Project Title: Value-added Utilization of GEM Normal and High-amylose Line Starch
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 starches. Two types of starch, high-amylose, ae
maize starch developed from GEM amylomaize project by Dr. Mark Campbell at Truman State
University, and normal maize grains of GEM lines supplied by the GEM Project Coordinator, Dr.
Mike Blanco, were the main genotypes used in this study. Three new GEM line high-amylose
maize starches, from GUAT209:S13 × (OH43ae×H99ae) B-B-4-1-2-1-1(designated as GOSH 1),
GUAT209:S13 × (OH43ae×H99ae) B-B-4-4-2-1-1(GOSH 2), and GUAT209:S13 × (OH43ae×H99ae)
B-B-4-4-2-1-2 (GOSH 3), have been identified having larger resistant starch (RS) contents
(37.3%-43.4%) than the inbred ae starches from H99ae, OH43ae, B89ae, and B84ae (10.6%14.1%). The RS contents are positively correlated with the apparent (r = 0.99) and the absolute
amylose contents (r = 0.96). The amylopectin, the large molecular-weight intermediate
components, and the small molecular-weight intermediate components were separated, and their
structures were determined. The small molecular-weight intermediate components consisted of
longer branch chain-length than the amylopectin and the large molecular-weight intermediate
components. The conclusion gelatinization temperatures of the new GEM line ae starches were
substantially higher than that of the inbred line ae starch samples.
To fully understand the molecular and granular structures of these three new GEM line ae
starches that consisted of large RS contents, we studied the morphology of the native starch and
the RS that remained after thermal stable enzyme hydrolysis at the water boiling temperature.
We also analyzed the molecular structures of the RS to reveal the mechanism of the enzyme
resistance. To study normal GEM line starches, selected normal GEM line starches were
subjected for enzyme digestibility tests to determine their suitability for small animal feed and
for ethanol production. Results showed substantial differences in enzyme digestibility both in
the cooked and uncooked dry-grind corn among different lines.
Objectives
Objectives of this research project are to identify starch lines of desirable characteristics
and to develop value-added utilization of GEM starch. Specific objectives of the study are:
1. To reveal the structures of the new GEM line high-amylose maize starch and understand the
mechanism of RS formation in the granule.
2. To produce easily digestible normal cornstarch to be used for feed of small animals and for
fuel ethanol production.
Progress Made in 2007
Objective 1. To reveal the structures of the new GEM line high-amylose maize starch and
understand the mechanism of RS formation in the granule
The RS contents of defatted and undefatted high-amylose maize starches were
determined and compared to reveal the impact of lipid contents. The results are given in Table 1.
The RS content of defatted starch of the new GEM ae-lines (27.8 – 28.9 %) was about 10-15%
less than that of undefatted starches (37.3 - 43.4%) (Table 1). The RS contents of defatted inbred
ae-line starches (9.3-11.0%), however, were slightly less than that of undefatted starches (10.614.1%) (Table 1). These results indicated that amylose-lipid complex contributed to the total RS
contents.
Table 1. RS contents of ae-maize starches
Sample
GOSH 1
GOSH 2
GOSH 3
H99ae
OH43ae
B89ae
B84ae
RS (%)
Before defatting
41.5±2.0
43.4±3.2
37.3±0.7
13.0±0.3
14.0±0.5
14.1±2.3
10.6±1.4
After defattinga
28.9±0.4
27.8±2.8
27.8±0.2
11.0±0.9
10.9±0.3
9.3±0.3
9.7±2.1
a
The native ae-mutant maize starches were defatted using methanol in a Soxhlet extractor
for 24h before resistant starch content was analyzed.
The RS, obtained after boiling the ae-mutant maize starch with thermal stable α-amylase
and incubating with protease and glucoamylase at 60°C, was subjected to thermal property
analysis using a differential scanning calorimeter. The results are shown in Table 2. All the
resistant starches gave similar onset (To, 107.3-114.4°C), peak (Tp, 118.3-122.4°C), and
conclusion (Tc, 125.9-134.0°C) gelatinization temperature (Table 2). The gelatinized resistant
starches were cooled down immediately to 25 °C at a cooling rate of 40°C /min and then
rescanned using the same parameter. The To, Tp, and Tc of the rescanned resistant starches were
86.3-94.8°C, 100.2-115.4 °C, and 115.5-126.1°C with substantially larger enthalpy changes
(Table 2). The increased enthalpy changes reflected that the resistant starch, having smaller
molecular weights, was much more prompt to recrystallization.
Table 2. Thermal properties of resistant starches (RS) from ae-maize starches a, b
Sample
GOSH 1
GOSH 2
GOSH 3
H99ae
OH43ae
B89ae
B84ae
a
Thermal properties of RS
Rescan of the RS
T0 (°C)
Tp (°C)
Tc (°C)
∆H (J/g)
T0 (°C)
Tp (°C)
Tc (°C)
∆H (J/g)
114.4±0.9
113.5±0.7
112.3±0.8
107.3±1.0
107.3±3.0
111.1±0.9
110.5±1.9
121.5±0.7
120.4±0.8
122.4±2.7
118.8±0.2
119.6±4.4
118.3±0.7
118.7±0.0
129.3±0.4
134.0±1.4
128.4±0.8
131.9±4.4
128.5±1.3
125.9±0.4
126.0±0.1
2.6±0.0.1
3.9±0.1
3.1±0.8
6.7±1.0
2.5±0.3
1.7±0.4
2.7±1.1
94.8±1.8
94.0±0.1
94.3±0.4
88.6±2.0
86.3±3.7
92.4±2.3
89.0±1.5
114.7±0.0
115.0±1.2
115.4±0.4
106.6±0.4
100.2±0.2
104.0±1.6
107.6±1.5
126.1±1.0
124.6±0.1
125.3±0.9
119.8±1.4
119.5±0.7
117.5±2.6
115.5±0.1
6.2±0.1
6.1±0.2
6.5±0.1
8.3±0.7
6.9±0.1
4.8±0.6
4.9±0.6
Samples (~6.0 mg, ~ 10% MC) and deionized water (~18.0 mg) were used for the analysis; T0, Tp, Tc and ∆H are onset, peak,
conclusion temperature, and enthalpy change, respectively.
b
Values were calculated from at least two replicates; ±Standard deviation.
Scanning electron micrographs (SEM) of representative ae-maize starches of the new
GEM lines and the inbred lines are shown in Fig. 1. All ae-maize starches contained two types of
starch granules, with spherical or rod/filamentous shapes. Starches of the GEM ae-lines
consisted of larger proportions of rod/filamentous granules (22.6-32%) (Fig. 1A) than that of
inbred ae-lines (5.2-7.7%) (Fig. 1B). The rod/filamentous starch granules consisted of rod,
filamentous, triangle, sock, and other shaped granules (Fig. 1A). Some filamentous granules
were more than 50 µm long. The SEM of RS of the new GEM ae-lines showed large numbers of
residual granules remained after boiling for 30 min with thermal stable α-amylase. The
remaining starch granules were found in the shapes of granule fragments and deformed granules
(Fig. 2A). The inbred line ae starches, however, were mostly dispersed to gels after the same
thermal and enzyme treatments (Fig. 2B). Shell-shaped fragments were observed in all ae-line
starches (Figs. 2A & B).
A
B
Fig. 1. Scanning electron micrographs of native starches at 1500X. A and B are native starches
of GSOH 2 and B84a, respectively.
A
B
Fig. 2. Scanning electron micrographs of resistant starches at 1500X. A and B are resistant
starches of GSOH 2 and B84a, respectively.
The polarized and phase-contrast light micrographs of native starches are shown in Fig. 3.
Comparison of polarized and phase-contrast light micrographs of native starches showed that
spherical starch granules displayed Maltese cross birefringence, some tadpole granules displayed
Maltese cross at the head but no birefringence along the tail, some rod granules displayed no
birefringence, some granules contained two Maltese cross, and some rod starch granules
displayed weak birefringence at the periphery (Fig. 3A, 3B, 3C & 3D).
A
B
C
D
Fig. 3. Light micrographs of native starches at 400x. polarized (A) and phase-contrast (B) light
micrographs of native starch of GSOH 1; polarized (C) and phase-contrast (D) light micrographs
of native starch of H99ae.
All ae- maize starches gave the B with some V type X-ray diffraction patterns. The
crystallinity of the new GEM ae-line starches (22.8-26.1%) was lower than that of the inbred aeline starches (27.5-33.0%). The RS obtained from the new GEM ae-line starches also displayed
the B with V type X-ray diffraction patterns with crystallinity ranged from 22.1 to 24.1%. It
indicated that the crystalline lamellar-structures remained after boiling with thermal stable αamylase.
The molecular-weight distributions of RS showed three distinct peaks, which were
designated as large (F1), medium (F2), and small (F3) molecular-weight fractions (Fig. 4). The
ratios of F1:F2:F3 are shown in Table 3. The RS from the new GEM ae-line starch showed a
larger proportion of F2 than that from the inbred ae-line starch (Fig. 4 and Table 3). The average
molecular weights of RS from the new GEM ae-line starches were 2.6 – 3.3 × 104 g/mol, larger
than those of RS from the inbred ae-line starches (1.4 – 1.7 × 104 g/mol). The molecular weights
of F2 and F3 ranged from 1.36 × 105 to 1.54 × 105 g/mol and from 0.96 × 104 to 1.20× 104 g/mol,
respectively (Table 3).
6
0.78
5
0.73
4
0.68
3
0.63
F1
0.58
F2
1
0.53
0
5
10
15
20
6
0.98
2
F3
7
1.03
RI signal (v)
0.83
B84ae
1.08
Log M w
RI signal (v)
7
25
0.93
5
0.88
4
0.83
0.78
3
0.73
0.68
F2
F1
0.63
Log M w
GSOH 1
0.88
2
F3
1
0.58
0
5
10
Elution volume (mL)
15
20
25
Elution volume (v)
Fig. 4. High performance size-exclusion chromatogram of RS determined using an HPSEC-MALLS-RI system.
Table 3. Molecular weights of fractions of the resistant starches a, b
% Ratio
Sample
GOSH 1
GOSH 2
GOSH 3
H99ae
OH43ae
B89ae
B84ae
a
F1
5.5
5.2
5.0
2.9
2.3
4.3
3.9
F2
36.9
37.9
30.8
20.2
19.2
18.2
14.7
F3
57.6
56.9
64.2
76.9
78.4
77.5
81.4
Molecular
weight of F2
×105 (g/mol)
1.54 ± 0.01
1.53 ± 0.00
1.40 ± 0.06
1.41 ± 0.05
1.41 ± 0.04
1.43 ± 0.02
1.36 ± 0.04
Molecular
weight of F3
×104 (g/mol)
1.17 ± 0.01
1.20 ± 0.01
1.12 ± 0.08
0.99 ± 0.01
1.02 ± 0.01
1.00 ± 0.01
0.96 ± 0.01
Average Mw of
F2 and F3
×104 (g/mol)
3.21 ± 0.14
3.32± 0.01
2.55 ± 0.26
1.72 ± 0.00
1.71 ± 0.01
1.66 ± 0.02
1.42 ± 0.01
The fully dispersed RS was injected into a high-performance size-exclusion (HPSEC)refractive index (RI) system; molecular weights were determined using pullulan standards.
b
F1, F2 and F3 were high, medium, and low molecular weight fractions of RS, respectively.
The F2 fractions isolated from the RS were subjected to debranching reactions using
isoamylase. The high-performance size-exclusion chromatograms of the F2 before and after the
debranching reaction are shown in Fig. 5. After debranching, all the F2 fractions showed a new
peak with chain-length of DP 84-112 in addition to the peak of large molecules with DP>250
except that of B84ae (DP 45). These results indicated that the F2 fraction was amylose molecules,
which was tightly packed in crystalline structures and could not be dispersed at the boiling
temperature and hydrolyzed by thermal stable enzymes
Peak1 DP 603
7500
6500
Peak2 DP 556
6400
5500
5400
4500
4400
3500
3400
2400
2500
1400
1500
8
11
14
17
20
23
El ut i on t i me ( mi n)
Peak2 DP 792
30000
RI si gnal ( uRI U)
20000
20000
Peak1 DP 773
15000
15000
10000
10000
5000
5000
0
8
13
18
23
El ut i on t i me ( mi n)
28
Peak2 DP 792
35000
30000
Peak3 DP 102
25000
20000
4800
2800
6300
18
23
El ut i on t i me ( mi n)
28
Peak1 DP 99
800
14800
Peak3 DP 84
12800
10800
5300
8800
4300
6800
3300
4800
2300
2800
8
15300
13300
25000
11300
13
18
23
El ut i on t i me ( mi n)
28
Peak2 DP 268
B89ae
800
19000
Peak3 DP 85
17000
15000
Peak1 DP 312
13000
9300
11000
7300
9000
5000
5000
3300
0
1300
28
13
Peak2 DP 252
10000
18
23
El ut i on t i me ( mi n)
8
OH43ae
10000
13
6800
2300
15000
8
8800
3300
15000
0
10800
4300
30000
20000
Peak1 DP 1339
12800
5300
1300
35000
GSOH 3
14800
6300
7300
0
16800
7300
8300
25000
Peak3 DP 112
18800
Peak3 DP 86
Peak2 DP 339
8300
1300
30000
GSOH 2
25000
RI si gnal ( uRI U)
26
9300
RI si gnal ( uRI U)
RI si gnal ( uRI U)
Peak3 DP 92
Peak1 DP 322
H99ae
10300
RI si gnal ( uRI U)
8400
7400
11300
8500
RI si gnal ( uRI U)
GSOH 1
9400
7000
5300
5000
3000
8
13
18
23
El ut i on t i me ( mi n)
28
1000
26500
B84ae
26200
Peak1 DP 56 Peak3 DP 45
RI si gnal ( uRI U)
21500
21200
16500
16200
Peak2 DP 322
11500
11200
6500
1500
6200
8
13
18
23
El ut i on t i me ( mi n)
28
1200
Fig. 5. High performance size-exclusion chromatograms of F2 (fractions 61-71) from resistant
starch determined using a HPSEC-RI system.
before debranching;
after debranching.
The branch chain-length distributions of F3 isolated from the RS are shown in Table 4.
Average branch chain-lengths varied between DP31.8 and 41.9. The longest detectable branch
chain-lengths ranged between DP85 and 100. In general, the F3 of the three new GEM ae-line
starches had longer average branch chain-lengths (DP39.2-41.9) than the F3 of the inbred ae-line
starches except that of B84ae (Table 4). Large proportions of long branch-chains (DP≥37) were
found in all the F3 fractions of RS. The F3 molecules were likely resulted from crystalline
intermediate components in the starch granules, which were resistant to enzyme hydrolysis.
Table 4. Branch chain-length distributions of F3 (fractions 72-85) from resistant starches a
Sample
GSOH 1
GSOH 2
GSOH 3
H99ae
OH43ae
B89ae
B84ae
DP≤12
4.5±0.2
2.9±0.2
3.4±0.0
7.7±0.1
5.4±0.2
6.5±0.3
5.6±0.1
DP13-24
22.1±0.2
19.0±0.2
26.7±0.6
35.1±0.1
30.7±0.2
23.0±0.7
20.1±0.1
DP25-36
22.5±1.1
22.4±0.7
23.0±0.6
24.7±0.5
25.9±0.4
26.6±0.2
20.0±0.6
DP≥37
50.8±1.2
55.5±0.6
47.6±1.6
32.5±0.3
38.0±0.0
43.5±1.4
54.3±0.6
Average
CL (DP)
40.3
41.9
39.2
31.8
34.4
35.7
40.2
Longest
detectable
DP
96
98
100
85
95
85
94
a
The branch chain-length distribution was analyzed using a fluorescence assistant capillary
electrophoresis (FACE) system.
Objective 2. To produce easily digestible normal cornstarch to be used for feed of small-animal
and for fuel ethanol production
Grains of selected normal GEM lines were analyzed for their starch contents. Starch content was
determined using total starch kits containing alph-amylase and glucoamylase, and glucose
determined using GOPOD method. The starch contents varied between 66.21 and 75.15% (Table
5). The grains were finely ground to pass a screen of 0.5mm pore size. The enzyme digestibility
of the dry-ground corn grains was analyzed in uncooked aqueous slurry using porcine pancreatic
α-amylase (PPA) for up to 48 hours and in cooked paste (at 85ºC) using thermal stable αamylase and incubated at 85ºC for up to 60 min. Results showed that in the paste form, the
enzyme digestibility at 60 min varied from 92.22 to 97.51% (Table 5), whereas in the uncooked
ground corn slurry, the digestibility after 48 hours varied from 75.21 to 82.69% (Table 5).
Grains of sample 05GEM02989 displayed the largest enzyme digestibility; the grains also had
the largest starch content.
Table 5. Starch content and enzyme digestibility of dry-grind corn samples
Inventory
05GEM03094
06GEM01721
06GEM01621
05GEM02989
06GEM02683
05GEM06031
05GEM02740
06GEM01778
05GEM06000
Pedigree/Characteristics
AR17056:N2025-574-001-B-BB-B
AR17056:N2025-574-001-B-BB-B-B
DKB844:S1601-289-001-B-B-BB-B
New-high starch, high density,
low prot.
New-high starch, low prot.
New-low Gel.Temp
New-high starch, high density,
low prot.
New-high prot, high oil, high
density
New-med prot, med oil, med
density
Starch
content
Enzyme digestibility*
Thermostable
PPA***
α-amylase**
(%, db)
30min
60 min
3h
24 h
****
48h
66.76
93.75
94.32
26.09
44.54
75.21
66.21
89.43
92.22
24.94
47.45
76.42
71.29
92.94
93.62
25.63
47.79
77.71
75.15
72.27
70.71
96.95
95.40
91.84
97.14
97.51
93.13
24.67
49.29
82.69
27.87
31.37
51.32
54.85
80.69
80.82
74.31
94.25
94.62
25.14
47.77
79.35
68.89
94.23
95.29
25.53
49.78
78.12
69.67
94.25
94.54
25.46
49.35
77.29
* per 100 g starch
** Flour slurry was pre-incubated at 85°C for 30 min and the hydrolyzed by thermostable αamylase (0.5 U/100 mg starch) at 85°C
*** Raw flour was hydrolyzed by PPA at 37°C (125 U/100 mg starch)
**** 125 U of new PPA was added to the slurry after 24 h