Project Title: Value-added Utilization of GEM Normal and High-amylose Line Starches
Prepared by Jay-lin Jane and Hongxin Jiang, Department of Food Science and Human Nutrition,
Iowa State University, Ames, IA 50011
Project Overview
This research project is aiming to characterize starches produced by the GEM projects
and to develop value-added utilization of these starch lines. Two types of starch, high-amylose
maize starch developed from GEM amylomaize project of Dr. Mark Campbell at Truman State
University (GEMS-0067 ae-lines), and normal maize starch extracted from GEM lines supplied
by the GEM Coordinator, Dr. Mike Blanco were the main genotypes used in the study. Three
GEMS-0067 ae-lines: GUAT209:S13 × (OH43ae×H99ae) B-B-4-1-2-1-1, GUAT209:S13 ×
(OH43ae×H99ae) B-B-4-4-2-1-1, and GUAT209:S13 × (OH43ae×H99ae) B-B-4-4-2-1-2 and four
existing ae-lines: H99ae, OH43ae, B89ae, and B84ae have been used in this study. The GEMS0067 ae-line starches displayed significantly larger RS contents (39.4-43.2%) than that of the
existing ae-lines (11.5-19.1%) determined using AOAC method 991.43 for total dietary fiber.
The GEMS-0067 ae-line starches had much larger amylose contents (83.1-85.6%) than did the
existing ae-line starches (61.7-67.7%). The resistant starch contents were positively correlated
with the apparent amylose content (r = 0.99), and the conclusion gelatinization temperatures of
the GEMS-0067 ae-line starches (122.0 – 130.0ºC) were substantially higher than that of the
existing ae-line starches (100.5 – 105.3º). The high gelatinization temperature of the GEMS0067 starch was likely to cause the large resistant starch content in the cooked starch.
Maize ae-starches are known for consisting of two types of starch granules: spherical
granules and rod/filamentous granules. The GEMS-0067 ae-line starches had substantially larger
proportions of rod/filamentous starch granules (22.6% - 32.0%) than did the existing ae-line
starches (5.2% - 7.7%). RS was concentrated in the rod/filamentous starch granules and on the
periphery of spherical starch granules. RS residues of the GEMS-0067 ae-line contained more
amylose-like molecules than did the existing ae-line RS residues. To understand RS formation in
the GEMS-0067 ae-line starch during maize-kernel development, we further studied molecularand granular-structures of the GEMS-0067 ae-line starches harvested at different maize
developmental stages.
Selectively crossed normal cornstarch lines aiming to improve enzyme digestibility of
cornstarch were provided by Dr. Blanco. The samples were tested for ethanol production using a
conventional method with cooked corn and a cold-fermentation method without cooking.
Results are shown in this report. One manuscript was published, another one is ready for
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submission, and two posters with abstracts were presented. The poster “Crystalline structure of
enzyme-resistant maize ae-mutant starches” by H. Jiang, et al., received an Outstanding Poster
Award from American Association of Cereal Chemist International Carbohydrate Division and
Corn Refiners Association, September 2008.
Publication:
L. Li, H. Jiang, M. Campbell, M. Blanco, and J. Jane, “Characterization of maize amyloseextender (ae) mutant starches. Part I: Relationship between resistant starch contents and
molecular structures” Carbohydrate Polymers 74 (2008) 396-404.
Presentations:
S. Srichuwong, J. Gutesa, M. Blanco, S. Duvick, J. Jane, “Effects of kernel composition and
starch structure on ethanol yield produced from dry-grind corn.” American Association of Cereal
Chemist International Annual Meeting, Honolulu, Hawaii, September 21 – 24, 2008.
H. Jiang, M. Campbell, and J. Jane, “Crystalline structure of enzyme-resistant maize ae-mutant
starches” American Association of Cereal Chemist International Annual Meeting, Honolulu,
Hawaii, September 21 – 24, 2008.
Objectives
The objectives of this research project were to characterize starches produced by the
Germplasm Enhancement of Maize (GEM) projects and to develop value-added utilization of
these starch lines.
Specific aims of the study in 2008 were:
1. To understand the mechanism of the RS formation in the GEMS-0067 ae-line starch
during maize-kernel development.
2. To produce easily digestible normal cornstarch to be used for fuel ethanol production.
Progress Made in 2008
Objective 1. To understand the mechanism of the RS formation in the GEMS-0067 ae-line starch
during maize-kernel development.
GEMS-0067 lines were planted in the field of North Central Regional Plant Introduction
Station (Ames, IA) in 2007 crop year. Maize kernels of self-pollinated inbred GEMS-0067 were
harvested on 15, 20, 25, 30, 35, 40, and 54 days after pollination (DAP). The endosperm was
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separated from the germ and pericarp following the method reported by Li, Jiang, Campbell,
Blanco, and Jane (2008).
The total starch and soluble sugar contents of GEMS-0067 endosperm are shown in
Table 1. The starch content of the endosperm increased from 36.6% on 15 DAP to 66.2% on 25
DAP, and maintained at ~70% up to 40 DAP. The starch content of the endosperm increased to
79.7% on 54 DAP (mature corn). The soluble sugar content of the endosperm decreased from
4.2% on 15 DAP to 0.3% on 54 DAP.
Table 1. Total starch and soluble sugar contents of GEMS-0067 endosperm harvested on
different DAP
Sample
Total starch
Soluble sugar
(% dry weight)
(% dry weight)
15 DAP
36.6 ± 1.3
4.2 ± 0.3
20 DAP
61.7 ± 2.8
3.0 ± 0.2
25 DAP
66.2 ± 2.4
1.8 ± 0.1
30 DAP
71.4 ± 0.5
1.1 ± 0.1
35 DAP
70.3 ± 0.3
0.9 ± 0.0
40 DAP
70.4 ± 0.2
0.6 ± 0.0
54 DAP (mature)
79.7 ± 2.7
0.3 ± 0.1
DAP = days after pollination
The endosperm starches were isolated from the GEMS-0067 maize kernels harvested at
different developmental stages. The resistant starch, amylopectin, amylose, and IC contents of
the GEMS-0067 endosperm starches are summarized in Table 2. The resistant starch contents
increased from 8.2 % on 15 DAP to 26.4 % on 20 DAP, and reached to 30.5% on 54 DAP with
fluctuation. The amylopectin content decreased from 44.8% on 15 DAP to 12.4 % on 54 DAP,
which had a negative correlation with resistant starch content (r = -0.95). The amylose content
increased from 27.2 % on 15 DAP to 73.7% on 40 DAP, and decreased to 69.5 % on 54 DAP.
The amylose content was positively correlated with the resistant starch content (r = 0.91). The IC
content varied between 14.9% and 28% during the kernel development.
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Table 2. Resistant starch, amylopectin, amylose, and intermediate component contents of
GEMS-0067 starch isolated from different kernel-developmental stages
Resistant starch b
Amylopectin c
Amylose c
IC c
(%)
(%)
(%)
(%)
a
d
15 DAP
8.2
27.2
28.0
44.8 ± 0.5
20 DAP
61.0
17.4
26.4 ± 0.1
21.6 ± 0.2
25 DAP
60.3
24.9
24.0 ± 0.9
14.8 ± 1.3
30 DAP
64.0
17.9
30.4 ± 1.9
18.1 ± 0.3
35 DAP
64.0
25.0
33.8 ± 0.4
11.0 ± 0.4
40 DAP
73.7
14.9
31.8 ± 2.8
11.4 ± 1.3
54 DAP
69.5
18.1
30.5 ± 2.1
12.4 ± 0.4
Correlation coefficient between RS%
-0.95
0.91
-0.63
and starch components
a
DAP = days after pollination. b Resistant starch content was determined using AOAC 991.43
method for total dietary fiber. c Amylopectin, amylose and intermediate component contents
were determined using Sepharose CL-2B gel permeation method. d Value was measured once.
Sample
DSC thermograms of starches isolated from kernels harvested at different developmental
stages are shown in Fig. 1, and the thermal properties are summarized in Table 3. Onset
gelatinization temperatures of starches ranged from 64.9 to 69.9 °C. Two thermal-transition
peaks of starch gelatinization were observed on the DSC thermograms of the starches harvested
on and after 20 DAP. The temperature of the first peak was ~79.2°C, which was the major peak
for samples of 15, 20, 25, and 30 DAP. The first-peak height decreased as the kernel maturity
increased and amylopectin content decreased (Fig. 1 and Table 2). The temperature of the second
peak was ~97.6 °C, which was the major peak for samples of 35, 40, and 54 DAP. The secondpeak height increased with the increase of kernel maturity and the amylose content (Fig 1 and
Table 2). The conclusion temperature was 105.0 °C for the sample of 15 DAP and increased to
124.1°C on 35 DAP, and then to 117.8°C on 54 DAP. The enthalpy changes decreased from 16.9
to 11.8 J/g with fluctuation. After defatting the GEMS-0067 starches with methanol, the secondpeak height decreased (Fig.1B), reflecting the presence of some amylose-lipid complex. The
results suggested that the first peak was mainly the gelatinization of amylopectin crystallites and
the second peak was the melting of amylose/IC crystallites and amylose-lipid complex.
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Table 3. Thermal properties of GEMS-0067 starches harvested on different DAP a, b
Sample
Thermal properties
Tp1 (°C)
Tp2 (°C)
Tc (°C)
To (°C)
ΔH (J/g)
15 DAP 69.5 ± 0.1
76.6 ± 0.6
Ndb
105.0 ± 0.7
16.9 ± 0.2
20 DAP 68.8 ± 1.0
78.5 ± 0.2
96.7 ± 0.2
119.3 ± 2.5
12.3 ± 0.6
25 DAP 69.4 ± 0.9
77.4 ± 0.1
98.6 ± 0.9
121.8 ± 0.4
11.4 ± 0.2
30 DAP 69.9 ± 0.5
79.5 ± 0.2
98.3 ± 0.6
122.2 ± 0.2
14.2 ± 0.8
35 DAP 66.1 ± 2.7
80.9 ± 0.1
98.0 ± 0.5
124.1 ± 1.3
10.9 ± 1.6
40 DAP 66.7 ± 2.4
80.4 ± 2.5
97.5 ± 0.0
122.2 ± 0.4
11.1 ± 0.4
54 DAP 64.9 ± 0.3
81.0 ± 0.6
96.2 ± 0.9
117.8 ± 1.9
11.8 ± 1.3
a
Samples (~6.0 mg, db) and deionized water (~18.0 μL) were used for the analysis; To, Tp, Tc
and ΔH are onset, peak, conclusion temperature, and enthalpy change, respectively.
b
DAP = days after pollination. nd = not detectable.
A
62. 6
15 DAP
61. 6
20 DAP
Heat Fl ow Endo Up
62. 1
25 DAP
61. 1
30 DAP
60. 6
35 DAP
40 DAP
60. 1
59. 6
54 DAP
40
50
60
70
80
90 100 110 120 130 140
Temperature (°C)
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Heat Fl ow Endo Up
62
B
20 DAP
61. 5
25 DAP
61
30 DAP
35 DAP
60. 5
40 DAP
60
59. 5
54 DAP
40
50
60
70
80
90
100 110 120 130 140
Temperature (°C)
Fig. 1. DSC chromatograms of native GEMS-0067 starches (A) and methanol-defatted GEMS0067 starches.
The X-ray diffraction patterns of GEMS-0067 starches harvested at different kerneldevelopmental stages are shown in Fig.2. All those GEMS-0067 starches displayed the B-type
X-ray diffraction pattern. The crystallinity of the starch decreased from 24.8% on 15 DAP to
17.6% on 54 DAP while the amylose content increased. These results suggested that amylose
was less crystalline than amylopectin and gave less diffraction intensity.
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15 DAP (24.8%)
I nt ensi t y
20 DAP (20.8%)
25 DAP (17.6%)
30 DAP (21.5%)
35 DAP (16.2%)
40 DAP (15.8%)
54 DAP (17.6%)
3. 00
8. 00
13. 00 18. 00 23. 00 28. 00
Two- Thet a ( degr ee)
33. 00
Fig. 2. X-ray diffraction patterns of GEMS-0067 starches at different kernel-developmental
stages. Crystallinity (%) is given in parentheses.
The scanning electron micrographs (SEM) of GEMS-0067 starches are shown in Fig. 3.
Starch harvested on 15 DAP, consisting of more amylopectin, had smaller, more spherical, and
fewer rod/filamentous granules than those harvested on later DAP. The small granules also
showed rough surface, which resembled the loosely packed starch structures around the hilum.
As the kernels further developed, the proportions of rod/filamentous granules increased with the
increase in the amylose content.
A
B
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C
D
E
F
Fig. 3. SEM of GEMS-0067 starches. A: 15 DAP, B: 20 DAP, C: 25 DAP, D: 30 DAP, E: 35
DAP, and F: 40 DAP.
Objective 2. To produce easily digestible normal cornstarch for fuel ethanol production
Corn inbred lines 05GEM06031, 06GEM01778, 05GEM02989 and 05GEM06000 were
provided by Dr. Blanco. Corn grains for ethanol fermentation experiment using the conventional
method were ground to pass through a 2-mm screen in a MicroHammer mill IV (GlenMills Inc.,
NJ, USA). Samples used for other analyses and cold fermentation were ground to pass through a
0.5 mm screen using a Cyclone Mill (UDY corp., CO, USA). Starch, lipid, and protein contents
of the four corn lines are listed in Table 4. Sample 05GEM02989 had the largest starch content
(75.2%) among the four samples. Sample 05GEM06031 possessed the amylopectin with the
shortest branch chain length (Table 5) comparing with others. Consequently, Sample
05GEM06031 displayed the lowest gelatinization temperature (Table 6). Because of the largest
starch content, Sample 05GEM02989 produced the largest ethanol yield, as shown in Table 7.
Sample 05GEM06031, however, displayed the greatest conversion rate of starch to ethanol
(Table 4), which could be attributed to its short amylopectin branch chains and its great enzyme
digestibility.
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Table 4 Composition of corn grains (g/100 g dry matter)
Sample
Starch
Lipid
Protein
05GEM06031
70.7 ± 0.66
3.2 ± 0.05
12.1 ± 0.07
06GEM01778
68.9 ± 0.53
4.7 ± 0.08
12.3 ± 0.19
05GEM02989
75.2 ± 0.39
2.8 ± 0.12
9.4 ± 0.14
05GEM06000
69.7 ± 0.20
3.0 ± 0.07
13.3 ± 0.12
Table 5. Amylose content, branch chain length distribution of amylopectin and thermal property
Sample
05GEM06031
06GEM01778
05GEM02989
05GEM06000
Amylose
content (%)
Avg. CL of
amylopectin
24.1
24.3
29.5
20.7
17.9
19.3
19.6
20.2
Amylopectin chain length distribution
DP6-12 DP13-24 DP25-36 DP>36
34.9
47.1
10.8
7.2
26.2
53.6
11.2
9.0
25.9
52.2
12.6
9.3
25.3
51.5
12.2
11.1
Table 6. Thermal properties of isolated starches
Thermal properties
Sample
To (°C)
Tp (°C)
Tc (°C)
ΔH (J/g)
05GEM06031
61.9 ± 0.3
68.3 ± 0.1
73.2 ± 0.4
11.4 ± 0.5
06GEM01778
66.1 ± 0.5
71.4 ± 0.3
76.3 ± 0.7
12.1 ± 0.9
05GEM02989
63.2 ± 0.5
69.1 ± 0.4
74.4 ± 0.1
11.8 ± 0.2
05GEM06000
65.3 ± 0.2
70.6 ± 0.2
75.0 ± 0.5
12.1 ± 0.6
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Table 7 Ethanol yield and conversion efficiency at 72 h fermentation
Sample
Ethanol yield
(g/100g dry corn)
Conversion
efficiency (%)
05GEM06031
36.2 ± 0.6
90.3 ± 1.6
06GEM01778
34.6 ± 0.2
88.4 ± 0.6
05GEM02989
38.0 ± 0.7
89.1 ± 1.7
05GEM06000
34.3 ± 0.9
86.8 ± 2.4
Additional 11 samples, Ames 2757, Ames 6048, NSL 34753, PI 172330, PI 221831, PI 451693,
PI 452052, PI 511664, PI 583352, PI 607516, and PI 645585, were also provided by Dr. Blanco
for ethanol production tests using a cold-fermentation process. The cold-fermentation process
was to hydrolyze starch granules in the dry-grind corn using granule-degrading amylases without
cooking corn to gelatinize starch. The ethanol yields of the 11 corn lines varied from 35.15g
ethanol/100g corn to 36.96g ethanol/100g corn as shown in Table 8.
Table 8. Ethanol yield and conversion efficiency of 11 corn lines using a cold-fermentation
technique
Sample
Ethanol yield
Conversion
(g/100g corn)
Efficiency (%)
Ames 2757
36.31
96.98
Ames 6048
35.15
92.72
NSL 34753
35.46
91.93
PI 172330
36.96
94.19
PI 221831
35.68
90.68
PI 451693
36.93
95.35
PI 452052
35.60
93.68
PI 511664
36.58
95.43
PI 583352
35.94
96.39
PI 607516
36.22
93.44
PI 645585
35.77
94.21
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In summary, during this report period, we have revealed the mechanism of
rod/filamentous granule formation in the amyloplast of GEM 0067 line. We also characterized
ethanol yields and starch conversion efficiency of different normal corn lines for breeding
program to produce new GEM lines, which will give superior ethanol yields.
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