol homeostasis toward a more satisfactory vitamer composition, increase of (absolute) vitamer content has been tackled in metabolic engineering approaches . Engineering the HGGT gene, catalyzing the committed step in tocotrienol biosynthesis (Figure 5), resulted in an increase in total tocochromanol content of maize kernels and up to 18-fold enhancement in tocotrienol accumulation (Dolde and Wang, 2011). Furthermore, engineering HPPD, a key enzyme in the biosynthesis of the tocochromanol precursor HGA (Figure 5), generated a massive accumulation of tocotrienols, provided that prephenate dehydrogenase (shikimate pathway) was also engineered to ensure sufficient flux toward tyrosine (Rippert et al., 2004). ...
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... Tansley Zhang et al., 2013) compared to overexpression of HGGT which significantly increased the concentrations of tocotrienols in maize and sorghum seeds (Cahoon et al., 2003;Dolde & Wang, 2011;Che et al., 2016). Increasing the flux through the methylerythritol 4-phosphate isoprenoid (MEP) pathway to provide more GGPP or PPP for tocotrienol and tocopherol biosynthesis, respectively, resulted in small, but measurable increases in tocochromanol concentrations (Est evez et al., 2001;Vom Dorp et al., 2015). ...
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