Master of Science / Department of Grain Science and Industry / Paul A. Seib / Yong Cheng Shi / Waxy wheat starch contains almost all amylopectin and is relatively new. Currently, advanced lines of hard winter waxy wheats are being bred through genetic elimination of waxy proteins. To realize the full potential of waxy wheat, the wet-milling of waxy wheat flour to produce gluten and waxy wheat starch was investigated. Flours of six advanced lines of waxy hard wheats and two normal hard wheats cultivars, Karl '92 and Trego, were fractioned by the dough-washing method. Doughs prepared from the waxy flours were found to be weaker than those of from normal wheats. All the waxy wheat and normal wheat flours were wet-milled by the dough-washing (Martin) process and the yield and recovery of starch and gluten were compared. One waxy wheat flour, NWX02Y2459, was sticky during the early stages of dough washing, and it gave relatively poor gluten and starch recoveries with low purity. By mixing the dough with 2% NaCl solution or by adding hemicellulase, the stickiness of the dough subsided during the washing step, and thereby recoveries of the gluten and starch fractions were improved. Waxy wheat starch offers unique functional properties. Waxy wheat starches gelatinize and cook at a relatively low temperature compared to maize starches, and their pastes retrograde more slowly and to a lower extent than waxy maize starch. Pasting curves showed that waxy wheat starch generated a much higher viscosity at a lower temperature, and a lower setback viscosity than normal wheat starch and waxy maize starch. Changes in the morphology of waxy and normal wheat starch granules were determined by using a hot-stage microscope, and those changes were related to their pasting properties. After waxy wheat starch was cross-linked in an aqueous slurry at about 37% starch solids with 0.01% phosphoryl chloride (starch basis), visco-amylograms showed that viscosity breakdown was eliminated and that the cooked paste became non-cohesive (less "stringy"). Increasing levels of phosphoryl chloride at 0.03% and 0.06% caused a steady decline in the peak and final paste consistencies of cross-linked waxy wheat starch, whereas the consistencies of waxy maize starch proceeded through an optimum. Waxy maize starch cross-linked with 0.03% phosphoryl chloride had a higher peak and final consistency at 7% solids than when cross-linked with 0.01% and 0.06% phosphoryl chloride.

Doctor of Philosophy / Department of Grain Science and Industry / Yong Cheng Shi / To realize the full potential of waxy wheat flours in food applications, six advanced hard waxy wheat lines were studied. Pasting properties of waxy wheat flours as well as factors governing the pasting properties were investigated. Waxy wheat starch granules swelled more extensively and were more prone to α-amylase degradation than normal wheat starch. A combination of endogenous α-amylase activity and protein matrix contributed to a large variation of pasting properties of waxy wheat flours. Bi-axial extension properties classified dough from waxy wheat as in-elastic. Waxy wheat flour had higher water absorption and lower mixing time than normal wheat flour. Waxy wheat starch affected protein hydration but not protein extractability after optimum dough mixing. Presence of some non-protein free thiol contents and some gliadins acting as chain terminators could be the underlying reasons for waxy wheat flours producing slack dough. In an effort to improve functionality of waxy wheat flours, hydro-thermal processing was used. Two temperatures (140 and 160°C), three moisture contents (0, 12.4 and 20%), and four exposure times (0, 5, 15, 30 and 60 min) were employed. Hydrothermal processing resulted in non-cohesive waxy wheat flours with high viscosity and greater acid stability than native waxy wheat flour. A closer investigation revealed the possible role of endosperm proteins in improving pasting properties of waxy wheat flours. Upon thermal processing, waxy wheat flours demonstrated a long hydration time before forming dough. Heating decreased protein solubility while no changes in starch molecular weight distribution were observed. Our results indicate that hydro-thermal processing results in increased starch protein interaction. As part of application of waxy wheat, bread was baked by replacing normal wheat flour with two hard waxy wheat flours at 15, 30, and 45% levels. Substitution with waxy wheat flour resulted in higher loaf volume and softer loaves. However, substitution at > 30% resulted in excessive post-bake shrinkage and a ‘key-hole’ shape with an open crumb structure. Bread crumb microstructure indicated a loss of starch granule rigidity and fusing of starch granules. Soluble starch content was significantly higher in bread 1-day old crumb containing waxy wheat flour than in control bread.

Master of Science / Department of Grain Science and Industry / David L. Wetzel / Isogenic waxy wheat lines differ from their non waxy (normal) parents in functionality, end use, and chemical (i.e. amylopectin/amylose, lipid) contents. Other investigations of waxy and parent wheats involved the carbohydrate and protein fractions. The goal of this work is to apply chemical images to discriminate between the waxy and parent wheats and define the contribution of contrasting lipid profiles. Recent waxy topics include current interest in plant breeding activity to develop new lines that incorporate desirable traits with advantageous success in baking and milling, and the differences needed in milling techniques for waxy versus normal wheats that may be associated with lipids. From our empirical preliminary success in sorting parent wheat kernels from waxy wheat full null specimens by nearIR chemical imaging it was anticipated that using fundamental vibrational spectra in the mid infrared would provide the chemical basis of discrimination. FT-IR microspectroscopic in situ probing and imaging of kernel frozen sections was applied to genetically pure, well documented isogenic breeding lines. With the use of high spatial resolution, elucidation of fundamental vibrations of mid IR provides chemical manifestation of the genetic expression that differentiates waxy wheats from their parent wheats. Comparison between numerous contiguous pixels, typically 3,000 for each type, establishes a consensus and a mean spectrum with characteristic bands for waxy and parent. Extractions with solvents of differing polarity were employed to aid in lipid extraction in situ and kernel extracted endosperm. Differences between kernel sections of waxy and parent are observed using FT-IR microspectroscopic imaging. However, revealing lipid class contribution to the molecular bands required infrared analysis after selective extraction. Triple mass spectrometry of lipid molecule ions was used for compositional analyses to enhance lipid class profile distinction. A normal and waxy advanced breeding line wheat were also analyzed via the same methods. It was noted that digalactoslydyglyercides are the most abundant lipids in all samples, however the relative lipid profiles of normal wheat versus waxy wheat differ as well as tetraploid versus hexaploid. It is observed that in the endosperm of all parent wheat versus waxy wheat specimens analyzed, all waxy wheat specimens contained higher lipid content. Methods were also applied to partial waxy isogenic cultivars to determine detection limits that correspond to the degree of waxy genetic expression.

Recent research has focused on the molecular characterization of null waxy (Wx), 'Wx-A1b, Wx-B1b', and 'Wx-D1b', alleles that produce no detectable Wx proteins in the endosperm starch of allohexaploid wheat (<i>Triticum aestivum</i> L.; 2n = 6x = 42; AABBDD). The major objectives of this thesis were to (1) isolate and characterize a Wx wheat cDNA and (2) to identify aberrant 'Wx' transcripts encoded by the null 'Wx-A1b' allele of CDC Wx2, a waxy hexaploid wheat line, which result in an absent Wx-A1 protein (~59 kD). In the first study, a cDNA library prepared from developing wheat kernels (cv. Fielder) was screened using a homologous PCR-digoxigenin labeled wheat cDNA probe. A 2.2 kb cDNA clone denoted GBSSIMMI (Accession no. Y16340) was sequenced and identified as encoding a Wx-D1 protein. The deduced amino acid sequence showed 94% similarity with a wheat Wx-A1 peptide, 96% similarity with a wheat Wx-B1 peptide, and 100% identity with two wheat Wx-D1 peptides. A 33-nucleotide deletion, encoding 11 amino acids (AMLCRAVPRRA), was detected within the GBSSIMMI cDNA relative to a previously isolated wheat cDNA (accession no. X57233). Complementation analysis using a glycogen synthase deficient 'E. coli' strain and an 'in vitro' starch synthase assay did not indicate that GBSSIMMI encoded a functional Wx-D1 protein. In the second study, two sister lines CDC Wx2 and CDC Wx6 were obtained by crossing lines Bai-Huo (carries null 'Wx-D1b' allele; lacks Wx-D1 protein) and Kanto 107 (carries null 'Wx-A1b' and -'B1b' alleles; lacks Wx-A1 and -B1 proteins). Waxy protein profiling, amylose concentration determinations, Northern blot analysis, and reverse transcriptase PCR (RT-PCR) analysis were conducted. Ten RT-PCR derived cDNA clones were selected from each genotype and characterized by DNA sequencing analyses. The waxy phenotype of CDC Wx2, lacking Wx-A1, -B1, and -D1 proteins and possessing a reduced amylose concentration ~4%), was associated with dramatically reduced levels of a 2.4 kb 'Wx' transcript when compared to the higher levels in a wildtype control line. DNA sequencing of clones from Kanto 107 and CDC Wx2 characterized two types of aberrant 'Wx' transcripts, one containing intron 1 and another containing introns 1 and 4. Intron 1 in both types of aberrant 'Wx' transcripts contained a premature stop codon which resulted in the translation of a truncated Wx protein ~4 or 11 kD). Analysis of CDC Wx6, lacking Wx-B1 and -D1 proteins and possessing a reduced amylose concentration (~14%) failed to reveal aberrant ' Wx' transcripts, suggesting that the RNA defects in this study were not responsible for the absence of the Wx-B1 or -D1 proteins. Thus, the aberrant Wx transcripts were encoded by the null 'Wx-A1b' allele. The presence of a premature stop codon in the 'Wx' transcripts encoded by the null 'Wx-A1b' allele explained the absence of the ~59 kD Wx-A1 protein in CDC Wx2 and its parental line Kanto 107.

Master of Science / Department of Grain Science and Industry / Yong Cheng Shi / Waxy wheat starch (WWS) containing little or no amylose has unique properties for food and industrial uses. Hydroxypropylation, a widely used means for modifying starches, could enhance functionalities of starch by substituting hydroxyl groups in starch. There are limited numbers of literatures regarding hydroxypropylation of WWS. WWS hydroxypropylated with 3.0-9.0% propylene oxide (PO) were prepared and compared to hydroxypropylated normal wheat starch (NWS) and hydroxypropylated waxy maize starch (WMS). The molar substitution (MS) of the resulting hydroxypropylated NWS, WWS and WMS was 0.055-0.151, 0.048-0.133, and 0.049-0.139, respectively. Gelatinization temperatures and enthalpy of hydroxypropylated starches were significantly lower than those of their unmodified counterparts and the extent of decrease was positively correlated to the MS. Differential scanning colorimetry (DSC) results showed that hydroxypropylation reduced the retrogradation of the starches during the storage after gelatinization but there were differences between the starches. For the same level of PO reaction (3 and 5% PO), hydroxypropylated WWS retrogradated less than hydroxypropylated WMS, suggesting that for the same level of cold storage stability, less PO is needed for WWS. After reacted with 6% PO, MS was 0.092 and 0.094, respectively, for WWS and WMS, and no retrogradation was observed for those hydroxypropylated starches, indicating that at MS ~0.094, hydroxypropyl groups prevents amylopectin chains from re-associating and forming crystalline structures. In contrast, a higher level of PO (9%) was needed to react with NWS to achieve the hydroxypropylated starch with an MS of 0.151 that gave no retrogradation. Consistent with microscopic observation results, Micro-Visco-Amylograph (MVA) analysis showed hydroxypropylated starches developed viscosity at lower temperatures and had improved hot and cold viscosities. The specific characteristics of hydroxypropylated starches are related to the degree of hydroxypropylation. Therefore, it is critical to determine the level of hydroxypropylation in modified starches. 1H NMR is a simple and rapid means of determining hydroxypropyl (HP) group in modified starches. In this study, a method to prepare a HP starch for NMR analysis was improved. The optimum parameters proposed to hydrolyze HP starches were 10% starch solid content, 3.5% (wt.%) DCl in D2O as the solvent, sodium acetate as an internal standard, heating at 90oC for 1 h. Optionally, 6% (v/v) trifluoroacetic acid-d as the water-peak shifting reagent. Six hydroxypropylated starch samples and two commercial cross-linked and hydroxypropylated starch samples were hydrolyzed with this modified method before 1H NMR analysis, and the results of HP group content in the samples were in agreement with the results from an enzyme-catalyzed method, which indicate that the improved acid hydrolysis method is applicable for both hydroxypropylated starch and cross-linked and hydroxypropylated starch.

Doctor of Philosophy / Department of Grain Science and Industry / Finlay I. MacRitchie / Novel genetic materials were used to deduce gluten protein composition-functionality relationships. The Pegaso bread wheat near-isogenic lines (NILs) included addition, variation and/or deletion of major loci coding for HMW-GS, LMW-GS and gliadins. The waxy wheat lines (Svevo and N11 set) included wild, partial and complete waxy lines. Triticale translocations include 1R.1D and 1A.1D lines (GDS7, Trim, Rhino and Rigel sets) with HMW-GS 5+10 and 2+12. The main goal of the study was to establish the usefulness of NILs as appropriate materials to investigate the structure-function relationships of wheat proteins and to evaluate the performance of unique triticale translocations and waxy wheat lines. Effect of genetic variation on phytochemical (phenolic acid and policosanol) contents was also studied. Innovative methods like MALLS, Lab-on-a-chip and micro (10 g) baking were utilized along with traditional analytical methods. Results confirmed the potential of using NILs in understanding the effects of certain proteins coded at specific loci that might often be targeted in breeding programs. Removal of expected chain terminators at Gli-1/Gli-2 loci causes a shift in MWD to higher values, reflected in higher UPP and dough strength. Lines with HMW-GS 5+10 were clearly separated from 2+12 lines in terms of dough strength and UPP. The present study obtained evidence that modified ω-gliadins acts as chain terminators and cause reduction of protein polymer size and thus shifts in MWD. Marked differences in terms of milling characteristics, protein composition and ultimately in end-use functionality were observed with various waxy wheat null lines. Loaf volumes with waxy wheat flour alone were higher than a 50% blend with commercial wheat; however, breads were unacceptable to consumers in all aspects. Poor milling quality, very low mixing times with low bread loaf volumes were typical of all the triticales studied. However, translocation of the HMW-GS from wheat chromosome 1D increased dough strength, particularly the HMW-GS 5+10. Among the phytochemicals studied, double nulls at Gli-1 loci of Pegaso NILs had the highest total policosanols and total phenolic acid contents.Slight variation to wheat phenolic acid composition and contents were observed with waxy wheat and triticale lines.