Dry‐Milling and Fractionation of Transgenic Maize Seed Tissues with Green Fluorescent Protein as a Tissue Marker

The efficiency of fractionating cereal grains (e.g., dry corn milling) can be evaluated and monitored by quantifying the proportions of seed tissues in each of the recovered fractions. The quantities of individual tissues are typically estimated using indirect methods such as quantifying fiber or ash to indicate pericarp and tip cap contents, and oil to indicate germ content. More direct and reliable methods are possible with tissue‐specific markers. We used two transgenic maize lines, one containing the fluorescent protein green fluorescent protein (GFP) variant S65T expressed in endosperm, and the other containing GFP expressed in germ to determine the fate of each tissue in the dry‐milling fractionation process. The two lines were dry‐milled to produce three fractions (bran‐, endosperm‐, and germ‐rich fractions) and GFP fluorescence was quantified in each fraction to estimate the tissue composition. Using a simplified laboratory dry‐milling procedure and our GFP‐containing grain, we determined that the endosperm‐rich fraction contained 4% germ tissue, the germ‐rich fraction contained 28% germ, 20% endosperm, and 52% nonendosperm and nonembryo tissues, and the bran‐rich fraction contained 44% endosperm, 13% germ, and 43% nonendosperm and nonembryo tissues. GFP‐containing grain can be used to optimize existing fractionation methods and to develop improved processing strategies.     

Distribution of Peroxidases in Durum Wheat (Triticum durum)

A biochemical study of the main durum wheat milling fractions (bran, embryo, and semolina) showed that peroxidases (POD) were present in multiple forms in the kernel and appeared to be tissue specific: one form for the embryo, one for the endosperm, one for the subaleuronic layer, and one for the outer layers. Large varietal differences were found regarding both the composition and the POD activity. POD activity, detected by diaminobenzidine, was found mainly in the cell wall of the subaleurone layer and inside some specific, differentiated cells of the embryo. Immuno‐localization with antibodies of durum wheat POD showed the presence of POD in several layers of the pericarp (epidermis) and the seed coat (testa), in the embryo, and also in the endosperm. In this latter tissue, the staining intensity decreased gradually from the outer layers toward the center of the kernel. The localization of POD in durum wheat kernel suggests specific functions for different forms.     

Prediction of relative tissue proportions in wheat mill streams by fourier transform mid-infrared spectroscopy

Fourier-transform mid-infrared (FTIR) spectroscopy was investigated as a method to quantify the relative wheat grain tissue proportion in milling fractions. Spectra were acquired with a FTIR spectrometer equipped with an attenuated total reflectance device on ground samples, and the relative tissue proportion was determined according to the biochemical marker methodology as the reference method. Partial least-squares models were developed independently to predict the amount of outer pericarp, aleurone layer, starchy endosperm, and an intermediate layer (made up of inner pericarp plus seed coat plus nucellar epidermis). Good quality of prediction was obtained regardless of the target tissue. The standard errors of prediction obtained for the outer pericarp, intermediate layer, aleurone layer, and starchy endosperm quantification were, respectively, 3.4, 1.3, 3.4, and 4.6%.     

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%).     

Evaluation and Strategies to Improve Fermentation Characteristics of Modified Dry‐Grind Corn Processes

New corn fractionation technologies that produce higher value coproducts from dry‐grind processing have been developed. Wet fractionation technologies involve a short soaking of corn followed by milling to recover germ and pericarp fiber in an aqueous medium before fermentation of degermed defibered slurry. In dry fractionation technologies, a dry degerm defiber (3D) process (similar to conventional corn dry‐milling) is used to separate germ and pericarp fiber before fermentation of the endosperm fraction. The effect of dry and wet fractionation technologies on the fermentation rates and ethanol yields were studied and compared with the conventional dry‐grind process. The wet process had the highest fermentation rate. The endosperm fraction obtained from 3D process had lowest fermentation rate and highest residual sugars at the end of fermentation. Strategies to improve the fermentation characteristics of endosperm fraction from 3D process were evaluated using two saccharification and fermentation processes. The endosperm fraction obtained from 3D process was liquefied by enzymatic hydrolysis and fermented using either separate saccharification (SS) and fermentation or simultaneous saccharification and fermentation (SSF). Corn germ soak water and B‐vitamins were added during fermentation to study the effect of micronutrient addition. Ethanol and sugar profiles were measured using HPLC. The endosperm fraction fermented using SSF produced higher ethanol yields than SS. Addition of B‐vitamins and germ soak water during SSF improved fermentation of 3D process and resulted in 2.6 and 2.3% (v/v) higher ethanol concentrations and fermentation rates compared with 3D process treatment with no addition of micronutrients.     

Surface Abrasion of Hulled and Hulless Barley: Physical Characterization of the Milled Fractions

Abrasion techniques were used to remove the hull and pericarp layers of barley kernels to obtain a smaller kernel enriched in endosperm. The objective of this study was to evaluate the fractions produced by two alternative abrading systems on four barley cultivars for potential use in fuel ethanol processes that feature an upstream (of the fermentation) dry fractionation system. Four barley cultivars, two hulled (Thoroughbred and Nomini) and two hulless (Doyce and Merlin), were scarified and whitened at 22 scarification times and three milling degrees (settings 2, 4, and 6), respectively. Three different abrasive surfaces (36, 40, and 50 grit) were used in the scarifier to determine the material removal ratio for each barley cultivar. Material balances and color analyses were conducted for all of the fractions produced. Three fractions were produced with the whitener at each milling degree: broken kernels, fine fractions >323 μm, and fine fractions <323 μm. Setting #2 seems to be the milling level that releases most of the hull in the hulled barley with the whitener. After 50 sec of scarification, rougher surfaces produced more fine material (<1,410 μm diameter) and consequently less coarse material (>1,410 μm diameter). A lower grit (36 grit) abrasive surface induced faster hull removal in hulled barley. Color parameters L* and b* were good indicators of the fine and coarse fractions produced by abrasive methods because they indicate the kernel layer removed and were modeled as a function of the fraction of the material produced. The information obtained in this study has application in designing processes capable of removing and recovering hull and pericarp layers of barley kernels and thereby producing smaller kernels or kernel pieces containing mainly endosperm tissue.     

Effect of Corn Milling Practices on Aleurone Layer Cells and Their Unique Phytosterols

Coarse and fine fiber fractions obtained from the corn wet‐milling processes, with and without steeping chemicals (SO₂ and lactic acid), were evaluated microscopically for structure and analytically for recovery of phytosterol compounds from the fiber oil. Microscopic results showed that wet milling, with and without chemicals during steeping, changed the line of fracture between pericarp and endosperm and therefore affected the recovery of the aleurone layer in coarse (pericarp) and fine (endosperm cellular structure) fiber. Analytical results showed that most of the phytosterols and mainly phytostanols in corn fiber are contributed by the aleurone layer. Hand‐dissection studies were performed to separate the two layers that comprise the wet‐milled coarse fiber, the aleurone, and pericarp layer. Analyses revealed that the aleurone contained 8× more phytosterols than the pericarp.     

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.     

Laboratory Measurement of Yield and Composition of Dry‐Milled Corn Fractions Using a Shortened,Single‐Stage Tempering Procedure

The objective was to describe a laboratory‐scale dry‐milling procedure that used single‐stage tempering and determine the effect of hybrid on yields and fraction compositions in milled corn. Samples of 11 commercially available hybrids were processed through a laboratory dry‐milling procedure that used 1 kg samples of corn to produce milling fractions of large grits, small grits, fines, germ, and pericarp. Compositions of milling fractions (protein, neutral detergent fiber, ash, and crude fat) were determined. The procedure used a single‐stage tempering step that increased corn moisture from 15 to 23.5% wb during an 18‐min tempering period. Germ were separated from endosperm particles using a roller mill followed by screening over a sieve with 1.68‐mm openings. Coefficients of variability were small, indicating acceptable repeatability. Overall yield means were 39.2, 25.3, 13.8, 78.2, 14.3, and 6.8 g/100 g (db) for large grits, small grits, fines, total endosperm, germ, and pericarp, respectively. There were effects due to hybrid (P < 0.05) on fraction yields and compositions of milling fractions. Correlations (r) among endosperm fractions (large grits, small grits, and fines) ranged from 0.54 to |–0.92|. Correlations among endosperm fractions and germ and pericarp were <0.68. The developed dry‐milling method estimated milling yields among hybrids with low standard deviations relative to the means and should be a useful tool for research and industry in measuring dry‐milling characteristics.     

Relationships Between the Microstructure,Physical Features,and Chemical Composition of Different Maize Accessions from Latin America

Chemical composition (moisture, total lipids, protein, and apparent amylose) and some physical features (1,000 kernel weight, hardness, and anatomical composition) were determined in 71 accessions representing races of maize from Latin America. Their microstructural characteristics (size and compaction of endosperm cell bodies, pericarp thickness, horny‐floury endosperm ratio, and morphology and size of starch granules) were also evaluated using environmental scanning electron microscopy (ESEM). Compaction was the most important microstructural feature of the maize kernels, representing kernel hardness. Highly compact kernels tended to be hard, with high protein, pericarp, and hard‐endosperm content and high pericarp thickness, but with low moisture, amylose content, and kernel weight and size. The opposite was observed in the least compact kernels. Highly compact kernels tended to have small, polygonal starch granules (<10 μm), while the least compact kernels contained large, spherical granules (>10 μm). These results suggest that microstructure is responsible for the physical features of maize kernels and that microstructure is related to chemical composition.     

Comparative Studies on Composition and Distribution of Phenolic Acids in Cereal Grain Botanical Fractions

The phenolic acid composition and concentration of four manually separated fractions (pericarp, aleurone layer, germ, and endosperm fractions) as well as whole grains of yellow corn, wheat, barley, and oats were analyzed by HPLC‐MS/MS following microwave‐assisted alkaline aqueous extraction. Phenolic acid compositions in whole grains and their fractions were similar, with minor differences among the grain fractions. Significant differences (P < 0.05), however, were observed in phenolic acid concentrations among cereal types, within cereal varieties, and among grain fractions, with yellow corn exhibiting the highest values. The concentrations of p‐coumaric and syringic acid in the pericarp were 10‐ to 15‐fold and 6‐ to 10‐fold higher, respectively, in yellow corn than in wheat, barley, and oats. In the aleurone layer, sinapic and vanillic acids in yellow corn were about 8‐ and 30‐fold more than in wheat. The germ fraction of wheat had 1.8 times more syringic acid than yellow corn germ. Grain fractions, excluding endosperm, had enhanced levels of phenolic acids compared with whole grain. Sinapic acid was more concentrated in the pericarp and germ of wheat, whereas isoferulic acid was concentrated in the germ of purple barley. Syringic and vanillic acids were concentrated in the pericarp and sinapic acid in the aleurone layer of yellow corn. These findings are important in understanding the composition and distribution of phenolic acids, and they act as a guide in identification of grain fractions for use as food ingredients. In addition, yellow corn fractions (aleurone and pericarp) may be potential alternative phenolic‐rich functional food ingredients in grain‐based food products.     

Study of Calcium Ion Diffusion in Components of Maize Kernels During Traditional Nixtamalization Process

Our report shows the calcium ion diffusion process through the different parts of maize kernels (pericarp, endosperm, and germ) during the traditional nixtamalization process as a function of steeping time (t) 0–24 hr. The cooking step of the nixtamalization process used 3 kg of maize kernels in 6L of water and 2% calcium hydroxide (w/w). The cooking temperature was 92°C for 40 min. The calcium content of the samples was measured using atomic absorption spectroscopy. We found that the whole instant corn flour, pericarp, endosperm, and germ, had a nonlinear relationship to steeping time, showing a local maximum at 9 hr. Analysis of the different parts of the nixtamalized kernels showed that in short steeping times (0–5 hr) calcium diffusion took place mainly in the pericarp. Calcium diffusion in the endosperm and germ occurred gradually over longer steeping times. However, the physical state of the kernels (broken kernels) accelerated the diffusion process. Calcium diffusion occurred first in the pericarp, followed by the endosperm and germ. Immediately after cooking (t = 0 hr), we found a 1.148% calcium content in the pericarp, 0.007% in the germ, and 0.028% in the endosperm. After 24 hr of steeping, the calcium contents were 2.714% in the pericarp, 0.776% in the germ, and 0.181% in the endosperm. In another study, the calcium content in the endosperm was measured by first separating the 10% from the outermost, followed by another 10% from the next endosperm tissue, and concluding with the remaining 80%. Calcium ions were present mainly in the outermost layers of the endosperm. The damaged kernels steeped for more than 5 hr showed greater calcium concentrations than the undamaged counterparts.     

玉米单倍体种子胚部特征提取及动态识别方法

为了实现基于机器视觉方法的玉米单倍体种子识别,该文研究了一种玉米单倍体种子胚部特征提取及动态识别方法。采用一种基于B通道平均像素值的胚部特征提取方法,提取了具有Navajo标记的玉米种子的胚部图像,基于此在RGB颜色空间内提取了样本的Navajo标记图像,从而得到一套玉米单倍体种子快速识别RGB组合算法。在玉米分选试验台上进行了动态分选试验。试验结果表明,该算法对LC09124-UH400品种玉米单倍体的识别正确率为98.04%,对杂合体的识别正确率为94.44%。该文提出的玉米单倍体种子RGB组合快速识别算法与玉米分选试验台结合形成的动态分选系统,有助于实现玉米单倍体种子的自动化分选。     

Zein Compositions of Mechanically Separated Coarse and Fine Portions of Maize Kernels

Coarse and fine kernel portions from 24 maize inbreds (six grown in two years) and four hybrids were separated by grinding and sifting. Zeins from both portions of all genotypes were analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Results agreed, in general, with those of a previous study in which endosperm separation was done manually, suggesting that endosperm fractionation can be done by mechanical means. Compositions of zeins, as revealed by RP-HPLC and SDS-PAGE, support the hypothesis that zeins help determine maize endosperm hardness.     

In‐Depth Study of Durum Wheat Grain Tissue Distribution at Milling

The starchy endosperm proportion in durum wheat grain and its ability to be isolated from the peripheral tissues appear as main intrinsic characteristics potentially related to the milling value but still difficult to assess. In this study, several durum wheat samples displaying distinct grading characteristics were analyzed and processed through a pilot mill. The histological composition of grains and milling fractions was monitored by using identified biochemical markers of each wheat grain tissue. Contrasted milling yields of semolina and flour were observed between samples, despite displaying a similar starchy endosperm proportion determined by hand dissection. These yields were related both to differences in the starchy endosperm extraction and to the presence of the aleurone layer, particularly its cellular content. Furthermore, two distinct types of fractionation behavior of the aleurone layer were distinguished depending on the wheat grain sample. Extraction of the envelopes and embryonic axis into semolina and flours were found negligible in comparison with the other tissues.     

A Radioisotopic Study of the Entry of Calcium Ion into the Maize Kernel During Nixtamalization

The entry of calcium ions from the nixtamalization solution into maize kernels over time was followed in model experiments using radiolabeled calcium ions, with autoradiographic evaluation of the kernels after different cooking and steeping times. Calcium ions immediately entered the pericarp and were rapidly fixed at the outer boundary of the endosperm, especially at the external surface of the germ. Entry of calcium into the endosperm occurred gradually after long steeping times, except in the case of broken kernels, for which massive invasion by calcium was observed. After extended steeping times, a moderate amount of calcium‐45 was evident in the germ. Specific perforation of the outer layers of the grains provided a defined route of facilitated entry of calcium into the endosperm. No fundamental difference with respect to penetrability by calcium ion was seen in a comparison between flint‐type grains and grains containing only floury endosperm.     

Protein Distribution Pattern in Floury and Vitreous Endosperm of Maize Grain

Alpha‐amino nitrogen compounds of floury and vitreous parts of hand‐dissected endosperm from eight maize (Zea mays L.) inbred lines, representing a broad range of vitreousness (42–95%), were isolated as nonprotein nitrogen, albumin‐globulins, zeins, and true glutelins. The three protein classes averaged, respectively, 13, 48, and 35% of total nitrogen in floury endosperm, and 4, 79, and 15% of that in vitreous endosperm. For six inbreds, floury endosperm was richer in 27 kDa γ‐zein than vitreous endosperm; the reverse was found for an Argentine flint inbred (ARGL 256), and only traces of 27 kDa γ‐zein occurred in both floury and vitreous endosperm of inbred F113. Results were compared with protein distribution patterns reported in the literature of whole endosperm of wild‐type and mutant genotypes of maize, and with wild relatives of maize, Tripsacum, and teosintes. When percentage of salt‐soluble nitrogen increased from 2% (Tripsacum) to 22% (in double mutant Oh43o2;bt2), zeins decreased from 87 to 22%, and true glutelins increased from 11 to 57%. The pattern of whole endosperm of Zea perennis was very similar to that of the vitreous endosperm of line ARGL 256. The mean pattern for whole endosperm of six o2 inbred lines was identical to that of floury endosperm of eight wild‐type lines, consistent with a lack of synthesis of α‐zeins due to the mutation in the O2 gene.     

Selective Nixtamalization of Fractions of Maize Grain (Zea mays L.) and Their Use in the Preparation of Instant Tortilla Flours Analyzed Using Response Surface Methodology

Using a continuous decorticating machine, white dent corn was efficiently separated, after brief steeping in water, into two fractions: the first (12.5%) consisting mainly of pericarp, germ, and tip cap (PGT); the second (87.5%) consisting of endosperm. Nixtamalization of the maize fractions in the presence of 0.6% (w/w) lime caused an increase in the hot‐paste viscosity at 90°C, while nixtamalization of PGT at lime inputs <0.6% (w/w) resulted in decreased viscosity. Three domains were found for the viscosity of nixtamalized endosperm at 90°C: lower concentrations of lime (< 0.15%, w/w) resulted in lower viscosity values; increased lime (0.15% – <0.3%, w/w) increased the viscosity values; and a lime concentration of 0.3% (w/w) resulted in a lower viscosity value. The response variables (water absorption index, water solubility index, initial viscosity, and viscosity at 90°C for nixtamalized PGT, and compression force and compression area of tortillas) indicated that the mathematical models fit the experimental data and the variance of the models was highly significant. Tortillas of good functional characteristics similar to tortillas produced by the traditional process were obtained when 5% nixtamalized fractions of PGT were blended with 95% nixtamalized endosperm.     

Differences in the Aleurone Layer Fate Between Hard and Soft Common Wheats at Grain Milling

In the milling process, efficient separation between the starchy endosperm and the other grain tissues is a key parameter estimated by ash measurement. Because this separation occurs near the aleurone layer interface, better understanding of this tissue fractionation is critical for a better analysis of the wheat milling behavior. Samples from hard and soft common wheat cultivars that had the same protein content were processed on a pilot mill, and whole grain meals or flour streams were analyzed for ash content. The para‐coumaric acid (p‐CA) and phytic acid flour contents were compared with ash measurement and used as markers of the aleurone cell walls or aleurone cell content, respectively. A greater amount of phytic acid in hard wheat flour compared with soft wheat flour was found and reveals a distinct milling behavior between those wheat classes, mainly at the breaking step. Therefore simple ash content measurement is not sufficient to analyze flour purity. At the reduction stage, quantity of phytic acid increases with the other markers and may result from the overall mechanical resistance of the aleurone tissue. As a consequence, wheat hardness not only determines grain milling behavior but also affects flour composition.     

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.