Project Title: Development of GEM line starch to improve nutritional value and biofuel production

Transcription

1 Project Title: Development of GEM line starch to improve nutritional value and biofuel production Prepared by Jay-lin Jane and Hanyu Yangcheng, Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 5 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 () characterize the molecular structure and properties of GEM corn starch to identify germplasm for high-digestibility and resistant starch; () characterize and develop utilizations of GEM corn starch to improve nutritional value to humans and animals; and (3) characterize GEM normal and waxy corn starch for biofuel production and to improve the yield of ethanol. For objective, two expired PVP (Ex-PVP) normal corn lines, G8 and LH8, were planted on May 8, at the North Central Regional Plant Introduction Station (Ames, IA). The corn lines were harvested on different dates (Oct.5 th and Oct.5 th ), and effects of the harvesting time on starch properties were analyzed. Starches of the corn lines harvested on Oct.5 th showed greater gelatinization conclusion temperatures than that harvested on Oct.5 th. For line LH8, starch of the corn harvested on Oct.5 th showed smaller gelatinization enthalpy change (ΔH=.J/g) and smaller molecular weight of amylopectin (M w =8. 8 g/mol) compared with that of the corn harvested on Oct.5 th (ΔH=.6J/g M w =9. 8 g/mol).there was no significant difference in the pasting properties of the starch of the corn harvested on different dates. For objective, starches of four ae wx double-mutant corn lines and one ae corn line were characterized to determine the slowly-digestible starch (SDS) and resistant- starch (RS) content. Starch of ae wx corn showed higher gelatinization onset temperature and greater enthalpy change but lower gelatinization conclusion temperature than the ae corn starch (Table 5). Enzymatic hydrolysis of the uncooked ae wx corn starch using porcine pancreas α-amylases showed similar enzymatic-hydrolysis kinetics to the uncooked ae corn starch. Analysis of starch digestibility showed that ae wx corn starch had lower RS content (4.-8.%) but higher SDS content ( %), compared with the ae corn starch (5.3% and 5.%, respectively). For objective 3, starch properties of four GEM normal corn lines grown in 9 and crop years were analyzed, and effects of starch properties on ethanol production were determined. The average starch-ethanol conversion efficiency (88.%) and percentage starch hydrolysis (77.4%) of the normal corn were substantially lower than that of the waxy corn (93.% and 95.%, respectively). The differences could be attributed to the greater amylose contents ( %) and longer average branch-chain-lengths of amylopectin (3.3 DP) of the normal corn starch compared with that of the waxy corn starch (.9-4.6% and. DP, respectively). The average gelatinization peak (T p, 69. C) and conclusion temperature (T c, 74.7 C) of the normal corn starch were significantly lower (p<.5) than that of the waxy corn starch (average T p =7. C and T c =77.4 C, respectively).the average gelatinization enthalpy-change of normal corn starch (.8J/g) was significantly (p<.) lower than that of the waxy corn starch (5.5J/g). Regression analyses showed that he amylose content of the starch showed significant negative-correlation with the starch-ethanol conversion efficiency (r= -.87, p<.). For the waxy corn samples, amylopectin branch-chain-lengths of the starch showed significant negative-correlations with the starch-hydrolysis rate and the ethanol yield.

2 Four waxy genotypes (two inbreds and two hybrids) grown in and eight genotypes (two waxy inbreds, three waxy hybrids, two normal inbreds and one normal hybrid) grown in were used for an ethanol cold-fermentation study. The ethanol production and corn kernel properties were compared between the inbred and hybrid corn. The hybrids showed similar starch contents and ethanol yields but slightly higher conversion efficiency compared with their parent inbred lines. -kernel weight of the hybrid corn were greater than that of their parent inbred lines, suggesting improved corn kernel yield of the hybrid corn. Publications and presentations: ) Yangcheng, Hanyu. Characterization of and Waxy Corn Starch for Bioethanol Production. Iowa State University,. ) H. Yangcheng, H. Jiang, M. Blanco and J. Jane. Characterization of and Waxy Corn Starch for Bioethanol Production. A manuscript submitted to Journal of Agricultural and Food Chemistry. 3) H. Yangcheng, H. Jiang, M. Blanco and J. Jane. Characterization of and Waxy Corn Starch for Bioethanol Production. Poster presentation to Corn Utilization & Technology Conference, Indianapolis, IN, June4-6,. Objectives Objective : Characterize the molecular structure and properties of GEM corn starch to identify germplasm for high-digestibility and resistant starch; Objective : Characterize and develop utilizations of GEM corn starch to improve nutritional values to humans and animals. Objective 3: Characterize GEM normal and waxy corn starch for biofuel production and to improve the yield of ethanol. Progress made in Objective : Characterize the molecular structure and properties of GEM corn starch to identify germplasm for high-digestibility and resistant starch; Starch physicochemical properties of GEM corn harvested on different dates Two Ex-PVP corn lines, G8 and LH8, planted in the field of the North Central Regional Plant Introduction Station (Ames, IA) in were used for a study to understand how harvesting dates impact starch structures and properties. The corn lines were planted on and pollinated at approximately the same date but harvested at different time points (Oct.5 th and Oct.5 th ). The harvested corn kernels were dried to % moisture content, and ground using a cyclone mill equipped with a.5mm screen. The dry-grind corn was analyzed for soluble sugars and starch content. Starches of the corn lines were isolated using a wet milling process, and the isolated starch was used for the analyses of starch thermal and pasting properties and amylopectin molecular weight. Soluble sugars and starch contents of kernel of the two corn lines are shown in Table. The dry-grind corn contained little soluble sugars for both G8 and LH8 (G8:.3-.4%;

3 LH8:.-.3%). Kernel starch contents of the corn were similar between the corn kernels harvested on different dates. Table. Soluble sugars and starch content of kernels of the corn lines harvested at different time Date of Soluble sugars Starch Line harvest (%) (%) Oct.5 th.4±. 73.7±. G8 Oct.5 th.3±. 73.4±.3 Oct.5 th.±. 7.8±.5 LH8 Oct.5 th.3±. 7.6±. Starch thermal properties are shown in Table. Starch of the corn lines harvested on Oct.5 th showed higher gelatinization conclusion temperature than that of the corn lines harvested on Oct.5 th. For line LH8, the sample of later harvesting time showed smaller gelatinization enthalpy change than the sample of early harvesting time. Table. Starch thermal properties of the corn lines harvested at different time Date of Line T a harvest o ( C) T p ( C) T c ( C) ΔH(J/g) Oct.5 th 65.±. 69.8±. 74.4±..±. G8 Oct.5 th 65.8±. 7.6±. 75.3±..±. Oct.5 th 64.±. 69.±. 73.8±..6±. LH8 Oct.5 th 64.±. 69.3±. 74.5±.4.±. a T o = onset gelatinization temperature, T p = peak temperature, T c = conclusion temperature, ΔH= enthalpy change. Starch pasting properties are shown in Figure, and the data is summarized in Table 3. There was no clear trend of changes in starch pasting properties for the samples harvested on different day. Table 3. Starch pasting properties of the corn lines harvested at different time Date of Pasting Peak Hold Final Breakdown Line harvest Temp.( C) (RVU a ) (RVU) (RVU) (RVU) G8 LH8 Setback (RVU) Oct.5 th 7.5± ±.6 9.3±.6 9.± ±. 99.7±.9 Oct.5 th 73.6±. 44.8±. 95.8±.4 9.± ±.5 94.±.6 Oct.5 th 7.9±. 48.6± ±.7 87.±.7 49.± ±. Oct.5 th 73.±. 48.9±.4 9.± ± ±. 96.8±. a RVU = Rapid visco unit 3

4 5 G8 8 RVU Temperature ( C) 5 5 Harvested on Oct.5th Harvested on Oct.5th Temperature 5 5 Time(min) LH8 8 RVU Temperature ( C) 5 Harvested on Oct.5th Harvested on Oct.5th Temperature 5 5 Time(min) Figure. Starch pasting profiles of the corn lines harvested at different time. Data of amylopectin molecular weights and gyration radii are shown in Table 4. For line LH8, the sample harvested on Oct.5 th showed smaller molecular weight of amylopectin (8. 8 g/mol) than that harvested on Oct.5 th (9. 8 g/mol), which might suggest degradation of amylopectin of the sample harvested on the later date. Table 4. Amylopectin molecular weights and gyration radii of the corn lines harvested at different time Date of M a Line w ( 8 ) R b harvest g/mol z (nm) G8 Oct.5 th 8.4±. 365.±. Oct.5 th 8.6± ±4.9 LH8 Oct.5 th 9.±. 37.5±.5 Oct.5 th 8.±. 36.±4. a Weight-average molecular weight b z-average radius of gyration 4

5 Objective : Characterize and develop utilizations of GEM corn starch to improve nutritional values to humans and animals. Characterization of ae wx double mutant corn starch Four ae wx double mutant GEM corn lines (59-, 67-, 88-3, and -) and one ae single mutant GEM corn line (48-3) were obtained from Truman State University. Isolated starch was characterized for physicochemical properties, including thermal properties, digestibility of the uncooked and cooked starch. Gel-permeation chromatography (GPC) profiles of the isolated starches are shown in Figure. All the ae wx corn starch showed single peak of amylopectin, whereas the ae corn starch showed peaks of amylopectin and amylose Blue Value Total Carb Blue Value Total Carb Blue Value Total Carb Blue Value Total Carb Blue Value Total Carb Figure. Gel-permeation chromatography (GPC) profiles of ae (48-3) and ae wx (59-, 67-, 88-3, and -) mutant corn starch. 5

6 Starch thermal properties of the mutant corn lines are shown in Table 5. Starch of ae wx corn showed lower gelatinization conclusion temperature ( C) but greater enthalpy change (8.6-. J/g) than the ae corn starch (4.4 C and. J/g, respectively). The differences were attributed to the crystalline structures of the mutant corn starch. It is known that amylopectin contributes to the starch crystallinity, whereas amylose molecules are present in an amorphous form (Jane 6). Starch of ae corn contained mainly amylose and a small proportion of amylopectin and, thus, had smaller gelatinization enthalpy change than the ae wx corn starch. Table 5. Starch thermal properties of the ae-containing mutant corn lines Gelatinization T o ( C) a T p ( C) T c ( C) H(J/g) - 69.±.7 79.± ±.3 8.7±.5 ae wx ± ±.9 95.±.8 8.6± ± ±. 9.±.5.± ± ±. 9.±. 8.9±. ae ±.5 8.6±.7 4.4±.3.±.4 a T o =onset gelatinization temperature, T p =peak temperature, T c =conclusion temperature, ΔH= enthalpy change. Enzymatic hydrolysis of the uncooked starch using porcine pancrease α-amylases is summarized in Table 6. Starch of a normal corn, line B73, was used as a control. After 48hr hydrolysis, 8.% of the normal corn starch was hydrolyzed to glucose, whereas less than 35% of the ae-containing mutant corn starch was hydrolyzed to glucose. The ae wx corn starch displayed similar hydrolysis-rates to the ae corn starch. Table 6. Percentages starch hydrolysis (%) of the ae-containing mutant corn and a normal corn 3h 6h h 4h 48h - 6.7±.3.±.5 4.8±.6 5.±.7 3.8±.7 ae wx ±..3±.3 4.8±.3 3.4±. 9.8± ±.4 4.±. 9.7±. 9.9±. 34.5± ±..±. 5.9±.3 6.9±.5 3.9±.6 ae ±. 3.6±.3 8.4±.5 9.5± ±.5 N. Maize B73 4.8±. 6.6±.3 4.±. 7.8±.3 8.±.5 6

7 Rapidly-digestible starch (RDS), slowly-digestible starch (SDS) and resistant-starch (RS) contents of the corn starch were analyzed following the method of Englyst, et al. (99), and the data are summarized in Table 7. After cooking, starch of B73 was almost all rapidly hydrolyzed in min (98.%). On the contrary, ae corn starch contained a large portion of RS (5.3%), and a significant portion of SDS (5.%). The RS content of ae corn starch was associated with its thermal properties. The T c of the ae corn starch (4.4 C, Table 5) was above water-boiling temperature, suggesting that the ae starch granules retained some of the B-type crystalline structures after heating in boiling water, which contributed to the resistance of ae starch to enzyme hydrolysis. Starch of ae wx corn had smaller RS contents (4.-8.%) but larger SDS contents ( %) compared with the ae corn starch (5.3% and 5.%, respectively). Table 7. RDS, SDS, and RS content of the mutant corn starch after cooking ae wx RDS% SDS% RS% ±.7 7.9±.6 4.6± ±. 7.9±.6 8.± ±. 7.6±. 4.± ±. 6.7±.9 5.8±.3 ae ±. 5.±. 5.3±. N. Maize B73 98.±.5.8±.8.±.6 Objective 3: Characterize GEM normal and waxy corn starch for biofuel production and to improve the yield of ethanol. Characterization of the normal and waxy corn starch for bioethanol production Twelve GEM corn lines (eight waxy and four normal lines) used for this study were planted in the field of the North Central Regional Plant Introduction Station (Ames, IA) in 9 and crop years. Previous results of kernel-starch contents and ethanol yields of the corn lines were reported in and some of the analyses were repeated in. Thus, data are included in the current report (Table 8) for the convenience of discussion. Starch physicochemical properties of the normal corn lines, including starch digestibility, amylose content, amylopectin branch-chain-length distribution, starch thermal and pasting properties, were analyzed and compared with that of the waxy corn lines. Effects of starch structures and properties on the ethanol production using the cold-fermentation process were determined. Enzymatic-hydrolysis rates of starch in dry-grind corn samples using the raw-starch hydrolyzing enzymes are shown in Table 9. The dry-grind waxy corn samples displayed substantially faster hydrolysis-rates than the normal corn samples. After 96h hydrolysis, more than 9% starch in the dry-grind waxy corn was hydrolyzed to glucose, whereas less than 8% starch in the dry-grind normal corn was hydrolyzed to glucose. 7

8 Table 8. Total starch content (%), ethanol titers, ethanol yields, and starch-ethanol conversion efficiencies of the normal and waxy corn a Line Starch content (%) 9 crop year crop year Ethanol concentration (ml/ml) 9 crop year crop year Ethanol yield (g/g dry grain) 9 crop year crop year Conversion efficiency (%) b 9 crop year crop year ±.3 7.3±. 8.7±. 8.9±. 37.6± ± ±.6 7.8±. 8.±. 8.4±. 36.3±. 37.± ± ±.7 8.±. 7.9±. 36.3±. 35.9± ±.6 69.±. 7.8±.4 7.9±. 35.7±.8 36.± Waxy ± ±. 7.6±. 7.6± ±. 35.3± ±. 65.3±. 7.±. 7.4± ± ± ± ±. 8.±.3 7.8±. 36.± ± ±. 66.8±.4 7.9±. 7.8±. 35.9±. 35.6± LSM±SEM c 68.3b ±.6 7.9a ±. 36.a ±.3 93.a ± ±.7 74.±.5 8.6±. 8.7±. 37.± ± ±.8 7.5±.4 7.5±.4 7.5±. 35.±.8 35.± ±.5 68.±. 7.±. 7.±.6 34.± ± ±.7 74.±.6 8.5±.3 8.4±.5 37.±.7 37.± LSM±SEM 7.9a ±.9 7.9a ±. 36.a ±.4 88.b ±.5 a Values are means ± standard deviations of two replicates. b Conversion efficiency (%) = ethanol yield (w/w)/theoretical yield of ethanol. c Least squares means (LSM) ± standard errors (SEM). Different letters following the LSM values within the same columns indicate statistically different mean values (p<.5). 8

9 Table 9. Percentages starch-hydrolysis (%) of the dry-grind grain a 9 crop year crop year Waxy Waxy Line 3h h 4h 7h 96h ±.3 47.±. 75.3± ± ± ±. 37.7±.3 67.±.5 94.± ± ±.3 4.±.4 67.±.5 9.± ±. 54.9±. 36.±. 64.3± ±.5 95.± ±. 4.±. 65.4± ± ± ±. 9.8±. 4.7±. 68.8±. 79.± ±. 5.±.6 36.±.3 64.±.4 7.7± ±. 35.5±. 48.±.4 7.±. 78.8± ±. 8.5±. 4.8±. 69.± ± ± ± ± ± ± ±. 44.4±. 7.9± ±.6 96.± ±. 48.±.6 75.±. 94.8±. 95.8± ± ±.3 7.5±.4 9.3±. 94.5± ±. 47.±. 7.±. 9.±.4 94.± ±. 8.9±. 4.±. 68.4± ± ±.3 9.7±.5 4.5± ±.6 75.± ±. 35.9±. 48.7±.7 7.± ± ±. 9.3±. 4.4±.6 69.± ±.4 LSM±SEM b Waxy 4.a ± a ±. 7.7a ±. 93.6a ±.6 95.a ±.6.5a ±. 3.4b ±. 4.9b ±. 68.8b ± b ±.7 a Values are means ± standard deviations of two replicates. Different letters within the same columns indicate statistically different mean values (p<.5). b Least squares means (LSM) ± standard errors (SEM). Different letters following the LSM values within the same columns indicate statistically different mean values (p<.5). 9

10 corn starch consisted of % amylose, determined using iodine potentiometric-titration, and % amylose, determined using gel-permeation chromatography (GPC) with total carbohydrate analysis (Table ). Amylose contents of waxy corn starch determined using the iodine potentiometric-titration ranged.9-4.6% but were not detectable using the GPC analysis. Differences in amylose contents of waxy and normal corn starch contributed to the different starch hydrolysis-rate during the cold-fermentation process. Differences in amylose contents of waxy and normal corn starch contributed to the different starch hydrolysis-rate during the cold-fermentation process. Amylose molecules of the normal corn starch are known to intertwine with amylopectin and restrict the swelling of the granule, and amylose molecules are more concentrated at the periphery of starch granules, developing a hard shell on the surface of the granule, which reduces the enzymatic-hydrolysis rate of normal corn starch (Jane 7). Table. Amylose content (%) of the normal and waxy corn starch a Amylose% Line Iodine titration b GPC c 536.9±. ND d 537.3±. ND Waxy 539.5±. ND 54.4±.4 ND 9 crop year 54.±. ND ±. 34.9± ±. 34.9± ±. 3.4± ±.6 3.± ±. ND 537.±. ND waxy 539.5±. ND 54.±. ND crop year ±.4 ND ±. 34.3± ±. 34.6± ±.5 3.± ±. 33.4±.3 LSM±SEM Waxy.b ±.5 8.5a ±.6 a Values are means ± standard deviations of two replicates. b Determined using iodine potentiometric-titration. c Determined using gel-permeation chromatography (GPC) followed by total carbohydrate analysis. d Not detectable

11 Table. Amylopectin branch-chain-length distribution a of the normal and waxy corn starch b 9 crop year crop year Waxy waxy Line DP< DP3-4 DP5-36 DP>37 ave. CL c 536.7± ±.7 4.±.7 5.5±.4.± ±. 45.9±.4 3.9±. 6.9±.7.5± ±. 48.±. 3.9±.5 6.±.4.6± ± ±.5 3.5±. 7.±..7±.5 54.±. 45.5±.3 3.5±.3 8.9±..4±. 47.8±. 38.4±.3 7.±..6±.4 3.± ±.3 4.6±.7 8.9±.6 4.±. 4.4± ±.5 45.±. 4.9±.6 9.6±.8.4±. 474.±.3 4.±. 5.3±.3.4±. 3.5± ± ±.6 5.±. 5.6±..4± ±. 47.±.5 3.9±. 6.4±.6.5± ± ±.4 4.8±.8 7.5±..±. 54.±. 45.±.6 4.5±..±.6 3.±. 54.6± ±3. 4.8±.6.±.8 3.± ±.5 4.5±. 7.7±.4 3.4±. 3.9±. 47.5±. 44.8±. 4.8±. 9.9±..7± ±. 43.3±. 5.5±.5.8±. 3.3±. 474.±.4 43.±.9 5.4±.8.6±.5.9±. Waxy.a ± a ±.5 4.b ±.3 7.4b ±.5.b ±. LSM±SEM 9.5b ±.6 4.5b ±.6 6.a ±.4.8a ±.6 3.3a ±. a Values are means ± standard deviations of two replicates. Different letters within the same columns indicate statistically different mean values (p<.5). b Molar basis. c Average branch-chain-length of amylopectin.

12 Amylopectin branch-chain-length distributions of the selected waxy corn and normal corn starches isolated from the corn kernels were analyzed in, and results are shown in Table. Amylopectin of the waxy corn starch displayed significantly (p<.) shorter average branch-chain-lengths (DP.) than that of the normal corn starch (DP 3.3). The difference in branch-chain-lengths between the normal and waxy corn starch resulted from that the amylopectin of the waxy corn starch consisted of significantly (p<.) smaller percentages of long branch-chains of DP>37 (mean=7.4%) and larger percentages of short branch-chains of DP< (mean=.%) than that of the normal corn starch (DP>37: mean=.8%, and DP<: mean=9.5%). For normal corn lines, there was no clear correlation between amylopectin branch-chain-lengths and starch-hydrolysis rates, indicating that amylopectin branch-chain-lengths of normal corn starch played a minor role on starch hydrolysis rates compared with the amylose content. For waxy corn lines, however, because of the little amylose content of the starch (Table ), amylopectin structures had significant effects on starch hydrolysis and ethanol yield. Regression analyses showed that average branch-chain-lengths of amylopectin had significant negative-correlations with the ethanol yield (r= -.9, p<.) and the percentages starch-hydrolysis (r= -.7, p<.5). In addition, percentages long-branch-chains of DP>37 negatively correlated with the ethanol yield (r= -.85, p<.) and the percentages starch-hydrolysis (r= -.63, p<.5) for the waxy corn samples. Thermal properties of selected waxy and normal corn starches are shown in Table. The average peak gelatinization temperature (T p, 7. C) and conclusion gelatinization temperature (T c, 77.4 C) of the waxy corn starch were significantly higher (p<.5) than that of the normal corn starch (T p =69. C and T c =74.7 C, respectively). The average gelatinization enthalpy-change of waxy corn starch (5.5J/g) was significantly (p<.) larger than that of the normal corn starch (.8J/g). Average percentage retrogradation of the waxy corn starch (4.4%) was significantly less than that of the normal corn starch (54.9%). The differences were attributed to the amylose molecules and long branch-chains of amylopectin (DP>37, Table ) in the normal corn starch granules, which restricted granule swelling and facilitated starch retrogradation (Jane 7). Regression analyses showed that percentages retrogradation of the waxy corn starch positively correlated with the average branch-chain-lengths of amylopectin (r=.85, p<.) and negatively correlated with the ethanol yield (r= -.76, p<.5). Pasting properties of selected waxy and normal corn starch are shown in Figure 3. Waxy corn starch displayed higher peak and break-down viscosities but lower set-back viscosities compared with the normal corn starch. Starch pasting properties are affected by the amylose content and amylopectin branch-chain-length distribution (Jane et al. 999). The waxy corn starch contained very little amylose. Thus, the swelling of starch granules was not restricted by amylose-lipid complex and displayed a higher peak viscosity than that of the normal corn starch. The amylose of the normal corn starch played a role on maintaining the integrity of swollen starch granules and decreased shear-thinning of starch paste, resulting in lower break-down viscosity and higher set-back viscosity of the normal corn starch.

13 Table. Thermal properties of the normal and waxy corn starch a 9 crop year crop year Waxy waxy Line Native starch Retrogradated starch Retro. c T o ( C) b T p ( C) T c ( C) ΔH(J/g) T o ( C) a T p ( C) T c ( C) ΔH(J/g) ±. 69.6±. 76.±.8 4.8±. 43.3±. 58.±. 67.5±.8 4.7±. 3.± ±. 69.8±.7 75.±.7 5.6±. 4.±.3 56.±.6 64.±. 5.± ± ± ± ±.3 5.7±. 4.±. 54.5±. 6.7±. 6.±. 39.± ±.4 7.±. 76.8±.3 5.5±. 4.3± ±. 6.9±. 6.5±. 4.9± ±.5 7.9±.4 79.±.3 5.9± 43.±.8 55.±. 64.3±.4 7.9± ± ±.9 66.±.3 7.±.4.±. 35.6± ±.8 6.9±.3 6.±. 53.± ± ± ±.4.3±. 37.7±.4 5.4±.7 6.±.4 7.3±. 59.3± ±. 69.6±. 74.5±..7±. 39.±. 5.6±.5 6.5±.3 6.6±. 56.4± ±. 67.±. 73.6±..8±. 39.8±. 5.7±. 6.6±.4 5.7±. 53.4± ±. 7.4±. 77.3±.3 5.7±.3 43.±.4 54.±. 6.4±. 6.5±.3 4.3± ±. 7.3±. 77.5±. 5.4±. 4.±. 54.7± ±. 6.6±. 43.± ±.4 7.4± ±.5 5.±.3 4.7± ±. 63.6±. 6.6±. 43.6± ± ± ±.3 5.±. 4.4± ±. 63.4±.3 6.8±. 44.8± ±.3 74.±. 79.±. 5.9±. 43.±. 54.9±. 64.±. 7.±. 45.4± ±. 69.±. 75.±..4±. 4.6±.8 5.±. 6.7±. 6.3±. 55.± ±. 7.8±. 76.±.3.3±. 4.7±. 5.3±. 63.±. 6.9±. 56.4± ±. 7.±. 76.9±..±. 4.7±.8 5.6±. 63.±.6 6.5±. 53.4± ±. 69.6±. 75.6±..±. 4.5±.6 5.6±. 63.±. 6.4±. 5.±. LSM± Waxy 63.9a ±. 7.a ± a ±. 5.5a ±. 4.4b ±.4 SEM d 6.5a ±. 69.b ±. 74.7b ±..8b ±. 54.9a ±.5 a Values are means ± standard deviations of two replicates. Different letters within the same columns indicate statistically different mean values (p<.5). b T o = onset gelatinization temperature, T p = peak temperature, T c = conclusion temperature, ΔH= enthalpy change. c Retrogradation(%) = ΔH of dissociation of retrograded starch / ΔH of starch gelatinization. (%) 3

14 Figure 3. Starch pasting profiles of the normal and waxy corn. A: Isolated starch of 9 samples; B: Isolated starch of samples. 4

15 Ethanol production of inbred and hybrid GEM corn lines Four GEM waxy genotypes (two inbreds and two hybrids) were grown in. One of the F hybrids included the two inbreds as parent lines. In, eight genotypes were grown which consisted of two waxy inbreds, three waxy F hybrids, two normal inbreds, and one normal hybrid. Two of the F waxy hybrids included the inbred waxy lines, and the normal F hybrid included the same normal inbreds as parental lines. All inbreds and F hybrids were self pollinated in and and seed harvested from 8 ears of each genotype. A cold-fermentation study was conducted to compare ethanol production between the inbred and hybrid corn. Kernel-starch content, -kernel weight, and ethanol yield of the corn lines are shown in Table 3 and Table 4. Table 3. -kernel weight, starch content, ethanol yield and conversion efficiency of cold fermentation of waxy inbred and hybrid corn grown in -kernel Starch Ethanol yield Conversion Pedigree weight (g) content (%) (g/g grain) efficiency (%) Waxy inbred Waxy hybrid GEMS ±.4 34.±. 93.5±.5 GEMN ±. 3.4± ±.4 GEMS-6/ GEMN-86 GEMS-85/ GEMN ± ±. 94.± ±. 34.±. 94.±.6 Table 4. -kernel weight, starch content, ethanol yield and conversion efficiency of cold fermentation of the inbred and hybrid corn grown in -kernel Starch Ethanol yield Conversion Pedigree weight (g) content (%) (g/g grain) efficiency (%) Waxy inbred Waxy hybrid inbred hybrid GEMS ±.7 3.5±.5 9.8±.4 GEMS ±. 33.6±.4 93.±. GEMS-6/ GEMN ±.3 3.8± ±.8 GEMS-85/ GEMN ± ±. 94.±.6 GEMS-3/ wx exp ± ± ±. GEMS ±. 33.7± ±.8 GEMN ±.7 N/A N/A GEMS-5/ GEMN ±.8 33.±.6 89.±.7 5

16 The hybrids showed similar ethanol yields but slightly higher starch-ethanol conversion efficiency compared with their parent inbred lines. Waxy F hybrids had similar kernel-starch contents to their parent inbred lines, whereas the normal F hybrid (GEMS-5/GEMN-56) had lower starch content than its two parent lines (GEMS-5 and GEMN-56). -kernel weights of the hybrids were greater than that of their parent lines (particularly for waxy), indicating improved corn kernel yield of the hybrid corn. In general, waxy genotypes had similar ethanol yield as the normal genotypes, although ethanol conversion-efficiency was greater for the waxy genotypes. Starch content of waxy genotypes was generally lower than normal genotypes and waxy hybrids did not have greater starch content than their corresponding parent lines. Starch enzymatic-hydrolysis rates of the corn lines grown in using the raw-starch hydrolyzing enzymes are shown in Figure 4. There was no significant difference in starch-hydrolysis rate between the waxy inbred and hybrid corn. Hydrolysis (%) GEMS-85 GEMN-86 GEMS-6/ GEMN-86 GEMS-85/ GEMN-86 Time (hr) Figure 4. Enzymatic hydrolysis of the starch in dry-grind waxy inbred and hybrid corn grown in. References. Englyst, H. N., Kingman, S. M., Cummings, J. H. Classification and measurement of nutritionally important starch fraction. Eur. J. Clin. Nutr., 99, 46, S Jane, J. Current understanding on starch granule structure. J. Appl. Glycosci. 6, 53, Jane, J. Structure of starch granules. Journal of Applied Glycoscience 7, 54, 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. 999, 76 (5),