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