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.     

Effects of Growing Location and Irrigation on Attributes and Ethanol Yields of Selected Grain Sorghums

Nine sorghum cultivars (five inbred lines and four hybrids) were grown in 2006 in three locations (Mount Hope, KS, Halstead, KS, and Plainview, TX) under different irrigation conditions and were evaluated for composition and ethanol fermentation efficiency. The objective was to study, in one growing season, the effects of genotype, growing location, and irrigation on the physical and chemical properties and fermentation efficiencies of grain sorghum. Genotype had a significant effect on chemical composition, physical properties, and ethanol yield. The cultivars showed a large variation in starch (61.0–74.8%), protein (7.56–16.35%), crude fat (2.79–4.77%), crude fiber (0.58–2.57%), ash (1.25–2.26%), kernel weight (20.0–35.9 mg), kernel hardness (49.6–97.5), and kernel size (1.9–2.7 mm) and were the most important factors affecting ethanol fermentation efficiency (87.5–93.9%). Starch and protein contents were significantly affected by growing location but not by irrigation. Environment had a significant effect on ethanol yields. Unexpectedly, irrigation somewhat reduced fermentation efficiency.     

Impacts of Kafirin Allelic Diversity,Starch Content,and Protein Digestibility on Ethanol Conversion Efficiency in Grain Sorghum

Seed protein and starch composition determine the efficiency of the fermentation process in the production of grain‐based ethanol. Sorghum, a highly water‐ and nutrient‐efficient plant, provides an alternative to fuel crops with greater irrigation and fertilizer requirements, such as maize. However, sorghum grain is generally less digestible because of extensive disulfide cross‐linking among sulfur‐rich storage proteins in the protein– starch matrix. Thus, the fine structure and composition of the seed endosperm directly impact grain end use, including fermentation performance. To test the hypothesis that kafirin (prolamin) seed storage proteins specifically influence the efficiency of ethanol production from sorghum, 10 diverse genetic lines with allelic variation in the β‐, γ‐, and (δ‐kafirins, including three β‐kafirin null mutants, were tested for ethanol yield and fermentation efficiency. Our selected lines showed wide variation in grain biochemical features, including total protein (9.96–16.47%), starch (65.52–74.29%), and free amino nitrogen (FAN) (32.84–73.51 mg/L). Total ethanol yield (ranging from 384 to 426 L/metric ton), was positively correlated to starch content (R² = 0.74), and there was a slight positive correlation between protein digestibility and ethanol yield (R² = 0.52). Increases in FAN content enhanced fermentation efficiency (R² = 0.65). The highest ethanol producer was elite staygreen breeding line B923296, and the line with the highest fermentation efficiency at the 72 h time point was inbred BT×623. A large‐seeded genotype, KS115, carrying a novel γ‐kafirin allele, was rich in FAN and exhibited excellent short‐term fermentation efficiency at 85.68% at the 20 h time point. However, the overall ethanol yield from this line was comparatively low at 384 L/metric ton, because of insufficient starch, low digestibility, and high crude protein. Multivariate analysis indicated an association between the β‐kafirin allele and variation in grain digestibility (P = 0.042) and FAN (P = 0.036), with subsequent effects on ethanol yield. Reversed‐phase HPLC profiling of the alcohol‐soluble kafirin protein fraction revealed diversity in protein content and composition across the lines, with similarities in peak distribution profiles among β‐kafirin null mutants compared with normal lines.     

Effect of Protease Treatment Before Hydrolysis with α-Amylase on the Rate of Starch and Protein Hydrolysis of Maize,Whole Sorghum,and Decorticated Sorghum

We studied the effect of sorghum decortication and protease treatment on starch hydrolysis before liquefaction with thermoresistant α-amylase and the generation of free amino nitrogen (FAN) in preparation for subsequent steps of ethanol production. A bifactorial experiment with a level of confidence of P < 0.05 was designed to study differences among maize, whole sorghum, and decorticated sorghum and the effectiveness of the protease treatment before starch liquefaction. Sorghum was decorticated 9.7% to remove most of the pericarp and part of the germ and increase starch concentration. Starch concentration increased in decorticated kernels, whereas total phenols, fiber, and fat decreased. The decorticated sorghum had significantly higher starch and protein hydrolysis compared with the whole kernel. Protease treatment before liquefaction improved the rate of starch hydrolysis, especially in mashes from whole and decorticated sorghums. Whole and decorticated sorghum hydrolyzates treated with protease contained ≈50% more reducing sugars than the untreated counterparts. Maize yielded hydrolyzates with the the highest amount of FAN, followed by decorticated and whole sorghums. The maize and both sorghum hydrolyzates treated with protease contained ≈60 and 30% more FAN compared with the untreated counterparts. Both sorghum decortication and protease treatments before hydrolysis with α-amylase are recommended to increase ethanol yields, save processing time (and therefore energy), and to produce mashes with higher FAN content, which is considered as an important yeast substrate.     

Germination‐Improved Ethanol Fermentation Performance of High‐Tannin Sorghum in a Laboratory Dry‐Grind Process

A high‐tannin sorghum cultivar with 3.96% tannin content was used to study the effects of germination on its ethanol fermentation performance in a laboratory dry‐grind process. High‐tannin sorghum sample was germinated for 3 and 4 days. Original and germinated samples were analyzed for tannin, starch, protein, free amino nitrogen (FAN), and glucose content. Endosperm structures and flour pasting properties of germinated and nongerminated sorghum samples were examined using a scanning electron microscope (SEM) and rapid visco analyzer (RVA). Germination reduced tannin content from 3.96% to negligible levels. The free fermentable sugars (glucose, maltose, and maltotriose) in the germinated samples were significantly higher than those in the nongerminated control. Judged by the starch (starch plus dextrin) and free amino nitrogen contents in the mashed samples, germination improved degree of hydrolysis for starch by 13–20% and for protein by 5‐ to 10‐fold during mashing. Germination significantly shortened the required fermentation time for ethanol production by 24–36 hr, increased ethanol fermentation efficiency by 2.6–4.0%, and reduced the residual starch content in the distillers dried grain with solubles (DDGS) compared to the nongerminated control. Ethanol yield for the 3‐day germinated samples was 2.75 gallons/bushel, which was 3.1% higher than the 2.67 gallons for the nongerminated control. Ethanol yield for the 4‐day germinated sorghum was 2.63 gallons/bushel due to excessive loss of starch during germination.     

Significance of Starch Properties and Quantity on Sponge Cake Volume

We evaluated the qualitative and quantitative effects of wheat starch on sponge cake (SC) baking quality. Twenty wheat flours, including soft white and club wheat of normal, partial waxy, and waxy endosperm, as well as hard wheat, were tested for amylose content, pasting properties, and SC baking quality. Starches isolated from wheat flours of normal, single‐null partial waxy, double‐null partial waxy, and waxy endosperm were also tested for pasting properties and baked into SC. Double‐null partial waxy and waxy wheat flours produced SC with volume of 828–895 mL, whereas volume of SC baked from normal and single‐null partial waxy wheat flours ranged from 1,093 to 1,335 mL. The amylose content of soft white and club wheat flour was positively related to the volume of SC (r = 0.790, P < 0.001). Pasting temperature, peak viscosity, final viscosity, breakdown, and setback also showed significant relationships with SC volume. Normal and waxy starch blends having amylose contents of 25, 20, 15, and 10% produced SCs with volume of 1,570, 1,435, 1,385, and 1,185 mL, respectively. At least 70 g of starch or at least 75% starch in 100 g of starch–gluten blend in replacement of 100 g of wheat flour in the SC baking formula was needed to produce SC having the maximum volume potential. Starch properties including amylose content and pasting properties as well as proportion of starch evidently play significant roles in SC baking quality of wheat flour.     

Germ‐Derived FAN as Nitrogen Source for Corn Endosperm Fermentation

Corn endosperm separated by dry fractionation could exhibit poor fermentation performance due to loss of germ components beneficial for yeast growth. Inorganic nitrogen and other nutritional supplementations are used to overcome slow fermentation rates. We investigated the use of a protease in generating free amino nitrogen (FAN) from germ as an alternative to exogenous nitrogen sources. Up to 300% more FAN can be generated from germ in 6 hr of incubation with protease than without protease. Protease incubation also resulted in higher dry solids (ds) and total glucose contents in the germ hydrolyzates. During fermentation without urea addition, ethanol yields were dependent on mash FAN concentrations. Ethanol yields increased to a maximum when FAN level was 80–90 mg of FAN/100 g ds. At half the optimal FAN level (≈40 mg of FAN/100 g ds), nitrogen limitation occurred, as indicated by high residual glucose concentrations. However, germ FAN did not increase the ethanol yields compared to urea supplementation, likely because germ FAN resulted in lower substrate consumption compared to urea supplementation. Lower substrate consumption correlated to the increase in residual maltose with increase in initial FAN. Ethanol productivity in 0–24 hr of fermentation was higher with germ FAN than with urea, thus decreasing overall fermentation time.     

Production of Bihon-type Noodles from Maize Starch Differing in Amylose Content

Maize starches extracted from selected maize cultivars with 0.2–60.8% amylose contents were used to produce bihon-type noodles. Starch dough using a pregelatinized starch binder was prepared and extruded through a laboratory-scale extruder simulating the traditional process of making bihon in the Philippines. The normal maize starches with amylose content of ≈28% were successfully used for bihon-type noodle production, but waxy maize starches with 0.2–3.8% amylose content and high-amylose maize starches with 40.0–60.8% amylose content failed to produce bihon-type noodles. Viscoamylograph profile parameters and swelling volume are significantly correlated to amylose content of maize starch samples evaluated. These physicochemical properties may be used to indicate that the starch samples at normal amylose levels may be used for bihon-type noodles. Starch noodles produced in the laboratory were not significantly different in terms of either cooking quality or textural properties from two commercially produced maize noodle samples, except for adhesiveness. The laboratory process and fabricated extruder can be used to produce bihon-type noodles.     

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.     

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.     

Ethanol‐Production Performance of Ozone‐Treated Tannin Grain Sorghum Flour

Ozone has been reported as being able to degrade macromolecules such as cellulose, starch, lignins, and tannins in the textile, pulping, and water‐treatment industries. Thus, we hypothesized that ozone treatment may also inactivate tannin activity and increase fermentation efficiency of tannin sorghum lines. The objective of this research was to study the physicochemical properties of ozone‐treated whole tannin grain sorghum flour and its fermentation performance in ethanol production. Results showed that the ethanol yields from ozone‐treated tannin grain sorghums were significantly higher than yields from the untreated flour. The fermentation efficiency of ozone‐treated tannin grain sorghum was approximately 90%, which was 8–14% higher than that of untreated samples at the 36th hr of fermentation. At the end of 72 hr of fermentation, the efficiencies of ozone‐treated sorghum flour were 2–5% higher than those of untreated samples. Measured tannin levels of ozone‐treated samples decreased significantly from 3.8 to 2.7%. Gel‐permeation chromatographic results indicated that both degradation and polymerization processes might have happened to starch molecules during ozone treatment. Rapid Visco Analyzer data showed that the setback of viscosity of ozone‐treated flour was lower than that of untreated flours. Distillers dried grains with solubles made from ozone‐treated sorghum were low in residual starch (<1%) and high in crude protein (≈35%). Therefore, ozonation could be a novel and useful method to improve ethanol yield and fermentation efficiency of tannin grain sorghum.     

Effect of Sorghum Decortication and Use of Protease Before Liquefaction with Thermoresistant α‐Amylase on Efficiency of Bioethanol Production

The aim was to study the dual effect of sorghum decortication and protease treatment before liquefaction with α‐amylase on the performance of subsequent steps of saccharification and fermentation. A bifactorial experiment with a level of confidence of P < 0.05 was designed to study differences among grains (maize, whole, and decorticated sorghum) and the effectiveness of the protease before liquefaction. Sorghum was decorticated to remove most of the pericarp and part of the germ and increase starch concentration of the feedstock. The decorticated sorghum had significantly higher starch hydrolysis during liquefaction compared with the whole kernel. These hydrolyzates contained ≈50% more reducing sugars than the untreated counterparts. At the end of saccharification, the final glucose concentration in hydrolyzates treated without protease was the highest for maize (180 mg/mL), followed by decorticated sorghum (165 mg/mL), and whole sorghum (145 mg/mL). Decortication and protease treatment had a significant effect on fermentation times. In decorticated sorghum mash treated with and without protease, fermentation times were 22 and 60 hr, respectively. The decorticated sorghum treated with protease yielded similar amounts of ethanol compared with maize and 44% more ethanol compared with the untreated whole sorghum. Both sorghum decortication and protease treatments before hydrolysis with α‐amylase are recommended to increase ethanol yields, lower yields of distilled grains, and save liquefaction, saccharification, and fermentation times.     

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.     

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.     

Effects of Amylose,Corn Protein,and Corn Fiber Contents on Production of Ethanol from Starch‐Rich Media

The effects of amylose, protein, and fiber contents on ethanol yields were evaluated using artificially formulated media made from commercial corn starches with different contents of amylose, corn protein, and corn fiber, as well as media made from different cereal sources including corn, sorghum, and wheat with different amylose contents. Second‐order response‐surface regression models were used to study the effects and interactions of amylose, protein, and fiber contents on ethanol yield and conversion efficiency. The results showed that the amylose content of starches had a significant (P < 0.001) effect on ethanol conversion efficiency. No significant effect of protein content on ethanol production was observed. Fiber did not show a significant effect on ethanol fermentation either. Conversion efficiencies increased as the amylose content decreased, especially when the amylose content was >35%. The reduced quadratic model fits the conversion efficiency data better than the full quadratic model does. Fermentation tests on mashes made from corn, sorghum, and wheat samples with different amylose contents confirmed the adverse effect of amylose content on fermentation efficiency. High‐temperature cooking with agitation significantly increased the conversion efficiencies on mashes made from high‐amylose (35–70%) ground corn and starches. A cooking temperature of ≥160°C was needed on high‐amylose corn and starches to obtain a conversion efficiency equal to that of normal corn and starch.     

Physical Properties of Poly(Lactic Acid) and Starch Composites with Various Blending Ratios

Two‐phase polymer blends of poly(lactic acid) (PLA) and corn or wheat starches at various ratios were prepared by using a laboratory‐scale twin‐screw extruder and compression molding. The blends were characterized for thermal transitions, mechanical properties, and water absorption. Starch and PLA were immiscible polymers, and the thermal behavior of PLA was not affected by starch. Crystallinity of the blends decreased in some degree as starch content increased 20–40%. Tensile strength and elongation of the blends decreased as starch content increased, but modulus increased as starch content increased up to 70%. As starch content increased to >60%, the PLA phase became discontinuous, and water absorption of the blends increased sharply. Blends made from wheat starch gave slightly better mechanical properties than those made from corn starch, and no differences in other properties were observed.     

Ethanol Production from Pearl Millet Using Saccharomyces cerevisiae

Four pearl millet genotypes were tested for their potential as raw material for fuel ethanol production in this study. Ethanol fermentation was performed both in flasks on a rotary shaker and in a 5‐L bioreactor using Saccharomyces cerevisiae (ATCC 24860). For rotary‐shaker fermentation, the final ethanol yields were 8.7–16.8% (v/v) at dry mass concentrations of 20–35%, and the ethanol fermentation efficiencies were 90.0–95.6%. Ethanol fermentation efficiency at 30% dry mass on a 5‐L bioreactor reached 94.2%, which was greater than that from fermentation in the rotary shaker (92.9%). Results showed that the fermentation efficiencies of pearl millets, on a starch basis, were comparable to those of corn and grain sorghum. Because pearl millets have greater protein and lipid contents, distillers dried grains with solubles (DDGS) from pearl millets also had greater protein content and energy levels than did DDGS from corn and grain sorghum. Therefore, pearl millets could be a potential feedstock for fuel ethanol production in areas too dry to grow corn and grain sorghum.     

Soft Wheat Starch Pasting Behavior in Relation to A‐ and B‐type Granule Content and Composition

Flours of two soft wheat cultivars were fractionated into native, prime, tailing, A‐, and B‐type starch fractions. Starch fractions of each cultivar were characterized with respect to A/B‐type granule ratio, amylose content, phosphorus level (lysophospholipid), and pasting properties to investigate factors related to wheat starch pasting behavior. While both cultivars exhibited similar starch characteristics, a range of A‐type (5.7– 97.9%, db) and B‐type granule (2.1–94.3%, db) contents were observed across the five starch fractions. Though starch fractions displayed only subtle mean differences (<1%) in total amylose, they exhibited a range of mean phosphorus (446–540 μg/g), apparent amylose (18.7–23%), and lipid‐complexed amylose (2.8–7.5%) values, which were significantly correlated with their respective A‐ and B‐type granule contents. A‐type (compared with B‐type) granules exhibited lower levels of phosphorus, lipid‐complexed amylose, and apparent amylose, though variability for the latter was primarily attributed to starch lipid content. While starch phosphorus and lipid‐complexed amylose contents exhibited negative correlation with fraction pasting attributes, they did not adequately account for starch fraction pasting behavior, which was best explained by the A/B‐type granule ratio. Fraction A‐type granule content was positively correlated with starch pasting attributes, which might suggest that granule size itself could contribute to wheat starch pasting behavior.     

Physicochemical Properties of Maize Starch Obtained from Intermittent Milling and Dynamic Steeping (IMDS) Under Various Steeping Conditions

Physicochemical properties of maize starch obtained under different steeping conditions by intermittent milling and dynamic steeping process (IMDS) were studied. Brazilian dent maize (hybrid XL 606) was milled using a 2×2×3 factorial experimental design with two lactic acid levels (0.0 and 0.55%, v/v), two SO₂ levels (0.05 and 0.1%, w/v), and three temperatures (52, 60, and 68°C). Properties of starch obtained by conventional wet‐milling process (36 hr at 52°C, 0.55% lactic acid, and 0.2% SO₂) were used for comparison. Starch protein content and solubility increased with presence of lactic acid, while swelling power decreased. Higher SO₂ concentration (0.1%) had the same effect as lactic acid on some properties. Steeping temperatures of 60 and 68°C increased solubility and most of the thermal properties but reduced swelling power, suggesting stronger starch annealing during IMDS at these temperatures. Some thermal changes on starch granules were visualized by scanning electron microscopy (SEM) at 60 and 68°C. Amylose content as well as pasting properties were affected by steeping factors and interactions. Starches from IMDS and conventional wet‐milling processes were similar in most properties, indicating that IMDS provides starch with quality similar to that from conventional milling.     

Granule Bound Starch Synthase I (GBSSI) Gene Effects Related to Soft Wheat Flour/Starch Characteristics and Properties

Eight soft spring wheat (Triticum aestivum L.) genotypes representing the four granule bound starch synthase I (GBSSI) classes were evaluated with respect to flour/starch characteristics and pasting behaviors. Native starch was isolated from genotype straight‐grade flours (94.8–98.1% of starch recovered) to approximate the starch populations of the parent flours. As anticipated, amylose characteristics varied among the genotypes according to GBSSI class and accounted for the primary compositional difference between genotypes. Total (TAM), apparent (AAM), and lipid‐complexed (LAM) amylose contents ranged from 1.0–25.5 g, 0.7–20.4 g, and 0.3–5.6 g/100 g of native starch, respectively, and gradually decreased with the progressive loss of active Wx alleles. In addition, genotype flour total starch (FTS) and A‐type starch granule contents, which ranged from 81.7–87.6 g/100 g of flour (db) and 61.6–76.8 g/100 g of native starch (db), respectively, generally decreased with an increase in waxy character in parallel with amylose characteristics, as likely secondary effects of Wx gene dosage. Though amylose characteristics predominantly accounted for the majority of genotype flour pasting properties, FTS content and ratios of A‐ to B‐type granules also exhibited significant influence. Thus, loss of one or more Wx genes appeared to induce measurable secondary effects on starch characteristics and properties.