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.     

Physical,Compositional, and Wet Milling Characteristics of Grain from Crosses of Corn Inbreds with Exotic and Nonexotic Background

Corn breeders have developed hybrids with enhanced compositional characteristics, but exotic germplasm represents little of the germplasm base used to produce these hybrids. Effects of the exotic germplasm on physical, compositional, and wet‐milling properties as well as the proximate composition of recovered fractions need to be determined before these materials are of value to the corn processing industry. Ten lines from the Germplasm Enhancement of Maize (GEM) project with exotic germplasm introgressed from Argentina, Chile, Uruguay, Cuba, and Florida were crossed to three adapted inbred lines (testers) and grain from the resulting 30 hybrids were evaluated for physical, compositional, and wet‐milling characteristics and the expression of heterosis in these variables. The B73xMo17 adapted public hybrid was used as control. Grain obtained by self‐pollination of the hybrid plants was analyzed using near‐infrared transmittance (NIT) technology and a 100‐g wet‐milling procedure. There was great variation among physical, compositional, and wet‐milling characteristics, and some of the experimental hybrids with exotic origin had better starch yield and starch recovery than B73xMo17, which suggests that wet‐milling characteristics of U.S. hybrids can be improved by breeding with exotic germplasm. In particular, GEM breeding crosses AR16035:S19, CH05015:N15, CUBA117:S1520, and FS8B(T):N1802 could be valuable germplasm sources to produce inbreds with good milling properties. Testers varied in ability to produce hybrids with good milling properties, indicating that choice of tester is an important factor when evaluating this end use. Although general trait trends for mid‐ and high‐parent heterosis were revealed, individual variation among hybrids and testers was large for most traits. This demonstrates the importance of analyzing individual hybrids that are intended for the wet‐milling industry when breeding with exotic introgressed lines.     

Thermal Starch Properties in Corn Belt and Exotic Corn Inbred Lines and Their Crosses

More knowledge is needed about variability of starch functional traits in adapted and exotic germplasm and possible genetic effects of these traits before conducting rigorous inheritance studies and breeding programs for starch quality. We studied and compared the range of variability for starch functional traits in a set of Corn Belt inbred lines with a set of exotic inbred lines from Argentina, Uruguay, and South Africa. Reciprocal hybrids of some of the lines within each set were compared with their parents. Functional traits were examined by using differential scanning calorimetry on starch extracted from single kernels of genotypes. The set of Corn Belt lines had a wider range of values for most traits than did the set of exotic lines. For both sets of lines, the maximum value for peak height index was as high as that previously reported for the waxy endosperm mutant. Although the Corn Belt lines exhibited a wider range of values for range of retrogradation than the exotic lines, the exotic lines showed a wider range of values for percentage retrogradation. Hybrid values were not consistently higher, lower, midpoint, or similar with respect to the values of their parents. This was true regardless of germplasm type or functional trait. Reciprocal cross values showed trends suggesting reciprocal differences, although there was no trend suggesting greater effect of the female parent. These traits seem to be controlled by many modifying effects in addition to major effects. Results indicate that sufficient variability exists in Corn Belt germplasm to conduct breeding and inheritance studies effectively and that there should be potential for breeding for functional traits.     

Gelatinization Properties of Starches from Three Successive Generations of Six Exotic Corn Lines Grown in Two Locations

The objectives of this research were to evaluate the intra‐ and interpopulation variability in gelatinization properties of starches from exotic corn lines and their derivatives when grown 1) during two successive years in the same location; and 2) in both temperate and tropical environments. Six novel exotic corn lines (two 100% exotic and four 25% exotic derived from a breeding cross developed by crossing an exotic hybrid with Corn Belt lines) were selected for this research because their starches have significantly different (and potentially useful) thermal properties from those found in starch from normal Corn Belt corn. The S_n (n = 3 for 25% exotic lines and n = 1 for 100% exotic lines) generations of the six exotic lines were self‐pollinated and grown in the winter nursery in Puerto Rico. Two successive generations (S_n+1 and S_n+2) of lines selected for low onset of gelatinization temperature were self‐pollinated and grown in the same environment near Ames, IA. To evaluate the effect of environment, the S_n+2 generation also was self‐pollinated and grown in the winter nursery in Puerto Rico. Thermal properties of starches from 10 single kernels from each line were analyzed by using differential scanning calorimetry (DSC) at a ratio of 4 mg of dry starch to 8 mg of distilled water. After subsequent generations, the differences in DSC gelatinization properties between selected kernels within each progeny line narrowed, suggesting increased homogeneity of starch structural properties within each line. Unusual thermal properties were fixed in some progeny lines. Environmental factors also affected the thermal properties of starch and a significant interaction between environment and genotype was observed. These results suggest that introgression of adapted germplasm with useful genes from exotic corn would increase the available genetic variability for starch functionality and allow the development of hybrids with important value‐added traits.     

Compositional,Physical, and Wet‐Milling Properties of Accessions Used in Germplasm Enhancement of Maize Project

Forty‐nine accessions used in the Germplasm Enhancement of Maize (GEM) project, two commercial hybrids (Pioneer Brand Hybrids 3394 and 3489), and two Corn Belt inbreds (B73 and Mo17) were evaluated for compositional, physical, and wet‐milling properties. GEM accessions had lower starch contents (65.9–69.1% vs. a mean of 72.2% for the commercial hybrids) and greater protein contents (12.0–14.4% vs. a mean of 8.2% for the commercial hybrids) than did the improved Corn Belt material. Absolute densities were consistently higher for the GEM accessions compared with the commercial hybrids (1.320 vs. 1.265 g/cm³, respectively). The wet‐milling characteristics of the GEM accessions were not nearly as good as for the commercial hybrids. Mean starch yields were only 54.3% for the GEM accessions versus 64.8% for the commercial hybrids. Residual protein levels in the starches recovered from the GEM accessions were much greater (0.45–2.03%) than for commercial corn hybrids (<0.3%).     

Effect of Stress Cracks on Corn Wet‐Milling Yields

U.S. No. 2 yellow dent corn was randomly probe‐sampled from rail cars being shipped to a wet‐milling plant from a Corn Belt local elevator. The probe samples were blended together and kernels were sorted into four levels of stress cracks (0, 1, 2, or multiple). Each level of stress cracking was then laboratory wet‐milled in triplicate. The only statistically observed differences were in total fiber and in protein content of the gluten meal fraction. The starch yield difference between zero stress cracked corn and multiple stress cracked corn was smaller (0.8%) than would be expected if stress cracking were an indicator of damage to the wet‐milling characteristics of the corn.     

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.     

Thermal Properties of Starch from Exotic‐by‐Adapted Corn (Zea mays L.) Lines Grown in Four Environments

The effect of four growing environments (two at Ames, IA; one at Clinton, IL; and one at Columbia, MO) on the thermal properties of starch from five exotic‐by‐adapted corn inbred lines (Chis37, Cuba34, Cuba38, Dk8, Dk10) and two control lines (B73 and Mo17) were studied using differential scanning calorimetry (DSC). The variations in thermal properties within environments were similar for the exotic‐by‐adapted lines and control lines. Missouri was the warmest environment and generally produced starch with the greatest gelatinization onset temperature (T_oG), the narrowest range of gelatinization (R_G), and the greatest enthalpy of gelatinization (ΔH_G). Illinois was the coolest environment and generally resulted in starch with the lowest T_oG, widest R_G, and lowest ΔH_G. These differences were attributed to higher temperatures in Missouri during grain‐filling months either increasing the amount of longer branches of amylopectin or perfecting amylopectin crystalline structure. The Ames 1 environment produced starch with thermal properties most similar to those of Illinois, whereas the Ames 2 environment produced starch with thermal properties most similar to those of Missouri. Ames 2, located near a river bottom where temperatures tend to be warmer, likely had temperatures most similar to those found in Missouri during grain filling.     

Laboratory Wet Milling of Corn: Milling Fraction Correlations and Variations Among Crop Years

Several coproducts result from fractionating corn in the wet‐milling process. Because small changes in product composition and milling characteristics can have a major impact on coproduct yields and values, testing is done to anticipate final product yields. Using small sample size and controlled conditions, a laboratory wet‐milling method proved to be a useful tool for wet milling and genetics industries. A wet‐milling process (100‐g batches) was used for data collection. Data collected during 11 years (1994–2004) were observed for samples used as benchmarks to verify process precision and accuracy and determine correlations among wet‐milling yields. More than 400 milling tests were performed on benchmark samples. Data from benchmark samples also were pooled. Coefficients of variation were low (<6%) for mean yields; year‐to‐year standard deviations of benchmark sample yield means were homogenous and implied precision of the procedure. Some differences were detected in mean yields among years (P ≤ 0.05) for benchmark data due to combined effects of hybrid and environment. A negative correlation (r = –0.58) was observed between starch and gluten yield for pooled benchmark data. Four years (2002–2005) of milling data from commercially available hybrids were analyzed using the milling procedure. For pooled commercial data, the correlation between starch and fiber yield was (r = –0.80); correlation between starch and gluten was (r = –0.76).     

Physical,Compositional, and Wet‐Milling Characteristics of Mexican Blue Maize (Zea mays L.) Landrace

Mexico has the largest diversity of genetic resources for maize in the world, with about 59 different landraces. However, little is known about their wet‐milling characteristics. The aim of this study was to determine whether 15 Mexican blue maize (Zea mays L.) genotypes of Elotero de Sinaloa landrace collected in the northwestern region of Mexico have suitable wet‐milling properties. Great variability of physical, compositional, and wet‐milling characteristics among these blue maize genotypes was observed. The FAUAS‐457 and FAUAS‐488 maize genotypes had similar starch yield and starch recovery as reported for the wet‐milling industry, which indicated that they may be useful as a source of extractable starch. Residual protein levels in the starch fractions were in the range of 0.39–0.68%, and total solids recovery exhibited a mean value of 98.8%, indicating acceptable efficacy of the wet‐milling process. This process afforded starches from blue maize genotypes with low protein contents. Wet‐milling fractions correlated with the physical and chemical properties of the kernels. Our results indicate that Mexican blue maize genotypes contain characteristics that make them appropriate and utilizable at the industrial level, and they can also be valuable for improving wet‐milling characteristics of maize through breeding programs.     

Effect of Laboratory Batch Steeping pH on Starch Yield and Pasting Properties of Selected Corn Hybrids

This study evaluated the effect of initial pH on percent of starch yield and pasting characteristics for a laboratory wet‐milling procedure. Four commercial hybrids, selected because they have significantly different starch yield values, were laboratory wet‐milled, and the pasting properties of the starch fractions were evaluated using a Rapid Visco Analyser (RVA). Percent starch yield (db) decreased when initial pH values were >4.0 but was unaffected by any lower initial pH values. The pasting properties of some of the selected hybrids were more sensitive to steepwater pH than others. There was an overall increase in peak, trough, and final viscosity as pH increased.     

Effect of Steeping Conditions (Sulfur Dioxide,Lactic Acid,and Temperature) on Starch Yield,Starch Quality,and Germ Quality from the Intermittent Milling and Dynamic Steeping Process (IMDS) for a Brazilian Corn Hybrid

Effect of lactic acid, SO₂, temperature, and their interactions were assessed on the dynamic steeping of a Brazilian dent corn (hybrid XL 606) to determine the ideal relationship among these variables to improve the wet‐milling process for starch and corn by‐products production. A 2×2×3 factorial experimental design was used with SO₂ levels of 0.05 and 0.1% (w/v), lactic acid levels of 0 and 0.5% (v/v), and temperatures of 52, 60, and 68°C. Starch yield was used as deciding factor to choose the best treatment. Lactic acid added in the steep solution improved the starch yield by an average of 5.6 percentage points. SO₂ was more available to break down the structural protein network at 0.1% than at the 0.05% level. Starch‐gluten separation was difficult at 68°C. The lactic acid and SO₂ concentrations and steeping temperatures for better starch recovery were 0.5, 0.1, and 52°C, respectively. The Intermittent Milling and Dynamic Steeping (IMDS) process produced, on average, 1.4% more starch than the conventional 36‐ hr steeping process. Protein in starch, oil content in germ, and germ damage were used as quality factors. Total steep time can be reduced from 36 hr for conventional wet‐milling to 8 hr for the IMDS process.     

Effect of Steeping Treatment on Pasting and Thermal Properties of Sorghum Starches

Chemical treatments in wet milling could improve the physico‐chemical properties of starch isolated from high‐tannin sorghums. Sorghums Chirimaugute (medium‐tannin), DC‐75 (high‐tannin), and SV2 (tannin‐free) were steeped in water, dilute HCl (0.9%, v/v), formaldehyde (0.05%, v/v), and NaOH (0.3%, w/v) solutions before wet milling and starch separation. Pasting, textural, and thermal properties of starch were determined. Steeping in NaOH resulted in starches with higher peak viscosity (PV), cool paste viscosity (CPV), and setback than when water, HCl, and formaldehyde were used. The time to PV (P_time) and PV temperature (P_temp) were markedly reduced by treatment with NaOH. NaOH could have caused a degree of pregelatinization. HCl treatment gave starches with higher P_temp and P _time, presumably due to delayed granule swelling. Gel hardness was largely determined by the starch amylase content. The low hardness of DC‐75 starch gels was slightly improved in NaOH‐treated grains. Gelatinization temperatures of sorghum starches were generally low, regardless of steeping treatment. Starch from NaOH‐treated grain generally had slightly higher gelatinization temperatures than when water, HCl, or HCHO was used. Chemical treatments during steeping of sorghum grains greatly affected starch properties. Dilute alkali steeping during wet milling could be used to improve properties of starch isolated from tannin‐containing sorghums.     

Pasting Properties and Surface Characteristics of Starch Obtained from an Enzymatic Corn Wet‐Milling Process

Recently, we reported the development of an enzymatic corn wet‐milling process that reduces or eliminates sulfur dioxide requirements during steeping, considerably reduces steep time, and produces starch yields comparable to that of conventional corn wet‐milling. The best results so far, using the enzymatic corn wet‐milling procedure, were achieved when a particular protease enzyme (bromelain) was used. In this study, pasting properties and surface characteristics of starch obtained from six different enzyme treatments (three glycosidases [β‐glucanase, cellulase, and xylanase] and three proteases [pepsin, acid protease, and bromelain]) using the enzymatic corn wet‐milling procedure were evaluated and compared with those from starch obtained using the conventional corn wet‐milling procedure. Significant effects from enzymatic milling were observed on all the three starch pasting properties (peak, shear thinning, and setback). The setback viscosities of starch from all enzyme treatments were significantly lower compared with those of the control sample, indicating that starch polymers from enzymatic corn wet‐milling do not reassociate to the same extent as with the control. Comparison between bromelain treatment and the control sample showed that starch samples obtained from bromelain treatment are very similar to control starch in water‐binding capacity, molecular breakdown, and time to swell when cooked in water. Significant effects from enzymatic milling were observed on the surface characteristics of starch granules. The glycosidase treatments, especially the β‐glucanase samples, showed holes in the starch granules. No visual differences were observed in starch granules between bromelain and control samples.     

A 10‐g Laboratory Wet‐Milling Procedure for Maize and Comparison With Larger Scale Laboratory Procedures

A very small scale laboratory procedure (≈10 g) is needed to test wet‐milling characteristics of corn when amounts of corn available for testing are quite limited. The objective of this study was to downscale 100‐g laboratory wet‐milling methods already widely used to measure wet‐milling properties of 10 g of corn. A Standard 100‐g procedure, a Modified 100‐g procedure, and an Experimental 10‐g procedure were compared using three corn hybrids with known differences in wet‐milling properties. All three procedures ranked most fraction yields (all except for germ) of the three hybrids the same. Germ separation was conducted differently for each procedure and probably accounts for these differences. Flotation and screening methods were likely affected by germ density and germ size, and hand‐picking the germ was efficient in recovering a pure germ fraction. The two 100‐g procedures were performed very similarly except for fiber recovery. The Modified 100‐g procedure was more efficient in recovering fiber because of intensive washing. Hybrid effects on the starch/gluten separation were more pronounced when the Experimental 10‐g procedure was used, which may allow for more discrimination among hybrids. Although most fraction yields are too small to run replicates for analytical tests, the Experimental 10‐g procedure will be useful in measuring milling efficiency of early generations of corn hybrids where limited samples are available, such as when valuable recombinant proteins are expressed for therapeutics and industrial enzymes.     

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.     

Resistant Starch and Starch Thermal Characteristics in Exotic Corn Lines Grown in Temperate and Tropical Environments

Corn as a food that is heated and cooled to allow starch retrogradation has higher levels of resistant starch (RS). Increasing the amount of RS can make corn an even healthier food and may be accomplished by breeding and selection, especially by using exotic germplasm. Sixty breeding lines of introgressed exotic germplasm backgrounds, selected for high yield, were grown in three tropical and temperate locations and analyzed for starch thermal characteristics and RS levels. Although actual values for all starch characteristics were within normal levels, most characteristics had significant genotypic effects, and all had significant location effects. Thermal properties of retrograded starch were more influenced by the environment than the thermal properties of raw starch, making retrograded starch traits more heritable than raw starch traits. This suggests that a breeding strategy based on retrograded starch traits will have a better chance of success than a breeding strategy based on raw starch traits. A significant genotype effect for RS levels indicates that genotypic selection to raise the level of RS and increase the healthful aspects of corn food should be successful. Significant location effects indicate that breeders using winter nurseries to accelerate their breeding progress need to be careful when making selections using RS data collected on seed grown in the tropics. A small but highly significant correlation between RS and some thermal characteristics, especially percentage of retrogradation, indicates that we may be able to select promising genotypes for RS selection based on our extensive database of thermal characteristics collected on a wide number of diverse corn lines.     

Fate of Bt Protein and Influence of Corn Hybrid on Ethanol Production

Corn hybrids were compared to determine the fate of recombinant Bt protein (CRY1Ab from Bacillus thuringiensis) in coproducts from dry grind and wet‐milled corn during production of fuel ethanol. Two pairs of Bt and non‐Bt hybrids were wet milled, and each fraction was examined for the presence of the Bt protein. Bt protein was found in the germ, gluten, and fiber fractions of Bt hybrids. In addition, one set of Bt and non‐Bt hybrids were treated by the dry‐grind ethanol process and Bt protein was monitored during each step of the process. The Bt protein was not detected after liquefaction. Subsequent experiments determined that the Bt protein is rapidly denatured at liquefaction temperatures. Finally, five hybrids were compared for ethanol yield after dry grinding. Analysis of fermentation data with an F‐test revealed the percent of total starch available for conversion into ethanol varied significantly among the hybrids (P < 0.002), indicating ethanol yield is not exclusively dependent on starch content. No difference, however, was observed between Bt and non‐Bt corn hybrids for either ethanol productivity or yield.     

Relationship Between Wheat (Triticum aestivum L.) Grain Hardness and Wet‐Milling Quality

Grain hardness variation has large effects on many different end‐use properties of wheat (Triticum aestivum). The Hardness (Ha) locus consisting of the Puroindoline a and b genes (Pina and Pinb) controls the majority of grain hardness variation. Starch production is a growing end‐use of wheat. The objective of this study was to estimate the differences in starch yield due to natural and transgenically conditioned grain hardness differences. To accomplish this goal, a small‐scale wet‐milling protocol was used to characterize the wet‐milling properties of two independent groups of isogenic materials varying in grain hardness and in Pin expression level. The first group of lines consisted of hard/soft near‐isogenic lines created in cultivars Falcon or Gamenya in which lines carried either the Pina‐D1a (functional) or the Pina‐D1b (null) alleles of Pina. The second group of lines consisted of Pina, Pinb, or Pina and Pinb overexpressing lines created in Hi‐Line, a hard red spring wheat. Soft near‐isogenic lines had higher starch extractability than the hard Pina null counterparts. This difference in starch extractability was more pronounced between Hi‐Line and its transgenic isolines, with highest levels of extractable starch observed in the transgenic isoline with intermediate grain texture. The results demonstrate that the Ha locus and puroindoline expression are both linked to wet‐milling starch yield and that selection for increased Ha function increases starch yield through the enhanced separation of starch granules and the protein matrix during wet milling.     

Effects of Maturity on Grain Quality and Wet‐Milling Properties of Two Selected Corn Hybrids

The effects of maturity on grain quality and wet‐milling properties were investigated for two hybrids of corn. Significant differences for hybrid and maturity were observed for all grain quality parameters. Test weight, absolute density, and thousand‐grain weight all increased as the corn matured. Kernel hardness increased and breakage susceptibility varied with increased maturity. Water uptake parameters decreased with maturity of the grain. The starch yield results from wet milling showed that the starch yield increased significantly within each cultivar in the early stages of grain maturity, but there were no significant differences between hybrids. Mathematical models using selected grain quality parameters accurately predicted trends in starch yield for the immature and mature corn samples in this study.