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.     

Comparative efficiencies of isopropyl and tert-butyl alcohols for extracting zeins from maize endosperm

Protein of endosperm of maize grains originating from three wild-type inbreds and their opaque-2 versions were solubilized in diverse extracts (E) by the sequential use of 0.5 M NaCl, water (E(1,2)), alcohol plus a reducing agent (E(3)), and salt plus a reducing agent (E(4)). Zeins were isolated in extracts E(3) and E(4) obtained by using 55% (w/w) isopropyl alcohol (i-PrOH) + 0.2% dithiothreitol (DTT) followed by 0.5 M NaCl + 0.2% DTT buffered at pH 10 or 60% tert-butyl alcohol (t-BuOH) + 0.2% DTT followed by 0.5% sodium acetate + 0.2% DTT in 30% t-BuOH. For a given genotype the percentage of extracted zeins was independent of the nature of the alcohol. The latter had a slight effect on the respective magnitude of E(3) and E(4): E(3) increased at the expense of E(4) when t-BuOH was substituted to i-PrOH for their isolation. The percentage of the total endosperm nitrogen present in E(3) + E(4) was identical to that of fractions F(II) + F(III) + F(IV) isolated according to the classical Landry-Moureaux extraction procedure. SDS-PAGE analysis revealed the presence of all types of zeins (alpha, beta, gamma, and delta) in E(3) and F(III), residual zeins in E(4) isolated with t-BuOH, and streaking only in E(4) and F(IV) isolated with NaCl at pH 10. The data together with those of the literature were discussed with regard to the influence of procedure on the yield of zeins using alcoholic extraction.     

Improved Method for Isolating and Quantitating α‐Amino Nitrogen as Nonprotein,True Protein,Salt‐Soluble Proteins,Zeins, and True Glutelins in Maize Endosperm

The conventional Landry‐Moureaux method for selective extraction of maize proteins was modified by reducing the contact time of meal with extractants and by removing 55% 2‐propanol as extractant. The new procedure, coupled with a method for quantitating protein at microgram level, was used for assessing the nitrogen distribution of four soluble protein fractions present in 100‐mg samples of endosperm originating from six maize inbreds and opaque‐2 versions. Proteins extracted with 55% 2‐propanol plus reductant were made up of α‐, β‐, γ‐, and δ‐zeins. Proteins extracted subsequently with salt plus reductant were minor and poor in lysine (1 mol%).They were associated with zeins. Comparison of present data with those available in the literature showed a close similarity for a given genotype between the percentage of total α‐amino nitrogen extracted by 2‐propanol plus reductant than by salt plus reductant under conditions of the modified procedure and that of total Kjeldhal nitrogen extracted by 2‐propanol with and without reductant, and by salt plus reductant, using the conventional procedure. A simplified protocol was described and tested for isolating and quantitating α‐amino nitrogen as nonprotein, true protein, salt‐soluble proteins, zeins, and true glutelins in any sample of maize endosperm.     

Protein Distribution in Gluten Products Isolated During and After Wet-Milling of Maize Grains

The protein distribution in five gluten samples isolated during and after wet-milling of maize grains (slurry before and after filtration, total industrial gluten meal, and coarse and fine fractions obtained after sieving) was investigated by sequential extraction. Six fractions (FI-FVI), including residue, were isolated. Heating filtered slurry to draw water away did not alter protein distribution. Compared with values reported in the literature for endosperm protein, we found a decrease in FI and FIV, respectively, extracted with salt alone and with reductant, due to proteolysis and partial elimination of nonprotein nitrogen during slurry filtration, and an increase in FII and decrease in FIII, alcohol-soluble proteins extracted without and with reductant, respectively, due to the presence of SO₂ in the steeping liquor. Gluten, with respect to the endosperm from which it originated, was richer in zeins (FII + FIII) and glutelins (FV + FVI) due to partial removal of salt-soluble proteins (FI + FIV) during the isolation process.     

Regulation of lysine metabolism and endosperm protein synthesis by the opaque-5 and opaque-7 maize mutations

Two high lysine maize endosperm mutations, opaque-5 (o5) and opaque-7 (o7), were biochemically characterized for endosperm protein synthesis and lysine metabolism in immature seeds. Albumins, globulins, and glutelins, which have a high content of lysine, were shown to be increased in the mutants, whereas zeins, which contain trace concentrations of lysine, were reduced in relation to the wild-type lines B77xB79+ and B37+. These alterations in the storage protein fraction distribution possibly explain the increased concentration of lysine in the two mutants. Using two-dimensional polyacrylamide gel electrophoresis of proteins of mature grains, variable amounts of zein polypeptides were detected and considerable differences were noted between the four lines studied. The analysis of the enzymes involved in lysine metabolism indicated that both mutants have reduced lysine catabolism when compared to their respective wild types, thus allowing more lysine to be available for storage protein synthesis.     

Effect of processing conditions on phytic acid, calcium, iron, and zinc contents of lime-cooked maize

Tortillas are made by cooking maize in a lime solution during variable times and temperatures, steeping the grain for up to 12 h, washing and grinding it to a fine dough, and cooking portions as flat cakes for up to 6 min. The effects of the main processing steps on the chemical composition, nutritive value, and functional and physicochemical characteristics have been areas of research. The present work evaluates the effect of lime concentration (0, 1.2, 2.4, and 3.6%) and cooking times (45, 60, and 75 min) on phytic acid retention of whole maize, its endosperm, and germ, as well as on the content of calcium, iron, and zinc on the same samples. The effects of steeping time and temperature and steeping medium on the phytic acid of lime-cooked maize were also studied. Finally, phytic acid changes from raw maize to tortilla were also measured. The results indicated that lime concentration and cooking time reduce phytic acid content in whole grain (17.4%), in endosperm (45.8%), and in germ (17.0%). Statistical analyses suggested higher phytic acid loss with 1.2% lime and 75 min of cooking. Cooking with the lime solution is more effective in reducing phytic acid than cooking with water. Steeping maize in lime solution at 50 degrees C during 8 h reduced phytic acid an additional 8%. The total loss of phytic acid from maize to tortilla was 22%. Calcium content increased in whole maize, endosperm, and germ with lime concentration and cooking and steeping times. The increase was higher in the germ than in the endosperm. The level, however, can be controlled if steeping of the cooked grain is conducted in water. Iron and zinc contents were not affected by nixtamalization processing variables but were affected in steeping.     

Fusarium Head Blight: Effect of Fungal Proteases on Wheat Storage Proteins

The effect of proteolytic enzymes, associated with Fusarium head blight, on wheat storage proteins and dough functionality was studied. Fusarium damaged kernels (FDK) and sound kernels were hand-picked from F. graminearum Schwabe and F. avenaceum (Fr.) Sacc. infected samples of bread and durum wheat. Scanning electron microscopy revealed significant degradation of endosperm protein in FDK. Storage proteins from FDK and sound kernels were analyzed by SDS-PAGE, RP-HPLC, and SE-HPLC. Total storage protein was lower in FDK but no significant qualitative differences in protein were detected by either RP-HPLC or SDS-PAGE. SE-HPLC was used to follow the hydrolysis of wheat storage protein by proteolytic enzymes found in FDK and a pure culture of F. graminearum. Selective inhibition of proteolytic activity by p-chloromercuribenzoate, and not soybean trypsin inhibitor or iodoacetic acid, suggests that the F. graminearum protease is an alkaline protease. Farinograph and extensigraph curves showed that the presence of FDK decreased dough consistency and resistance to extension. The presence of FDK in flour resulted in a substantial reduction in loaf volume. The loss of dough functionality and loaf volume potential was attributed to the presence of fungal proteases.     

Isolation and quantitation of zeins in waxy and amylose-extender and wild flint and dent maize endosperm using a new solvent sequence for protein extraction

Protein distribution in endosperm of maize grains differing by their texture, flint or dent, and by their genotype, wild or waxy or amylose-extender, was examined by the successive use of 0.5 M NaCl, 0.5 M NaCl plus 0.6% 2-mercaptoethanol (2ME) at neutral and then alkaline pH, and 55% 2-propanol plus 0.6% 2ME as extractants. Proteins extracted in the presence of 2ME were characterized by their size polymorphism and amino acid composition. Proteins isolated with NaCl plus 2ME at neutral pH corresponded with a mixture of gamma-zein (27 kDa) and glutelin-like proteins. Proteins isolated with NaCl plus 2ME at pH 10 were a mixture of gamma-zeins (27 and 16 kDa) and beta-zeins (14 kDa). Alcohol-soluble proteins consisted of alpha-, beta-, and delta-zeins, alpha subunits being predominant. Zein quantitation was improved by weighing the nitrogen percentage of extracts by their zein content, as estimated from the data on amino acid composition. The data reported by Wolf et al. (Cereal Chem. 1975, 52, 765) were integrated to the results of this work to suggest the occurrence of an inverse correlation between amylose in starch and zeins in proteins.     

Applicability of the quantification of genetically modified organisms to foods processed from maize and soy

The applicability of quantifying genetically modified (GM) maize and soy to processed foods was investigated using heat treatment processing models. The detection methods were based on real-time quantitative polymerase chain reaction (PCR) analysis. Ground seeds of insect resistant GM maize (MON810) and glyphosate tolerant Roundup Ready (RR) soy were dissolved in water and were heat treated by autoclaving for various time intervals. The calculated copy numbers of the recombinant and taxon specific deoxyribonucleic acid (DNA) sequences in the extracted DNA solution were found to decrease with time. This decrease was influenced by the PCR-amplified size. The conversion factor (Cf), which is the ratio of the recombinant DNA sequence to the taxon specific DNA sequence and is used as a constant number for calculating GM% at each event, tended to be stable when the sizes of PCR products of two DNA sequences were nearly equal. The results suggested that the size of the PCR product plays a key role in the quantification of GM organisms in processed foods. It is believed that the Cf of the endosperm (3n) is influenced by whether the GM originated from a paternal or maternal source. The embryos and endosperms were separated from the F1 generation seeds of five GM maize events, and their Cf values were measured. Both paternal and maternal GM events were identified. In these, the endosperm Cf was lower than that of the embryo, and the embryo Cf was lower than that of the endosperm. These results demonstrate the difficulties encountered in the determination of GM% in maize grains (F2 generation) and in processed foods from maize and soy.     

Characterization of kafirin and zein oligomers by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Quantitative and qualitative analysis of uncooked zein and kafirin fractions were performed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretic profiles. Kafirins and zeins present the same oligomer and monomer compositions with the exception of a 66 kDa oligomer that is only present in kafirins. The quantitative analysis showed differences between zein and kafirin. The composition of each oligomer was established via preparative SDS-PAGE. Part of the cooked oligomers resists reduction; the presence of those oligomers could be related to the decrease on protein digestibility with the cooking process.     

Proteolysis in model sourdough fermentations

Model wheat doughs started with six different lactic acid bacteria (LAB), with or without a commercial baker's yeast culture, were used to study proteolysis in sourdough fermentations. Cell counts, pH, and free amino acid concentration were measured. Sequential extraction of dough samples was performed to separate wheat proteins. The salt-soluble protein fraction (albumins and globulins) was analyzed by RP-HPLC and SDS-PAGE, whereas propanol-soluble (gliadins) and insoluble (glutenins) protein fractions were analyzed by SDS-PAGE only. Multivariate statistical methods were used for the analysis of results. The presence of yeasts and LAB affected RP-HPLC and SDS-PAGE patterns of the salt-soluble fraction in a complex way. The only changes in the gluten proteins that could be related to the presence of LAB were the appearance of new protein fragments (20 and 27 kDa) from gliadins and the degradation of high molecular weight glutenin subunits.     

Physical and biochemical properties of maize hardness and extrudates of selected hybrids

Protein and starch determinants of maize kernel hardness and extruded products were characterized to better define the role of endosperm texture during extrusion. Maize physical properties were correlated with total proteins and zein subclasses (p < 0.01). The extrusion process significantly altered protein solubility and increased protein fragmentation as measured by RP-HPLC and size exclusion chromatography. Harder grits and extrudates demonstrated higher amylose content, lower degree of starch damage, and fragmentation at different screw speeds than softer grits and extrudates. Differences in extrudate expansion ratio, water absorption index, water solubility index, oil absorption capacity, and breaking stress between harder and softer hybrids were related to protein aggregation and fragmentation as well as starch damage and fragmentation.     

Fractionation of zein by size exclusion chromatography

Zein is a group of alcohol-soluble corn proteins, which consists of several individual proteins. A single-step gel filtration chromatography method was developed to fractionate individual zeins from ethanol extracts of whole corn. A Superdex prep 75 column was used with different mobile phases to fractionate the zeins, which were analyzed by SDS-PAGE and UV spectrophotometry. With 70% aqueous ethanol as the mobile phase, fractions containing a mixture of alpha-zein/beta-zein and alpha-zein/delta-zein were obtained. With ammonium bicarbonate added to the 70% ethanol mobile phase, it was possible to obtain beta-zein and delta-zein fractions devoid of other proteins. However, all fractions containing alpha-zein also contained minor amounts of delta-zein and/or beta-zein. Almost all fractions also contained non-protein impurities.     

A Study of Maize Endosperm Hardness in Relation to Amylose Content and Susceptibility to Damage

The relative amounts of amylose and amylopectin in maize starch were determined in samples representing hard and soft endosperm. Although differences were small, amylose content differed significantly (P < 0.001 and P < 0.05) between the two types of endosperm, with hard endosperm containing a higher percentage of amylose. Scanning electron microscopy was used to determine that the surface appearance of starch granules from hard and soft endosperm differed. Starch granules from soft endosperm had randomly distributed pores on their surfaces, which had a rough appearance. Few pores were observed on granules from hard endosperm. A fairly common occurrence with starch granules from soft endosperm was the development of wrinkles or fissures upon prolonged exposure to the beam of the electron microscope. Thus, a correlation existed between endosperm hardness, amylose content, and susceptibility to wrinkling and fissures. The granules of the soft endosperm of maize, presumably less mature than the granules of the hard endosperm, have a lower amylose content (20.5 ± 1.9% vs. 23.0 ± 1.0%), exhibit more surface pores, and are more susceptible to wrinkling in an electron beam, compared with granules of the hard endosperm. Results suggested that the composition and internal architecture of the starch granule differ depending on the hardness of the endosperm from which it was obtained.     

Changes in chymotrypsin hydrolysis of beta-lactoglobulin A induced by high hydrostatic pressure

Beta-lactoglobulin (beta-Lg) was subjected to high pressures up to 400 MPa and proteolysis with chymotrypsin. The hydrolysates were analyzed by SDS-PAGE and RP-HPLC, and the fragments obtained were identified by ESI-MS/MS. The results obtained showed that beta-Lg was hydrolyzed by chymotrypsin in a "progressive proteolysis" manner at either atmospheric or high pressure. Proteolysis during or after high-pressure treatment showed longer and more hydrophobic peptides than proteolysis at atmospheric pressure. Chymotrypsin showed a behavior similar to that of trypsin, with some differences, probably related to the orientation of the target residues specific for each enzyme. The similarities between proteolytic fragments produced by the two enzymes support that proteolysis enhancement under high pressure depends on the substrate changes rather than the enzyme. High pressure seemed to accelerate the first steps of proteolysis, probably through dimer dissociation, while leaving portions of the molecule more resistant to the enzyme.     

Characterization of protein fractions from Bt-transgenic and non-transgenic maize varieties using perfusion and monolithic RP-HPLC. Maize differentiation by multivariate analysis

Protein fractions from transgenic Bt and non-transgenic maize varieties, extracted by the Osborne solvent fraction procedure, were characterized for the first time by perfusion and monolithic RP-HPLC in very short analysis times. Albumins and globulins from different transgenic Bt maizes as well as from their non-transgenic isogenic varieties were eluted in four peaks using perfusion RP-HPLC, whereas prolamins and glutelins were separated in seven peaks. Monolithic RP-HPLC enabled the separation of maize proteins in a large number of peaks showing 6 and 10 main peaks for albumins and globulins, respectively. Prolamins migrated at retention times higher than 5 min as seven peaks, whereas glutelins were separated in three main peaks appearing at retention times higher than 6.0 min. Moreover, chromatograms of the whole protein extracts showed 8 and 11 components for perfusion and monolithic RP-HPLC, respectively. A comparison of the chromatograms of the whole protein extracts relative to transgenic and non-transgenic varieties evidenced quantitative differences on the percentages of area, mainly for peaks 2 and 3 by perfusion RP-HPLC and for peaks 3 and 7 by monolithic RP-HPLC. A discriminant analysis based on these proteic profiles was carried out to classify and predict transgenic Bt maize lines, achieving 100% correct classification using perfusion RP-HPLC.     

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.     

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.     

Evaluation of Quality Factors in Argentine Maize Races

Samples of Argentine maize from 12 landraces were analyzed for starch and amylose content and were evaluated for hardness parameters. Amylose contents of these Argentine landraces were generally higher than for typical dent hybrids grown in the United States. Hardness, as estimated by near‐infrared reflectance; from wavelength shift of the near‐infrared transmittance spectrum absorbance maximum (λ_max) in the 610–680 nm region; and by percentage of γ‐zein in zein‐2 (determined by reversed‐phase HPLC), correlated well with classical definitions of endosperm type and with amylose content. Starch content correlated negatively with hardness. Flint landraces varied substantially in amylose and starch content. The strong correlation between amylose content and maize endosperm hardness confirms and strengthens previous observations, and may provide a compositional basis for endosperm quality.