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 / Grain Science and Industry / Scott R. Bean / Hulya Dogan / The role of sorghum protein cross-linking into high M[subscript w] polymeric groups in grain hardness was investigated using a number of protein analytical techniques to study the protein composition (reduced and non-reduced) of isolated vitreous and floury endosperm. The relative molecular weight distributions of polymeric proteins within two of three differentially extracted fractions were determined by size exclusion chromatography (SEC). The proteins in vitreous endosperm showed more protein cross-linking and a larger M[subscript w] distribution than found in the floury endosperm. An improved method for fractionating sorghum proteins designed to obtain intact disulfide linked protein polymers was developed. Three protein fractions obtained by application of the method represented proportionally different protein polymer contents as evidenced by comparative SEC and provides an improved tool for polymeric protein content comparison and measurement. The improved method was applied to a highly diverse non-tannin wild-type sorghum sample set spanning a range of in-vitro protein digestibility (IVPD) values to determine polymers involved with and influencing IVPD. Grain traits other than cross-linked proteins were also investigated for significant relationships to IVPD. Three protein fractions (F1, F2, F3) containing intact protein polymers were obtained for analysis by SEC and RP-HPLC. Proteins represented by four of five individual SEC peaks from F3 were significantly negatively correlated to IVPD, with three of the correlated peaks being polymeric. A 2-dimensional (2-D) technique involving peak collection after size exclusion chromatography followed by reverse phase high performance liquid chromatography (SEC x RP-HPLC) of the collected peaks was applied to protein polymers previously determined to be correlated with IVPD. RP-HPLC chromatogram patterns unique to each collected SEC peak from three selectively extracted protein fractions allowed qualitative and quantitative comparisons of protein polymer components. A pair of early eluting peaks appearing in the [gamma]-kafirin region of 2nd-dimension RP-HPLC chromatograms from a protein fraction with the largest M[subscript w] distribution were significantly correlated to IVPD. The correlated peak of interest was collected and characterized using SDS-PAGE and was preliminarily identified as 27kDa [gamma]-kafirin. By combining techniques using differing selectivity’s (solvent based, molecular size based, hydrophobicity based), it was possible to disassemble and compare components of protein polymers significantly correlated to IVPD.

Doctor of Philosophy / Department of Grain Science and Industry / Yong-Cheng Shi / Wheat bran is high in dietary fiber. Resistant starch (RS) is considered a source of dietary fiber. Wheat bran and RS have different functional properties and may not have the same nutritional properties. This dissertation covers two areas of importance in cereal chemistry and human nutrition: wheat bran and RS. Wheat bran chemical and physical influence of nutritional components Wheat bran has become a hot topic due to positive nutritional correlations, and industrial /humanitarian needs for healthy ingredients. Evolving wheat bran into a demanded product would impact the industry in a positive way. The overall aim of this research was to understand chemical and structural composition, to provide avenues for wheat bran development as a healthy food ingredient. To achieve this goal, antioxidant properties in dry wheat milling fractions were examined, effects of wheat bran particle size on phytochemical extractability and properties were measured, and substrate fermentation was investigated. It was noted that mixed mill streams, such as mill feed, have antioxidant properties (0.78 mg FAE/g; 1.28 mg/g total antioxidant capacity; 75.21% DPPH inhibition; 278.97 [mu]mol FeSO[subscript]4/g) originating from the bran and germ fractions. Additionally, extraction of reduced particle size whole wheat bran increased measured values for several assays (185.96 [mu]g catechin/g; 36.6 [mu]g/g; 425 [mu]M TE), but did not increase volatile fatty acid production during in vitro rumen fermentation over unmilled bran. RS digestion, glycemic response and human fermentation In vitro action of enzymes on digestion of maize starches differing in amylose contents were studied. The objectives of this study were to investigate the exact role of [alpha]-amylase and amyloglucosidase in determining the digestibility of starch and to understand the mechanism of enzymatic actions on starch granules. Starch digestibility differed (30-60%) without combination of enzymes during in vitro assay. Further investigations utilized human glycemic response and fermentation with consumption of a type 3 RS without dietary fiber (AOAC method 991.43). Blood glucose response provided lower postprandial curves (glycemic index value of 21) and breath hydrogen curves displayed low incidences fermentation (40%) with consumption of the type 3 RS, due to the structure of starch and digestion by enzymatic action.

Doctor of Philosophy / Department of Chemical Engineering / Jennifer L. Anthony / Ionic liquids are low melting salts composed of an organic cation and an inorganic or organic anion. Ionic liquids are of interest for their wide range of applications and unique properties, such as the negligible vapor pressure of some types of ionic liquids, and the ability to modify ionic liquid properties by selection of the cation or anion. It has been hypothesized that over one million binary ionic liquids (meaning a single cation/anion pair) are possible. Due to the vast number of potential combinations, it should be possible to design ionic liquids specifically for an application of interest. One potential application is their use as protein solvents. However there is little understanding of how ionic liquids affect proteins. This research examined the solubility and secondary structure of the hydrophobic corn protein zein in seven ionic liquids and three conventional solvents as a function of temperature and solvent properties. Zein’s solubility in the solvents was measured gravimetrically from 30 to 60 degrees Celsius. Solubility was then related to solvent properties to gain an understanding of what solvent properties are important, and how to design an ionic liquid to dissolve zein. It was found that a good solvent for zein has a small molecular volume, a low polarity, and is a weak hydrogen bond acceptor. Infrared spectroscopy with curve fitting was used to examine the secondary structure of zein as a function of both solvent and temperature from 25 to 95 degrees Celsius. It was found that most of the ionic liquids change zein’s secondary structure, but those secondary structure changes were not affected by temperature. Aprotic ionic liquids increase the amount of β-turn secondary structure through non-polar interactions between the mixed aromatic-alkyl imidazolium cations and the non-polar portions of the zein. Strong hydrogen bond accepting molecules were found to increase the amount of β-turn secondary structure. It is hypothesized from this research that suitable solvents for zein will have a small molar volume, low polarity, and be poor hydrogen bond acceptors. This combination of properties will enhance zein’s solubility and limit secondary structure changes that can harm protein properties.

Master of Science / Department of Grain Science and Industry / Rebecca Miller / Wheat flour tortillas are the second most consumed bread product after white pan bread. Commercial tortillas are formulated with highly viscoelastic hard wheat flours selected and grown for breadmaking. However, the inherent properties of bread flours require costly formula adjustments to enhance dough extensibility necessary for tortilla production. The objective of this study was to identify the biochemical and physical factors in wheat affecting wheat tortilla quality. Six popular hard winter wheat cultivars (1863, Armour, Clara CL, Denali, Everest, Tiger) were grown in five locations in Kansas. Wheat and flour properties were characterized using approved AACCI methods. Protein composition was determined using size-exclusion high performance liquid chromatography. Flour particle size and starch granule size were measured with laser diffraction. Tortillas were made with a laboratory hot press method. Tortilla shelf-stability over 14 days, opacity, appearance, dough machinability and specific volume were measured. Data collected from flour and tortilla tests were analyzed using ANOVA and means were compared with Tukey-Kramer HSD. In general, the flours did not differ significantly in flour or tortilla properties. Regression analysis (Pearson) showed flour protein content was highly and significantly correlated with tortilla opacity (r=-0.81), L color value (r=-0.79), a color value (r=0.80), and day 14 shelf-stability (r=0.76). Flour water absorption showed highly significant correlations with tortilla opacity (r=-0.81), L color value (r=-0.79), a color value (r=0.77) and day 14 shelf-stability (r=0.73). Tortilla opacity was highly correlated with B-type starch granules (r=-0.83). This study showed that starch granule size, flour protein content and flour water absorption appeared to influence tortilla appearance. However, repeating the study with a larger and more diverse sample set is recommended.

Seeds are complex entities, within which the intricate processes of germination and early growth occur. We describe here a novel technique of our group in 2012 which is capable of simultaneous solution-, gel-, and solid-state analysis. CMP-NMR was applied to intact seeds where all components are studied and differentiated in situ. Characterization, germination and early growth of seeds were studied by variety of 1D and 2D 1H and 13C CMP-NMR experiments. Various metabolites, lipids, carbohydrate biopolymers and structural carbohydrates were first identified and further studied in germination and early growth stages. This research demonstrates the utility of CMP- NMR as a powerful tool to better understand the composition of seeds and processes underlying early seed growth.

Seeds are complex entities, within which the intricate processes of germination and early growth occur. We describe here a novel technique of our group in 2012 which is capable of simultaneous solution-, gel-, and solid-state analysis. CMP-NMR was applied to intact seeds where all components are studied and differentiated in situ. Characterization, germination and early growth of seeds were studied by variety of 1D and 2D 1H and 13C CMP-NMR experiments. Various metabolites, lipids, carbohydrate biopolymers and structural carbohydrates were first identified and further studied in germination and early growth stages. This research demonstrates the utility of CMP- NMR as a powerful tool to better understand the composition of seeds and processes underlying early seed growth.

Doctor of Philosophy / Grain Science and Industry / Praveen V. Vadlani / Lignocellulosic biomass feedstocks are a sustainable resource required for rapid growth of bio-based industries. An integrated approach, including plant breeding, harvesting, handling, and conversion to fuels, chemicals and power, is required for the commercial viability of the lignocellulosic-based biorefineries. Optimization of conversion processes, including biomass pretreatment and hydrolysis, is a challenging task because of the distinct variations in composition and structure of biopolymers among biomass types. Efficient fermentation of biomass hydrolyzates comprising of different types of sugars is challenging. The purpose of this doctoral research was to evaluate and optimize the various processing steps in the entire the biomass value chain for efficient production of advanced biofuels and chemicals from diverse biomass feedstocks. Our results showed that densification of bulky biomass by pelleting to better streamline the handling and logistic issues improved pretreatment and hydrolysis efficiencies. Alkali pretreatment was significantly more effective than acid pretreatment at same processing conditions for grass and hardwood. The ethanol-isopropanol mixture, and glycerol with 0.4% (w/v) sodium hydroxide were the promising organic solvent systems for the pretreatment of corn stover (grass), and poplar (hardwood), respectively. None of the pretreatment methods used in this study worked well for Douglas fir (softwood), which indicates a need to further optimize appropriate processing conditions, better solvent and catalyst for effective pretreatment of this biomass. The brown midrib (bmr) mutations improved the biomass quality as a feedstock for biochemicals production in some sorghum cultivars and bmr types, while adverse effects were observed in others. These results indicated that each potential sorghum cultivar should be separately evaluated for each type of bmr mutation to develop the best sorghum line as an energy crop. Development of an appropriate biomass processing technology to generate separate cellulose and hemicellulose hydrolyzates is required for efficient 2,3-butanediol (BD) fermentation using a non-pathogenic bacterial strain, Bacillus licheniformis DSM 8785. This culture is significantly more efficient for BD fermentation in single sugar media than Klebsiella oxytoca ATCC 8724. Though K. oxytoca is a better culture reported so far for BD fermentation from diverse sugars media, but it is a biosafety level 2 organism, which limits its commercial potential.

Master of Science / Department of Chemistry / Christopher T. Culbertson / Capillary isoelectric focusing (cIEF) is one of several electrophoretic separation techniques for proteins and various other bio - molecules widely used in biochemistry laboratories. A wide range of analytes separable by the different modes of Capillary Electrophoresis (CE) includes from a small organic or an inorganic molecule to the complex bio-molecules such as protein, peptides, cell organelles, and live microorganisms (e.g. bacteria and viruses). Of the various modes of electrophoresis, Isoelectric focusing (IEF) is a good method for the separation of large amphoteric molecules such as peptides and proteins because of the attainment of overall surface charge depending up on its environment pH. This thesis mainly focuses on application of cIEF for proteins separation and viruses’ detection, which is one of the biggest concerns of human and animal health because of viral outbreak causing loss of thousands of lives and property every year. In chapter one of this thesis, the principles and mechanisms of separation of CE, cIEF, comparative advantages of dynamic coatings over static coating, and advantages of Whole Column Imaging Detection (WCID) over On - olumn Single Point Detection have been discussed. Chapter two includes experimental procedure and calculations for EOF determination. The results of cIEF experiments with standard proteins to develop calibration curve followed by UV absorbance detection of two bacteriophage viruses TR4 and T1 are presented in the chapter three. Final chapter four includes the conclusion and discussion on future direction for the project. The main motivation for this work was to develop a method which is less labor intensive and requires shorter detection time compared to traditional detection methods such as virus culture in serology (7days), polymerase chain reaction (PCR) and gel electrophoresis (6hrs to 2days). A commercially available dynamic coating reagent, EoTrol LN® copolymer used our CE experiments found to be more convenient and efficient than commonly used surface modifiers for example silane-based reagents. Preliminary determination of the pIs of these T1 and TR4 by cIEF was 3.1 ± 1.0 and 6.8 ± 1.0 respectively. The pI of viruses can differ by their strains and the phase of virus - growth. The viruses, though closely related, are easily distinguishable by their different pIs.

Master of Science / Department of Animal Sciences and Industry / KC Olson / Condensed tannins (CT) in plants are phenolic compounds with relatively high binding affinities for proteins. In ruminants, dietary CT limit DM intake and digestibility, and ruminal protein degradation by forming CT-protein complexes. Effects of dietary CT, animal species, prior dietary CT exposure, and antimicrobial inclusion on 48-h rate and extent of digestion were measured in two in vitro experiments. Cattle, sheep, and goats (n = 3 / species) were used in a 2-period, randomized complete-block experiment with a 2 × 3 × 2 × 3 factorial arrangement of treatments. Factor 1 was substrate: tannin-free or high-CT. Factor 2 was source of ruminal fluid inoculum: cattle, sheep, or goat. Factor 3 was prior animal exposure to a high-CT diet: non-exposed or exposed. Factor 4 was inclusion of antimicrobials: no antimicrobial, penicillin + streptomycin to suppress bacterial activity, or cycloheximide to suppress fungal activity in the fermentation. Tannin-free or high-CT substrates were incubated in vitro using ruminal fluid from animals either not exposed (period 1) or exposed to dietary CT (period 2). Periods consisted of an adaptation to tannin-free (10 d) or high-CT diets (21 d) and a 15-d period of ruminal-fluid collection via stomach tube. The presence of CT or penicillin + streptomycin in in vitro fermentation reduced (P < 0.001) total gas pressure, DM disappearance, and total VFA, acetate, propionate, butyrate, valerate, and branched-chain VFA concentrations. We concluded that: 1) CT had negative effects on fermentation, 2) prior exposure to dietary CT attenuated some but not all negative effects, and 3) CT effects were similar to the effects of penicillin + streptomycin.