Evaluation of Nebraska Waxy Sorghum Hybrids for Ethanol Production

To evaluate the ethanol production performance of waxy sorghum hybrids and the effects of location and harvest year on ethanol yield, samples of four waxy sorghum hybrids collected from two Nebraska locations (Mead and Lincoln) in both 2009 and 2010 were tested for ethanol production in a dry‐grind process. No significant difference (P = 0.216) in starch contents was observed among the four hybrids, but starch contents of the hybrids were significantly affected by growth location (P = 0.0001) and harvest year (P = 0.0258). Location, hybrid, and harvest year all had significant effects on ethanol fermentation efficiency in the dry‐grind process. Lincoln sorghum samples showed higher (P = 0.022) ethanol fermentation efficiency (90.4%) than did Mead sorghum samples (90.0%). Sorghums harvested in 2010 had higher (P < 0.001) ethanol fermentation efficiency (91.1%) than those harvested in 2009 (89.3%). The 2009 sorghum flours had more amylose‐lipid complexes than the 2010 samples did, and amylose‐lipid complexes as previously reported had adverse effects on ethanol fermentation. Residual starch contents in distillers dried grains with solubles (DDGS) were significantly affected by hybrid and harvest year (P < 0.0001), but we observed no difference in protein content in DDGS from the four hybrids.     

Maize Proximate Composition and Physical Properties Correlations to Dry‐Grind Ethanol Concentrations

Dry‐grind ethanol plants incur economic losses because of seasonal variations in ethanol yields. One possible cause associated with ethanol yield variability is incoming grain quality. There is little published information on factors causing variation in dry‐grind ethanol concentrations. The objective of this study was to determine relationships between rapidly measurable corn quality attributes (physical parameters and chemical composition) and dry‐grind ethanol concentrations. Corn samples obtained from a Midwestern ethanol plant were analyzed for physical quality parameters (test weight, kernel weight, true density, percent stress cracks, and moisture content) and composition (starch, protein, oil, and soluble sugars contents) and then processed with a laboratory‐scale dry‐grind procedure. There were significant (P < 0.05) variations in corn quality parameters and ethanol concentrations. Correlation coefficients were significant (P < 0.05) but low (–0.50 < r < 0.50) between starch content and final ethanol concentrations (72 h) and total soluble sugar content and ethanol concentrations at 72 and 48 h. Ethanol concentrations (at 24, 48, and 72 h) were predicted as a function of a combination of grain quality factors using multiple regression methods; however, the R² values obtained were low. Variations in ethanol concentrations were not related to physical and chemical composition quality factors. Other factors, such as structural and physiologic attributes of corn grain, need to be evaluated.     

Hydrolysis and Fermentation of Pericarp and Endosperm Fibers Recovered from Enzymatic Corn Dry‐Grind Process

A modified dry‐grind corn process has been developed that allows recovery of both pericarp and endosperm fibers as coproducts at the front end of the process before fermentation. The modified process is called enzymatic milling (E‐Mill) dry‐grind process. In a conventional dry‐grind corn process, only the starch component of the corn kernel is converted into ethanol. Additional ethanol can be produced from corn if the fiber component can also be converted into ethanol. In this study, pericarp and endosperm fibers recovered in the E‐Mill dry‐grind process were evaluated as a potential ethanol feedstock. Both fractions were tested for fermentability and potential ethanol yield. Total ethanol yield recovered from corn by fermenting starch, pericarp, and endosperm fibers was also determined. Results show that endosperm fiber produced 20.5% more ethanol than pericarp fiber on a g/100 g of fiber basis. Total ethanol yield obtained by fermenting starch and both fiber fractions was 0.370 L/kg compared with ethanol yield of 0.334 L/kg obtained by fermenting starch alone.     

Effect of Lactic Acid on Protein Solubilization and Starch Yield in Corn Wet‐Mill Steeping: A Study of Hybrid Effects

To better understand the role of lactic acid (LA) in corn wet‐milling, steeping studies were performed on different yellow dent corn hybrids using four different solutions containing LA, sulfur dioxide (SO₂), a combination of LA and SO₂, or no added chemicals. Although there was variation in protein solubilization among the hybrids, protein release was consistently higher when LA was included in the steepwater than when it was excluded (both with and without SO₂). Several groups have reported that starch recoveries are improved when steepwater contains LA. To explore the relationship between protein solubilization and starch yield as effected by LA, several yellow dent hybrids were steeped in 0.20% SO₂ and 0.50% LA‐0.20% SO₂ solutions and milled to recover starch by a 100‐g laboratory corn wet‐milling procedure. In all instances, both starch yields and protein solubilization were enhanced in solutions containing LA. These results support the hypothesis that direct dissolution of the endosperm protein matrix by LA contributes to the improved starch recoveries.     

Wet‐Milling Characteristics of 10 Lines from Germplasm Enhancement of Maize Project Compared with Five Corn Belt Lines

The use of corn (Zea mays L.) hybrids with high grain yield and starch extractability has steadily increased in the processing industry. In light of widespread corn seed industry participation in the Germplasm Enhancement of Maize Project (GEM), which seeks to enhance exotic germplasm, future hybrids may contain more exotic sources in genetic backgrounds. It is necessary to establish and monitor physical, compositional, and milling characteristics of the new exotic breeding materials to determine the processing value. The present study was conducted to determine the wet‐milling characteristics of a set of GEM lines compared with typical Corn Belt lines. Ten GEM lines introgressed with exotic materials from Argentina, Chile, Cuba, Florida, and Uruguay and previously identified as having different starch yields, three commercial inbred lines, and two public inbred lines (B73 and Mo17) were analyzed using both near‐infrared transmittance (NIT) and a 100‐g wet‐milling procedure. There were statistical differences (P < 0.05) in the yield of wet‐milled fractions (starch, fiber, gluten, and germ). The GEM lines AR16035:S19‐227‐1‐B and CUBA117:S1520‐562‐1‐B had similar or better starch yield and starch recovery than B73 and the other adapted inbred lines, indicating that they may be useful in improving the proportion of extractable starch present in kernels of hybrids. Residual protein levels in the starch and gluten fractions were 0.26–0.32% and 38–45%, respectively. The starch yield of GEM lines from wet milling correlated positively with starch content from NIT and was negatively correlated with protein content of the corn kernels. Oil content in the germ varied from 50 to 60%. Our results indicate that incorporating GEM lines in a breeding program can maintain or even improve wet‐milling characteristics of Corn Belt materials if lines with appropriate traits are used.     

Comparison of Enzymatic (E‐Mill) and Conventional Dry‐Grind Corn Processes Using a Granular Starch Hydrolyzing Enzyme

A new low temperature liquefaction and saccharification enzyme STARGEN 001 (Genencor International, Palo Alto, CA) with high granular starch hydrolyzing activity was used in enzymatic dry‐grind corn process to improve recovery of germ and pericarp fiber before fermentation. Enzymatic dry‐grind corn process was compared with conventional dry‐grind corn process using STARGEN 001 with same process parameters of dry solid content, pH, temperature, enzyme and yeast usage, and time. Sugar, ethanol, glycerol and organic acid profiles, fermentation rate, ethanol and coproducts yields were investigated. Final ethanol concentration of enzymatic dry‐grind corn process was 15.5 ± 0.2% (v/v), which was 9.2% higher than conventional process. Fermentation rate was also higher for enzymatic dry‐grind corn process. Ethanol yields of enzymatic and conventional dry‐grind corn processes were 0.395 ± 0.006 and 0.417 ± 0.002 L/kg (2.65 ± 0.04 and 2.80 ± 0.01 gal/bu), respectively. Three additional coproducts, germ 8.0 ± 0.4% (db), pericarp fiber 7.7 ± 0.4% (db), and endosperm fiber 5.2 ± 0.6% (db) were produced in addition to DDGS with enzymatic dry‐grind corn process. DDGS generated from enzymatic dry‐grind corn process was 66% less than conventional process.     

Effects of Different Mill Types on Ethanol Production Using Uncooked Dry‐Grind Fermentation and Characteristics of Residual Starch in Distiller's Dried Grains (DDG)

This study aimed to investigate impacts of milling methods on ethanol production using an uncooked dry‐grind (cold fermentation) process and characterize residual starch in the distiller's dried grains (DDG) coproduct. Four corn lines with different chemical compositions were ground with cyclone, ultra‐centrifugal, or hammer mills equipped with a screen of 0.5 mm opening and used for the cold fermentation process. Greater starch hydrolysis and ethanol yield were obtained from cyclone‐milled corn, resulting from larger damaged starch contents and smaller particle sizes of the ground corn. Corn grains and ground corn after five‐month storage showed less starch hydrolysis than the freshly ground counterpart. Residual starch (2.8–8.0%) with large proportions of intact amylopectin contents (up to 42.5%) was found in the DDG from all types of milling. The results suggested that the entrapment of starch granules in ground corn and a low activity of amylolytic enzymes at a high ethanol concentration were accountable for the remaining of starch in the DDG.     

Comparison of Raw Starch Hydrolyzing Enzyme with Conventional Liquefaction and Saccharification Enzymes in Dry‐Grind Corn Processing

In a conventional dry‐grind corn process, starch is converted into dextrins using liquefaction enzymes at high temperatures (90–120°C) during a liquefaction step. Dextrins are hydrolyzed into sugars using saccharification enzymes during a simultaneous saccharification and fermentation (SSF) step. Recently, a raw starch hydrolyzing enzyme (RSH), Stargen 001, was developed that converts starch into dextrins at low temperatures (<48°C) and hydrolyzes dextrins into sugars during SSF. In this study, a dry‐grind corn process using RSH enzyme was compared with two combinations (DG1 and DG2) of commercial liquefaction and saccharification enzymes. Dry‐grind corn processes for all enzyme treatments were performed at the same process conditions except for the liquefaction step. For RSH and DG1 and DG2 treatments, ethanol concentrations at 72 hr of fermentation were 14.1–14.2% (v/v). All three enzyme treatments resulted in comparable ethanol conversion efficiencies, ethanol yields, and DDGS yields. Sugar profiles for the RSH treatment were different from DG1 and DG2 treatments, especially for glucose. During SSF, the highest glucose concentration for RSH treatment was 7% (w/v), whereas for DG1 and DG2 treatments, glucose concentrations had maximum of 19% (w/v). Glycerol concentrations were 0.5% (w/v) for RSH treatment and 0.8% (w/v) for DG1 and DG2 treatments.     

Yield,Protein Content,and Viscosity of Starch from Wet‐Milled Corn Hybrids as Influenced by Environmentally Induced Changes in Test Weight

Forty‐three yellow dent corn samples of five different hybrids varying in test weight and moisture content were obtained from 14 different locations in 1993. The locations for acquired samples were selected randomly to cover a wide range of test weights based on preliminary data from eight states of the corn belt where 94% of the U.S. corn crop was produced in 1993. Samples were wet‐milled using a 100‐g standard laboratory‐scale wet‐milling procedure. Protein content in starch and starch viscosity were determined. Starch yield, protein content in starch, and starch viscosity were not affected significantly by test weight.     

Effect of Alternative Milling Techniques on the Yield and Composition of Corn Germ Oil and Corn Fiber Oil

The effects of alternative corn wet‐milling (intermittent milling and dynamic steeping (IMDS), gaseous SO₂ and alkali wet‐milling) and dry grind ethanol (quick germ and quick fiber with chemicals) production technologies were evaluated on the yield and phytosterol composition (ferulate phytosterol esters, free phytosterols, and fatty acyl phytosterol esters) of corn germ and fiber oil and compared with the conventional wet‐milling process. Small but statistically significant effects were observed on the yield and composition of corn germ and fiber oil with these alternative milling technologies. The results showed that the germ and fiber fractions from two of the alternative wet‐milling technologies (the gaseous SO₂ and the IMDS) had, for almost all of the individual phytosterol compounds, either comparable or signficantly higher yields compared with the conventional wet‐milling process. Also, both of the modified dry grind ethanol processes (the quick germ and quick fiber) with chemicals (SO₂ and lactic acid) can be used as a new source of corn germ and fiber and can produce oils with high yields of phytosterols. The alkali wet‐milling process showed significantly lower yields of phytosterols compounds in germ but showed significantly higher yield of free phytosterols, fatty acyl phytosterol esters and total phytosterols in the fiber fraction.     

Protease Treatment to Improve Ethanol Fermentation in Modified Dry Grind Corn Processes

To improve fractionation efficiency in modified dry grind corn processes, we evaluated the effectiveness of protease treatment in reducing residual starch in endosperm fiber. Three schemes of protease treatment were conducted in three processes: 1) enzymatic milling or E‐Mill, 2) dry fractionation with raw starch fermentation or dry RS, and 3) dry fractionation with conventional fermentation or dry conv. Kinetics of free amino nitrogen production were similar in both dry and wet fractionation (E‐Mill), indicating that proteolysis was effective in all three schemes. At the end of fermentation, endosperm fiber was recovered and its residual starch measured. Using protease treatment, residual starch in the endosperm fiber was reduced by 1.9% w/w (22% relative reduction) in dry conv and 1.7% w/w (8% relative reduction) in dry RS, while no reduction was observed in the E‐Mill process. Protease treatment increased ethanol production rates early in fermentation (≤24 hr) but final ethanol concentrations were unaffected in both dry RS and E‐Mill. In dry conv, the addition of protease resulted in a decline in final ethanol concentration by 0.3% v/v, as well as a higher variability in liquefaction product concentration (higher standard deviations in the glucose and maltose yields). Protease treatment can be used effectively to enhance modified dry grind processes.     

Use of Pigmented Maize in Both Conventional Dry‐Grind and Modified Processes Using Granular Starch Hydrolyzing Enzyme

In the dry‐grind ethanol process, distillers dried grains with solubles (DDGS) is the main coproduct, which is primarily used as an ingredient in ruminant animal diets. Increasing the value of DDGS will improve the profitability of the dry‐grind ethanol process. One way to increase DDGS value is to use pigmented maize as the feedstock for ethanol production. Pigmented maize is rich in anthocyanin content, and the anthocyanin imparts red, blue, and purple color to the grain. It is reported that anthocyanin would be absorbed by yeast cell walls during the fermentation process. The effects of anthocyanin on fermentation characteristics in the dry‐grind process are not known. In this study, the effects of anthocyanin in conventional (conventional starch hydrolyzing enzymes) and modified (granular starch hydrolyzing enzymes [GSHE]) dry‐grind processes were evaluated. The modified process using GSHE replaced high‐temperature liquefaction. The ethanol conversion efficiencies of pigmented maize were comparable to that of yellow dent corn in both conventional (78.4 ± 0.5% for blue maize, 74.3 ± 0.4% for red maize, 81.2 ± 1.0% for purple maize, and 75.1 ± 0.2% for yellow dent corn) and modified dry‐grind processes using GSHE (83.8 ± 0.8% for blue maize, 81.1 ± 0.3% for red maize, 93.5 ± 0.8% for purple maize, and 85.6 ± 0.1% for yellow dent corn). Total anthocyanin content in DDGS from the modified process was 1.4, 1.9, and 2.4 times of that from the conventional process for purple, red, and blue maize samples, respectively. These results indicated that pigmented maize rich in anthocyanin did not negatively affect the fermentation characteristics of the dry‐grind process and that there was a potential to use pigmented maize in the dry‐grind process, especially when using GSHE.     

Ethanol Fermentation of Starch from Field Peas

Field peas (Pisum sativum) were evaluated as a potential feedstock for ethanol production. Ground peas were dry‐milled and separated into starch, protein, and fibrous fractions by air classification. Starch‐enriched fractions prepared from whole peas and dehulled peas contained 73.7% wt and 77.8% wt starch, respectively, a nearly two‐fold enrichment compared with whole peas. The fractions were liquefied and saccharified using industrial α‐amylase and glucoamylase at recommended enzyme loadings. A final ethanol concentration of 11.0% (w/v) was obtained in 48–52 hr, with yields of 0.43–0.48 g of ethanol/g of glucose. Starch present in whole ground peas was also saccharified and fermented, with 97% of the starch fermented when an autoclaving step was included in the liquefaction stage.     

Ethanol Production from Modified and Conventional Dry‐Grind Processes Using Different Corn Types

Different corn types were used to compare ethanol production from the conventional dry‐grind process to wet or dry fractionation processes. High oil, dent corn with high starch extractability, dent corn with low starch extractability and waxy corn were selected. In the conventional process, corn was ground using a hammer mill; water was added to produce slurry which was fermented. In the wet fractionation process, corn was soaked in water; germ and pericarp fiber were removed before fermentation. In the dry fractionation process, corn was tempered, degerminated, and passed through a roller mill. Germ and pericarp fiber were separated from the endosperm. Due to removal of germ and pericarp fiber in the fractionation methods, more corn was used in the wet (10%) and dry (15%) fractionation processes than in the conventional process. Water was added to endosperm and the resulting slurry was fermented. Oil, protein, and residual starch in germ were analyzed. Pericarp fiber was analyzed for residual starch and neutral detergent fiber (NDF) content. Analysis of variance and Fisher's least significant difference test were used to compare means of final ethanol concentrations as well as germ and pericarp fiber yields. The wet fractionation process had the highest final ethanol concentrations (15.7% v/v) compared with dry fractionation (15.0% v/v) and conventional process (14.1% v/v). Higher ethanol concentrations were observed in fractionation processes compared to the conventional process due to higher fermentable substrate per batch available as a result of germ and pericarp fiber removal. Germ and pericarp yields were 7.47 and 6.03% for the wet fractionation process and 7.19 and 6.22% for the dry fractionation process, respectively. Germ obtained from the wet fractionation process had higher oil content (34% db) compared with the dry fractionation method (11% db). Residual starch content in the germ fraction was 16% for wet fractionation and 44% for dry fractionation. Residual starch in the pericarp fiber fraction was lower for the wet fractionation process (19.9%) compared with dry fractionation (23.7%).     

Reducing Steep Time by Adding Lactic Acid During Countercurrent Steeping of Corn with Different Initial Moisture Contents

Reducing corn steep time by adding lactic acid instead of relying on in situ fermentation was studied. Corn at two initial moisture levels (15 and 20%) was steeped for 18 hr in a countercurrent steep system. The initial SO₂ target concentration in steepwater was 2,000 or 3,000 ppm, while the initial lactic acid concentration in steepwater was 0, 0.28, or 0.55%. Adding lactic acid under all steeping conditions decreased steepwater pH, accelerated SO₂ absorption, and increased the amount of solids released from corn. Adding lactic acid during steeping also increased the first grind slurry density and made germ skimming easier than when no lactic acid was added. Starch yields for the hybrid used in this study under all steep conditions were comparable to those from 24‐hr steeping, except when steeping corn with an initial moisture content of 15% in ≈2,000 ppm of SO₂ alone. For the 20% moisture corn, adding lactic acid to fresh steepwater significantly improved the starch yield at ≈2,000 ppm of SO₂ for 18‐hr steeping. At ≈3,000 ppm of SO₂, adding lactic acid did not increase the starch yield for the hybrid used. The protein content in starch was significantly lower when lactic acid was added. Pasting properties of starch were not affected by adding lactic acid. The hybrid used in this study had an initial moisture content of 20% and could be wet‐milled without affecting starch yield, starch protein content, and pasting properties.     

Effect of Harvest Moisture Content on Selected Yellow Dent Corn: Dry‐Grind Fermentation Characteristics and DDGS Composition

Efficiently utilizing the nongrain portion of the corn plant as ruminant food and the grain for ethanol will allow the optimization of both food and fuel production. Corn and corn stover could be more effectively used if they were harvested earlier before dry down. Corn harvested at different moisture contents (MCs) may exhibit different processing characteristics for the ethanol industry, because of differences in physical and chemical properties. Therefore, the objective of this study was to investigate the effect of corn harvest MC on dry‐grind fermentation characteristics and dried distillers grains with solubles (DDGS) composition. Pioneer hybrid 32D78 was harvested at seven different dates from August 21 to November 23, 2009, with harvest MCs ranging from 73 to 21% (wb). The corn samples with different harvest MCs were evaluated by a conventional dry‐grind process. Final ethanol concentration from the corn with harvest MC of 54% (kernel dent stage) was 17.9% (v/v), which was significantly higher (0.5–1.2 percentage points) than the mature corn with lower harvest MCs (P < 0.05). Ethanol conversion efficiencies for the corn with harvest MCs of 73 and 54% (wb) were 98.5 and 93.2%, respectively, whereas ethanol conversion efficiencies for the corn with lower harvest MCs were significantly lower (P < 0.05), ranging between 83.2 and 88.3%. For DDGS composition, with corn harvest MC decreasing from 73 to 21% (wb), the residual starch concentration increased from 7.7 to 15.2%, the crude protein concentration decreased from 29.4 to 24.9%, and the neutral detergent fiber concentration decreased from 26.6 to 20.6%.     

Effects of Ground Corn Particle Size on Ethanol Yield and Thin Stillage Soluble Solids

The effects of ground corn particle size on ethanol yield and soluble solids in thin stillage was evaluated using a 2‐L laboratory dry‐grind procedure. The procedure was optimized for grinding, liquefaction, sacchari‐fication, and fermentation parameters. The optimized procedure was reproducible with a coefficient of variation of 3.6% in ethanol yield. Five particle size distributions of ground corn were obtained using a cross‐beater mill equipped with five screens (0.5, 2, 3, 4, and 5 mm). Particle size had an effect on ethanol yield and on soluble solids concentration in thin stillage. The highest ethanol yield of 12.6 mL/100 mL of beer was achieved using a 0.5‐mm screen in the cross‐beater mill. Treatment using the 0.5‐mm mill screen resulted in soluble solids concentration of 25.1 g/L and was higher than soluble solids concentrations obtained with other screens. No differences in soluble solid concentrations were observed in samples of thin stillage obtained from 2, 3, 4, and 5‐mm screens which had a mean yield of 16.2 g/L. By optimizing particle size for maximum ethanol yield and minimum solids in thin stillage, dry‐grind corn plants could realize reduced capital and operating costs.     

Evaluation of Waxy Grain Sorghum for Ethanol Production

The objective of this research was to investigate the fermentation performance of waxy grain sorghum for ethanol production. Twenty‐five waxy grain sorghum varieties were evaluated with a laboratory dry‐grind procedure. Total starch and amylose contents were measured following colorimetric procedures. Total starch and amylose contents ranged from 65.4 to 76.3% and from 5.5 to 7.3%, respectively. Fermentation efficiencies were in the range of 86.0–92.2%, corresponding to ethanol yields of 2.61–3.03 gallons/bushel. The advantages of using waxy sorghums for ethanol production include easier gelatinization and low viscosity during liquefaction, higher starch and protein digestibility, higher free amino nitrogen (FAN) content, and shorter fermentation times. The results showed a strong linear relationship between FAN content and fermentation rate. Fermentation rate increased as FAN content increased, especially during the first 30 hr of fermentation (R² = 0.90). Total starch content in distillers dried grains with solubles (DDGS) was less than 1% for all waxy varieties.     

Influence of Stenocarpella maydis Infected Corn on the Composition of Corn Kernel and Its Conversion into Ethanol

Widespread epidemics of Stenocarpella ear rot (formerly Diplodia ear rot) have occurred throughout the central U.S. Corn Belt in recent years, but the influence of S. maydis infected grain on corn ethanol production is unknown. In this study, S. maydis infected ears of variety Heritage 4646 were hand‐harvested in 2010 from a production field in central Illinois and segregated into one of five levels of ear rot severity based upon visual symptoms. The concentration of ergosterol, a sterol produced by fungi but not plants, was observed to increase with the severity of ear rot (127–306.5 μg/g), and none was detected in the control corn. Corn test weight declined with progression of the disease and was 42.6% lower for the most severely rotted grain from ears infected early in their development. Accompanying changes in composition were also apparent. Crude fat and oil contents decreased (from 4.7 to 1.5%) and fiber increased (from 6.6 to 9.6%), but starch content remained largely invariant. Oil composition also varied among the infected samples. Control and infected corn samples were subjected to ethanol fermentation with a laboratory‐scale corn dry‐grind ethanol process. Ethanol yields for control and infected samples were similar on an equivalent weight basis (2.77–2.85 gal/bu). In comparison with the control, S. maydis infection altered the distillers dried grains with solubles (DDGS) properties, wherein the crude protein was significantly higher and oil significantly reduced, and ash, fiber, and yield per ton were not significantly different. Based upon these results, we conclude that Stenocarpella ear rot has the potential to affect DDGS composition but not ethanol yield on an equivalent weight basis.     

Resistant Starch Escaped from Ethanol Production: Evidence from Confocal Laser Scanning Microscopy of Distiller's Dried Grains with Solubles (DDGS)

The origin of resistant starch (RS) in distiller's dried grains with solubles (DDGS) of triticale, wheat, barley, and corn from dry‐grind ethanol production was studied. A considerable portion of starch (up to 18% in DDGS) escaped from either granular starch hydrolysis or conventional jet‐cooking and fermentation processes. Confocal laser scanning microscopy revealed that some starch granules were still encapsulated in cells of grain kernel or embedded in protein matrix after milling and were thus physically inaccessible to amylases (type RS1). The crystalline structures of native starch granules were not completely degraded by amylases, retaining the skeletal structures in residual starch during granular starch hydrolysis or leaving residue granules and fragments with layered structures after jet‐cooking followed by the liquefaction and saccharification process, indicating the presence of RS2. Moreover, retrograded starch molecules (mainly amylose) as RS3, complexes of starch with other nonfermentable components as RS4, and starch–lipid complexes as RS5 were also present in DDGS. In general, the RS that escaped from the granular starch hydrolysis process was mainly RS1 and RS2, whereas that from the jet‐cooking process contained all types of RS (RS1 to RS5). Thus, the starch conversion efficiency and ethanol yield could be potentially affected by the presence of various RS in DDGS.