Project Title: Development of GEM line starch to improve nutritional value and
bio-fuel production
Prepared by Hanyu Yangcheng, Michael Reed and Jay-lin Jane, Department of Food Science
and Human Nutrition, Iowa State University, Ames, IA 50011
Project Overview
This report serves to document research conducted under a cooperative agreement
between ARS and Iowa State University. Specific objectives of this research project are
to (1) characterize the molecular structure and starch granule formation and develop
analytical methods to identify germplasm for high-digestibility and resistant starch, (2)
characterize and develop utilizations of GEM line starch to improve nutritional value to
humans and animals, and (3) characterize GEM line normal and waxy starch for biofuel
production and to improve the yield of ethanol.
For Objectives 1 & 2, three GEM lines, 06GEM02119 (normal), 03GEM00138 (high
protein), and 09GEM07913 (high oil), planted in the field of the North Central Regional
Plant Introduction Station (Ames, IA) in 2010, were used for this study. The kernel starch
contents of these three lines were 60.1%, 62.2%, and 62.5%, respectively. The amylose
contents of the starch samples, analyzed using iodine potentiometric titration, were
30.8%, 31.8%, and 27.7%, respectively. The protein contents were 12.5%, 11.2%, and
9.9%, respectively. The high-oil line, 09GEM07913, had a lipid content of 5.5%, which
were greater than the other two lines, 06GEM02119 (3.3%) and 03GEM00138 (4.0%).
The starch hydrolysis of the dry-ground corn and the isolated starch was analyzed using
porcine pancreatic alpha-amylase after cooking. The dry-grind kernels of the high-oil
09GEM07913 line showed the slowest starch-hydrolysis rate among the three, which
could result from the formation of an amylose-lipid complex during cooking. There was
no difference in the rate of starch hydrolysis between samples of the isolated starch.
For Objective 3, thirteen GEM lines of the 2009 and 2010 crop years, including four
normal GEM lines and nine waxy GEM lines, were selected for ethanol production
studies using a cold-fermentation process. The largest ethanol yield using a raw-starch
fermentation technique of waxy corn was 37.9g/100g (line 08GEM05036), which was
greater than the largest ethanol yield of the normal corn (line 08GEM04701, 37.5g/100g).
The total starch contents of the waxy lines, ranging from 65.3% (line 08GEM05042) to
72.8% (line 08GEM05037), were proportional to their ethanol yields (R2=0.76, p<0.01).
The average starch-to-ethanol conversion efficiency of the waxy corn (93.6%) was
substantially greater than that of the normal corn (88.1%). Starch of six waxy lines
(08GEM05036, 05037, 05039, 05040, 05041 and 05042) was isolated for chemical and
structural analysis, including branch-chain length distribution of amylopectin, thermal and
pasting properties, and the enzymatic hydrolysis of raw starch. The enzymatic hydrolysis
of isolated starch showed different kinetics from that of starch in the dry-grind corn,
resulting from effects of cell wall structure and the protein matrix in the dry-grind corn.
Publications and Presentations
1) S. Srichuwong and J. Jane, “Characterization of residual starch in distiller’s dried
grains with solubles (DDGS).” Cereal Chemistry, 88 (2011) 278-282.
2) H. Jiang and J. Jane, “Analysis of resistant starch content using differential
scanning calorimetry.” A keynote lecture to North America Thermal Analysis Assoc.
Des Moines, Iowa, August 7-10, 2011
3) J. Jane, “Structures and properties of resistant starch and its health benefits.” A
plenary lecture to the 2nd EPNOE International Polysaccharide Conference,
Wageningen, the Netherlands, August 29 – September 2, 2011.
4) J. Jane, “Structures and applications of Type-2 and Type-5 resistant starch,” An
invited lecture to Starch Roundtable Conference, Palm Springs, CA, October
12-14, 2011.
5) J. Medic, S. Setiawan, Y. Ai, C. Hurburgh, C. M. L. Franco, and J. Jane, “Effects of
postharvest-processing conditions on endogenous amylase activities of cereal.”
An invited lecture to American Assoc, of Cereal Chemists International, Palm
Springs, CA, October 15-19. 2011.
Objectives
Objective1: Characterize the molecular structure and starch granule formation and
develop analytical methods to identify germplasm for high digestibility and resistant
starch.
Objective 2: Characterize and develop utilizations of GEM line starch to improve
nutritional value to humans and animals.
Objective 3: Characterize GEM line normal and waxy starch for biofuel production and to
improve the yield of ethanol.
Progress made in 2011
Objective 1&2
Three GEM lines, 06GEM02119 (normal), 03GEM00138 (high protein), and
09GEM07913 (high oil) planted in the field of the North Central Regional Plant
Introduction Station (Ames, IA) in 2010 were used for this study. Corn kernels of the lines
were ground using a cyclone mill equipped with a 0.5mm screen. The dry-grind corn was
analyzed for starch, lipid and protein contents, and the data is shown in Table 1. Amylose
content of the starch isolated from the GEM lines was analyzed using iodine
potentiometric titration.
The starch content of the three GEM lines was 60.1% (line 06GEM02119), 62.2%
(line 03GEM00138) and 62.5% (line 09GEM07913), respectively. The greatest oil content
(5.5%) was in the 09GEM07913 line, and the 06GEM02119 line showed the greatest
protein content (12.5%) among the three GEM lines.
Table 1. Kernel Composition and of the GEM lines and amylose content of the starch
06GEM02119
Starch
(%)
60.1±0.2
Lipid
(%)
3.3±0.0
Protein
(%)
12.5±0.2
Amylose (%) of the
Starch
30.8±0.3
03GEM00138
62.2±0.1
4.0±0.0
11.2±0.1
31.8±0.3
09GEM07913
62.5±1.0
5.5±0.5
9.9±0.0
27.3±0.0
Sample
Starch hydrolysis (%)
The starch hydrolysis rate of the dry-grind corn and the isolated starch was analyzed
using porcine pancreatic α-amylase (PPA) after cooking. Dry-grind corn of the
09GEM07913 line had the largest lipid content and showed the slowest rate of hydrolysis.
Dry-grind corn of the 06GEM02119 line (normal) showed the greatest rate of hydrolysis,
and that of the 03GEM00138 line (high-protein) was in between (Figure 1). Starch
hydrolysis of the isolated starches using PPA, however, showed no difference among the
three lines (data not shown).
100
90
80
70
60
50
40
30
20
10
0
06GEM02119 (normal)
03GEM00138 (high-protein)
09GEM07913 (high-oil)
0
20
40
60
80
Time (min)
100
120
140
Figure 1. Enzyme hydrolysis of starch in dry-grind kernel of GEM lines.
Pasting properties of the starch isolated from the GEM lines were analyzed using a
Rapid Visco Analyzer (RVA). The peak viscosities of starches of the corn 06GEM02119,
03GEM00138, and 09GEM07913 lines were 158.1RVU, 136.1 RVU, and 135.8 RVU,
respectively, and the setback viscosities were 90.2 RVU, 87.8 RVU, and 81.3 RVU,
respectively. Starch from the 09GEM07913 line had the greatest peak viscosity (158.1
RVU) and the least setback viscosity (81.3 RVU), which could be attributed to its smaller
amylose content (27.3%).
200
100
180
160
Viscosity (RVU)
120
60
100
80
40
06GEM02119 (normal)
60
03GEM00138 (high protein)
40
09GEM07913 (high oil)
20
Temp
20
0
0
‐2
Temperature ºC
80
140
3
8
Time (min)
13
18
23
Figure 2. Starch pasting properties of the GEM lines. The pasting properties of the
isolated starches were analyzed using a Rapid Visco-Analyzer with 8% (w/w, dsb) starch
concentration.
Thermal properties of starches isolated from the three GEM lines are summarized in
Table 2 and Table 3. Onset gelatinization temperature (To) of the 09GEM07913 line
starch (63.2⁰C) was higher than that of the 03GEM00138 line (62.8⁰C) and the
06GEM02119 line (61.3⁰C) starches. Gelatinization enthalpy changes for the
06GEM02119, 03GEM00138, and 09GEM07913 starches were 52.8 J/g, 55.3 J/g, and
54.1 J/g, respectively. The enthalpy changes of dissociation of the amylose-lipid complex
of the 06GEM02119 and 03GEM00138 starches were higher (1.3 J/g) than that of the
09GEM07913 starch (0.6 J/g) (Table 3). This difference could be attributed to the lesser
amylose content of the 09GEM07913 starch.
Table 2. Starch thermal properties of the GEM line starch
Sample
Gelatinization of Starch
To (⁰C)
a
Tp (⁰C)
Tc (⁰C)
Dissociation of Retrograded Starch
∆H (J/g)
To (⁰C)
Tp (⁰C)
Tc (⁰C)
∆H (J/g)
R(%)b
06GEM02119 61.3±0.3 75.2±0.2 78.1±0.2 12.0±0.1
40.4±0.1 51.6±0.1 62.8±0.2
6.6±0.1
52.8
03GEM00138 62.8±0.3 72.7±0.1 78.1±0.1 11.2±0.1
41.9±0.4 53.4±0.2 63.1±0.1
6.2±0.1
55.3
09GEM07913 63.2±0.2 72.2±0.0 74.7±0.0 12.2±0.1
43.2±0.1 53.9±0.0 81.8±0.1
6.6±0.2
54.1
a
To = onset temperature, Tp = peak temperature, Tc = conclusion temperature, R% = percentage retrogradation, and ∆H =
enthalpy change.
b
% retrogradation = 100 × ∆H of dissociation of retrograded starch / ∆H of starch gelatinization.
Table 3. Thermal properties of dissociation of amylose-lipid complex
Sample
a
Raw starch
To(⁰C)a
Tp(⁰C)
Tc(⁰C)
Retrograded starch
∆H(J/g)
To(⁰C)
Tp(⁰C)
Tc(⁰C)
∆H(J/g)
06GEM02119
87.8±0.2
96.7±0.7 103.0±0.2
1.3±0.1
93.2±0.4
98.2±0.1 103.2±0.2
1.0±0.1
03GEM00138
85.6±1.0
95.4±0.2 101.2±0.7
1.3±0.1
91.1±0.1
97.9±0.5 101.8±0.3
0.5±0.0
09GEM07913
85.9±0.6
94.7±0.2 100.9±0.2
0.6±0.1
92.5±0.4
96.1±0.4 100.1±0.0
0.3±0.0
To = onset temperature, Tp = peak temperature, Tc = conclusion temperature, and ∆H = enthalpy change.
Objective 3
Thirteen GEM lines used for this study were planted in the field of the North Central
Regional Plant Introduction Station (Ames, IA) in 2009 and 2010 crop years. The
dry-grind corn was subjected to simultaneous raw-starch saccharification and ethanol
fermentation for 96hr using raw-starch hydrolyzing enzymes (Novozyme 5009,
Novozyme, Franklinton, NC). The supernatant was collected for ethanol yield analysis.
Ethanol yields for the 13 GEM lines grown in crop years of 2009 and 2010 are shown in
Table 4. The ethanol yields of the four normal lines of 2009 crop year ranged from 34.2%
(34.2g/100g dry matter) for the 08GEM04703 line to 37.2% for the 08GEM04701 line.
The data from the 2010 crop year ranged from 34.7% (the 08GEM04703 line) to 37.5%
(the 08GEM04701 line). For the nine GEM waxy lines, the 08GEM05041 line showed
the lowest ethanol yield of 33.1% among the 2009 samples, and the 08GEM05042 line
showed the lowest yield of 34.8% among the 2010 crops. The data of the 2010
08GEM05041 crop was not available because of insufficient quantity of corn kernels
for ethanol fermentation. The line 08GEM05036 showed the largest ethanol yield
among the waxy lines for both 2009 (37.5%) and 2010 (37.9%) crop years.
Table 4. Ethanol yield (g/100g dry matter) of raw-starch fermentation of GEM lines
2009 crop year 2010 crop year
Normal
Waxy
08GEM04701
37.2±0.9
37.5±0.3
08GEM04702
35.2±0.8
35.1±0.3
08GEM04703
34.2±0.7
34.7±0.7
08GEM04704
37.2±1.0
36.8±1.2
Average yield
36.0
36.0
08GEM05036
37.5±0.3
37.9±0.0
08GEM05037
36.3±0.3
37.2±0.1
08GEM05038
36.6±0.1
36.8±0.3
08GEM05039
35.7±0.2
36.0±0.2
08GEM05040
35.4±0.5
35.4±0.5
08GEM05041
33.1±0.7
N/A
08GEM05042
34.6±0.6
34.8±0.2
08GEM05043
36.1±0.3
35.6±0.6
08GEM05044
35.9±0.7
35.6±0.2
Average yield
35.7
36.2
There was no significant difference in ethanol yield between the GEM waxy and
normal lines. This resulted from a broad range of the starch content of the GEM waxy
lines (Table 5). Among the GEM waxy lines, the line 08GEM05036 (2009) showed the
greatest ethanol yield and had the largest starch content (71.1%), whereas the line
08GEM05041 (2010) had the smallest ethanol yield and the least starch content
(62.3%). The total starch contents of the GEM waxy lines were positively correlated to
the ethanol yields of both the 2009 (R2=0.84, p<0.001) and 2010 crops (R2=0.76,
p<0.01). The average conversion efficiency from starch to ethanol of GEM waxy lines
was 93.2% and 93.6% for the 2009 and 2010 crops, respectively, whereas the average
conversion efficiency of the normal lines was 87.9% (2009) and 88.1% (2010). The
waxy lines showed significantly greater conversion efficiencies than that of the normal
lines.
To understand how starch structure and properties affect the ethanol conversion
efficiency, six waxy GEM lines (08GEM05036, 5037, 5039, and 5040-5042) of the
2010 samples were selected for starch characterization. Corn kernels were
degerminated and decorticated, and endosperm starches were isolated following the
method reported by Li, et al. (2008).
Table 5. Starch content (%) and starch-to-ethanol conversion efficiency of GEM lines
Starch content (%)
Conversion efficiency
2009 crop
2010 crop
2009 crop
2010 crop
year
year
year
year
08GEM04701
74.3±0.7
74.1±0.5
0.88
0.89
08GEM04702
71.5±0.8
70.5±0.4
0.87
0.88
Normal
08GEM04703
68.6±0.5
68.1±0.0
0.86
0.9
08GEM04704
74.1±0.7
74.1±0.6
0.89
0.88
Average
72.1
71.7
0.88
0.89
08GEM05036
71.1±0.3
72.3±0.2
0.93
0.92
08GEM05037
71.0±0.6
72.8±0.0
0.9
0.9
08GEM05038
67.5±0.5
67.3±0.7
0.96
0.96
08GEM05039
68.0±0.6
69.2±0.0
0.93
0.92
08GEM05040
66.6±0.3
66.4±0.2
0.94
0.94
Waxy
a
08GEM05041
62.3±0.2
0.94
N/A
N/A
08GEM05042
64.1±0.0
65.3±0.1
0.95
0.94
08GEM05043
68.9±0.7
66.7±0.2
0.92
0.94
08GEM05044
Average
68.1±1.0
67.5
66.8±0.4
68.4
0.93
0.93
0.94
0.94
Starch hydrolysis rates of uncooked isolated starch samples and the dry-grind corn
kernels using Novozyme 5009 are shown in Figure 3. The waxy dry-grind corn kernels
and isolated starches displayed substantially greater hydrolysis rates than the
dry-grind B73 normal line as the reference. Most of the dry-grind corn samples
displayed greater hydrolysis rates during the first 3-6hr hydrolysis time than the
isolated starches, but the rate was reduced after 6hr hydrolysis. The differences were
attributed to the presence of endogenous amylases and damaged starch present in the
dry-grind corn. For the 2010 corps, the percentage hydrolysis of the dry-grind corn
positively correlated with ethanol yields (R2=0.93, p<0.01).
100
A
B
90
80
80
70
60
50
08GEM05036
08GEM05037
08GEM05039
08GEM05040
08GEM05041
08GEM05042
B73
40
30
20
10
0
Time(h) 0
100
Starch hydrolysis (%)
Starch hydrolysis (%)
90
12
24
36
48
60
70
60
50
30
10
72
0
Time(h) 0
12
24
36
48
60
72
100
C
90
90
80
80
70
70
60
08GEM05036
50
08GEM05037
40
08GEM05039
30
08GEM05040
08GEM05041
20
24
36
48
60
60
50
08GEM05036
40
08GEM05037
30
08GEM05039
08GEM05040
08GEM05042
10
B73
12
D
20
08GEM05042
10
0
Time(h) 0
08GEMS5036
08GEMS5037
08GEMS5039
08GEMS5040
08GEMS5041
08GEMS5042
B73
40
20
Starch hydrolysis (%)
Starch hydrolysis (%)
100
72
0
Time(h) 0
B73
12
24
36
48
60
72
84
96
Figure 3. Enzymatic hydrolysis of isolated starch and starch in dry-grind kernels of selected GEM waxy lines. The B73 normal corn was used as the control. A:
Isolated starch of the 2009 crop year; B: Dry-grind corn of the 2009 crop year; C: Isolated starch of the 2010 crop year; D: Dry-grind corn of the 2010 crop year.
Amylopectin branch-chain length distributions of the six GEM waxy lines are
shown in Table 6. Average branch-chain lengths of the waxy corn amylopectin were
negatively correlated with the ethanol yields for both the 2009 (R2=0.98, p<0.01) and
the 2010 crops (R2=0.95, p<0.01).
Table 6. Amylopectin branch-chain length distribution of the selected GEM waxy lines
DP6-12 DP13-24 DP25-36 DP>37
ave. CL
08GEM05036 23.8±1.4 49.4±1.8 12.9±1.0 13.8±2.1 21.4±0.7
08GEM05037 22.5±0.3 47.7±0.1 13.5±1.3 16.3±0.9 22.4±0.4
08GEM05039 22.9±0.0 49.0±0.4 12.1±0.6 16.0±1.0 22.5±0.4
2009
crop year 08GEM05040 23.1±0.3 46.9±1.5 12.5±0.1 17.5±2.0 23.0±0.6
08GEM05041 22.3±0.2 45.3±1.2 11.8±0.6 20.5±2.0 24.3±1.0
08GEM05042 22.4±0.4 47.1±0.1 11.6±0.7 18.9±1.1 23.4±0.6
08GEM05036 25.7±0.6 48.7±0.7 13.1±0.7 12.5±0.7 21.1±0.4
08GEM05037 25.1±2.0 47.7±0.2 13.3±0.7 13.8±1.0 21.6±0.6
08GEM05039 21.4±0.1 47.4±0.2 14.8±0.1 16.4±0.4 23.1±0.0
2010
crop year 08GEM05040 22.2±0.2 48.2±0.6 13.6±0.2 16.1±0.6 23.1±0.2
08GEM05041 21.1±0.0 47.2±0.2 14.0±0.1 17.7±0.3 23.9±0.1
08GEM05042 21.1±0.2 48.0±0.4 14.4±0.2 16.6±0.4 23.5±0.0
a
Molar basis
b
Average branch-chain lengths of amylopectin
Thermal properties of the six GEM waxy line starches are shown in Table 7. All the
waxy line starch had higher gelatinization peak temperature, 69.6-72.9°C (2009) and
71.3-74.1°C (2010), and conclusion temperature, 74.9-79.1°C (2009) and 77.3-80.5°C
(2010), than that of the B73 normal corn (68.0°C and 73.2°C, respectively). The
percentage retrogradation was positively correlated with the average branch-chain
length of amylopectin (R2=0.88, p<0.01 for the 2009 crops, and R2=0.86, p<0.01 for the
2010 crops). These results were in agreement with previously reported data that
amylopectin with long-branch chains had a greater tendency to retrograde (Jane, et al.,
1999).
Summary and future studies
Dry-grind kernels of the GEM high-amylose corn line showed a slower starch
hydrolysis rate than the normal and high-protein lines. The high-oil corn line can be
developed for health food application. Raw-starch ethanol fermentation yields of the
GEM waxy lines were positively correlated with the starch contents and the starch
hydrolysis percentages using raw-starch hydrolyzing enzymes. GEM waxy lines
displayed greater average starch-to-ethanol conversion efficiencies than the normal
lines in the raw-starch fermentation process, although the average ethanol yield of the
waxy and normal lines were similar.
For the future work, studies will be conducted to develop health food products
using the GEM high-oil corn. The amylose content and amylopectin branch-chain
length distribution of the starches of the four GEM normal lines will be analyzed. The
information would be useful to determine how the structural properties of the starch
affect the ethanol yields of the GEM normal lines.
References
1. Li, L.; Jiang, H.; Campbell, M.; Blanco, M.; Jane, J. Characterization of maize
amylose-extender (ae) mutant starches. Part I: Relationship between resistant starch
contents and molecular structures. Carbohydr.Polym 2008, 74, 396-404.
2. Jane, J.; Chen, Y. Y.; Lee, L. F.; McPherson, A. E.; Wong, K. S.; Radosavljevic, M.;
Kasemsuwan, T. Effects of amylopectin branch chain length and amylose content on the
gelatinization and pasting properties of starch. Cereal Chem. 1999, 76 (5), 629-637.
Table 7. Starch thermal properties of the selected GEM waxy lines of 2009 and 2010 crop years
Native
Retrograded
Tc(°C)
To(°C)
Tp(°C)
Tc(°C)
Tp(°C)
H(J/g)
H(J/g)
To(°C)a
43.3±2.1 58.0±0.2 67.5±0.8 4.7±0.1
08GEMS05036 62.3±0.2 69.6±0.1 76.1±0.8 14.8±0.1
08GEMS05037 63.6±0.2 69.8±0.7 75.0±0.7 15.6±0.1
42.2±0.3 56.0±2.6 64.2±2.1 5.2±1.3
2009
08GEMS05039 64.1±0.7 69.7±0.8 74.9±1.3 15.7±0.1
41.2±0.1 54.5±0.1 62.7±0.0 6.1±1.2
crop
08GEMS05040 64.3±0.4 70.2±0.1 76.8±0.3 15.5±0.0
40.3±1.7 54.8±0.2 62.9±0.0 6.5±0.1
year
08GEMS05041 65.8±0.2 71.9±0.1 77.8±0.9 16.4±0.1
42.2±0.3 55.1±0.1 64.3±0.7 8.5±0.7
43.0±0.8 55.1±0.0 64.3±0.4 7.9±0.4
08GEMS05042 65.0±0.5 72.9±0.4 79.1±0.3 15.9±02
08GEMS05036 65.1±0.2 71.4±0.1 77.3±0.3 15.7±0.3
43.1±0.4 54.0±0.2 61.4±0.1 6.5±0.3
08GEMS05037 65.4±0.0 71.3±0.0 77.5±0.0 15.4±0.1
42.0±0.2 54.7±0.4 64.6±0.2 6.6±0.1
2010
08GEMS05039 64.5±0.4 71.4±0.5 77.7±0.5 15.2±0.3
41.7±0.7 54.3±0.0 63.6±0.2 6.6±0.1
crop
08GEMS05040 57.7±0.3 69.6±0.9 79.9±0.3 15.1±0.0
41.4±0.3 54.6±0.0 63.4±0.3 6.6±0.1
year
08GEMS05041 63.3±0.1 72.0±0.0 80.5±0.9 16.2±0.1
42.6±0.4 54.3±0.0 63.0±0.1 7.4±0.2
08GEMS05042 66.6±0.3 74.1±0.0 79.2±0.1 15.9±0.1
43.0±0.2 54.9±0.0 64.0±0.0 7.2±0.0
B73
63.6±0.1 68.0±0.2 73.2±0.1 12.1±0.1
41.3±0.3 51.9±0.1 61.4±0.2 6.5±0.1
a
To= onset gelatinization temperature, Tp= peak temperature, Tc= conclusion temperature, H= enthalpy change.
b
Retrogradation (%)=100 × ∆H of dissociation of retrograded starch/∆H of starch gelatinization
Retrogradation(%)b
32.0±0.1
33.5±1.3
39.0±1.2
41.9±0.1
51.6±0.7
49.8±0.4
41.3±1.4
43.0±0.9
43.6±0.1
43.8±0.0
45.8±1.5
45.4±0.0
53.6±1.4