Browsing by Subject "protein"
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Item Aquatic Plants from Minnesota Part 4 - Nutrient Composition(Water Resources Research Center, University of Minnesota, 1973-04) Goodrich, R.D.; Linn, J.G.; Meiske, J.C.; Staba, E. JohnSamples of 22 freshwater aquatic plants were analyzed to determine their potential feeding value for ruminants. Proximate analyses (mean +/- SD), on a dry matter basis were: crude protein, 12.7+/-4. 4%; either extract, 1.46+/-.98%; crude fiber, 19.2+/-6.4%; ash, 2.05+/-1.24%; and NFE 64.6+/-6.5%. NDF, ADF, and ADL contents averaged 41.6+/-13.4%; 32.0+/-9.6% and 6.35+/-2.76%, respectively. Mineral contents (mean +/-SD) of the 22 aquatic plants were: P,o.25+/-0.19%; Ca, 1.83+/-1.68%; K, 1.54+/-.92%; Na, 0.30+/-.25%; mg, 0.31+/-.16%; Fe 924=?-730 ppm; Zn, 80.6+/-96.6 ppm; Cu, 13.8+/- 34.0 ppm; Mo, 19.7+/-9.7 ppm and Mn, 269+/-152 ppm. Van Soest's estimated apparent digestibility averaged 63.0+/-8.3%.Item Characterizing mVenus adsorption to photodegraded polyethylene using circular dichroism and fluorescence spectroscopy(2022-08) Amaris, AltheaDue to their versatility and relative cost-effectiveness, plastics as a material have gained increasing popularity and are heavily utilized by almost every major industry in the modern day. Their exponential rate of production coupled with a lack of proper disposal methods, however, have resulted in the global environmental issue of plastic pollution. Upon entering the ecosystem, plastic surfaces can act as a foundation for the formation of microbial communities known as biofilms. An initial key step to biofilm growth is the attachment of bacterial surface proteins onto the polymer. In this study, we examine structural changes of a “hard” model protein in the presence of environmentally relevant plastics. Using the intrinsic probes of the mVenus protein, a model yellow fluorescent protein (YFP), we study its structural response to variably photo-aged polyethylene (PE) through circular dichroism (CD) and tryptophan (W)/YFP-fluorescence spectroscopy. Upon binding to aged PE, mVenus undergoes mild secondary structure rearrangement. Interestingly, a forbidden transition in W-fluorescence is observed, evolving from the interaction between the sole tryptophan in mVenus and the increasingly hydrophilic surface of PE as the polymer is progressively photo-oxidized. The beta barrel and beta sheet structure of mVenus retains the overall stability of the protein, whereas the local structure and turn regions accommodate the protein-polymer interactions based on polymer surface chemistry. We can therefore start to predict that proteins bind variably during the initial docking of cells as the secondary structure behaves distinctly based on the age of the film to which it attaches. The dependence of protein docking on the extent of PE-irradiation reveals that film age, polymer type, and structural stability can either accelerate or inhibit biofilm growth.Item Effect of the Thermodynamic and Physical State of the Freeze-Concentrate on Protein Stability(2017-12) Jena, SampreetiIn this dissertation research, specific interactions (excipient-excipient, excipient/protein-ice, protein-excipient) governing protein conformational stability and crystallization behavior of excipients in the freeze concentrate, were explored. Furthermore, the effects of formulation composition (type and mole fractions of excipients in the formulation) on afore-mentioned interactions, during freeze-thaw and freeze-drying of protein formulations, was investigated. Concentration dependent effects of excipients including the bulking agent, lyo/cryo-protectant and surfactant on the nucleation and growth of crystalline phases in the freeze concentrate were characterized and quantified. Changes in the secondary and tertiary conformations of model proteins (such as Bovine Serum Albumin and Immunoglobulin) due to crystallization of excipients, were determined as a function of formulation composition during freeze-thaw and freeze-drying. Infrared (IR) Spectroscopy was used to detect onset of crystallization the bulking agent and lyo/cryo-protectant. X-Ray Diffractometry (XRD) was used to characterize the polymorphic form of crystalline phases. Far UV circular Dichroism (CD) was used to characterize secondary conformation of protein in thawed and reconstituted (freeze-dried) formulations. IR Spectroscopy was used to characterize secondary conformation of protein in frozen and freeze-dried formulations. A bulking agent – lyo/cryo-protectant – protein system, a typical freeze-drying formulation, was chosen for characterization of frozen and freeze-dried formulations. It was observed that high concentrations of non-crystallizing components such as the protein and lyo/cryo-protectant (usually a disaccharide such as trehalose) inhibited crystallization of the (otherwise readily crystallizing) bulking agent (such as mannitol) and vice versa. At low concentrations, surfactants such as Polysorbate 20, prevented growth of crystalline phases due to amphiphilic interface coverage, but when their concentrations exceeded the critical micelle concentration (CMC), they enhanced degree of crystallinity in the formulation. Structural unfolding of the protein was detected upon crystallization of the lyo/cryo-protectant and micelle formation (when surfactant concentration exceeded CMC). Detection of protein aggregates in reconstituted solutions, confirmed that unfolding induced during freezing, thawing and drying processes, did not reverse upon reconstitution. Presence of ice surfaces and other crystalline interfaces (such as those introduced by the bulking agent) significantly contributed to protein degradation. In our model system, thawing induced stresses such as recrystallization were found to be more detrimental than the stresses induced by freezing and desiccation and hence, freeze-drying yielded better structural recovery of the protein than freeze-thaw in our model system. Secondary relaxations arising from the flexible polar groups on the protein surface (millisecond time scales) and dynamic ring flips of the monosaccharide units about the glycosidic linkage (microsecond time scales) of disaccharides (indicating flexibility of glycosidic linkage) were detected in our model freeze-dried system using Frequency Domain Dielectric Spectroscopy. In the presence of protein, flexibility of the glycosidic linkage was decreased and likewise, presence of disaccharides slowed down the dynamics of flexible protein groups, up to a critical protein to disaccharide mass ratio (= 0.5). Surfactant and higher protein to disaccharide mass ratios (≥ 0.5) produced the opposite effect. These secondary relaxations govern conformational stability of the protein and propensity of the disaccharide to crystallize during storage below the Tg. In the final part of the thesis, effects of slow freezing on lyo/cryo-protectant-protein formulations during cryo-vitrification was investigated. Chemical toxicity of cell penetrating lyo/cryo -protectants such as Dimethyl Sulfoxide (DMSO), frequently used for cryo-vitrification of organs and tissues, was shown to be dictated by their hydrogen bonding behavior (characterized by IR Spectroscopy). At temperatures where hydrogen bonding interactions between lyo/cryo-protectant and water were unfavorable, the lyo/cryo-protectant directly partitioned in the hydration shell of the protein and caused unfolding of the protein, potentially due to hydrophobic interactions. It was also ascertained that when the freeze concentrate is vitrified during freezing, rapid thawing is a necessity to minimize ice recrystallization during devitrification to minimize the damage to the proteins. This dissertation research has enhanced an overall understanding of interactions between the excipients, protein and crystalline interfaces (of ice and crystalline excipients such as bulking agent) as well as protein dynamics in the freeze-concentrate. This information is needed to identify stresses arising in the protein micro-environment that lead to conformational destabilization (and loss of activity) during preservation of protein formulations and is currently absent in literature.Item Enhancement of Pea Protein Solubility and Thermal Stability for Beverage Applications via Endogenous Maillard-Induced Glycation and Chromatography Purification(2022-05) Schneider, AlissaGrowing demands for non-soy, plant protein sources have guided the rapid expansion of the pea protein ingredient market in recent years. Pea protein has emerged as the most prominent alternative to soy protein, as pea is not a major allergen, non-GM, sustainable, and widely available. Accordingly, a variety of pea protein products are now commercially available, suited for a number of different applications. Pea protein generally exhibits inferior functionality compared to soy protein, however, as a result of its intrinsic protein profile and structure, especially following commercial processing. Namely, pea protein exhibits inferior solubility and thermal stability compared to whey and soy proteins, limiting its application in high-protein, RTD beverages. Enhancement of pea protein solubility under acidic conditions and following thermal treatments is, therefore, of interest.Controlled, Maillard-induced glycation is a protein modification technique that has the potential to improve pea protein solubility and thermal stability. While glycation of pea protein has been reported, this process has not been developed for an industrial scale, unreacted carbohydrates are rarely removed, and all previous studies have utilized exogenous saccharides (e.g., pectin, gum Arabic, and corn maltodextrin), presenting concerns regarding application in “clean label” products. The starch-rich by-product of pea protein extraction may be further processed to produce an endogenous reducing saccharide, such as maltodextrin, which may react with protein under controlled glycation conditions. Glycation coupled with purification of partially-glycated pea protein has the potential to produce a highly soluble and thermally stable protein ingredient with added-value, having potential for RTD beverage applications. Therefore, the main objective of this work was to enhance pea protein solubility and thermal stability by producing an endogenously and partially-glycated pea protein (PG-PP) ingredient by completing several goals: (1) develop a method to produce maltodextrin from pea starch with a specific reducing power, (2) initiate and control the early stage of the Maillard reaction to partially-glycate pea protein isolate (PPI) with pea maltodextrin, (3) remove unreacted maltodextrin from the PG-PP via hydrophobic interaction chromatography (HIC) to produce a purified, PG-PP concentrate or isolate, and (4) characterize the effect of glycation coupled with purification on protein structure and the consequent impact on solubility and thermal stability. A method to produce maltodextrin from the pea starch-rich by-product obtained during the production of native pea protein isolate (nPPI) was developed by monitoring maltodextrin dextrose equivalent (DE) in response to hydrolysis time, small saccharide removal, and centrifugation to remove large molecular weight residual starch and fiber. The resulting chain-length distribution was evaluated. Maillard-induced glycation was then confirmed and monitored by assessing changes in color, free amino groups, and protein/glycoprotein profiles upon incubation of the produced maltodextrin with nPPI. Next, removal of unreacted maltodextrin by HIC was confirmed by monitoring maltodextrin elution. The purified PG-PP, along with purified PPI controls and reference samples (nPPI and commercial PPI), were then characterized by assessing their composition and protein/glycoprotein profile. Protein thermal denaturation properties, surface properties (surface hydrophobicity and zeta potential), and secondary structures were evaluated, as well. Lastly, protein solubility and thermal stability and protein digestibility were assessed. The starch-rich by-product was partially hydrolyzed, with hydrolysis conditions optimized to produce maltodextrin with targeted characteristics (DE 15.7, average degree of polymerization 8.3). PPI and maltodextrin were incubated under mild conditions, which initiated and controlled the Maillard reaction to the early stage. PG-PP was formed with minimal browning and protein polymerization, along with moderate free amino group loss. Additionally, PG-PP was purified by removing the majority of unreacted carbohydrates and polymerized proteins via HIC, resulting in a purified water fraction (PW-PG-PP) with a protein content of nearly 60%, reduced surface hydrophilicity, and increased solubility (up to ~91%) and thermal stability at conditions relevant to RTD beverages. Protein digestibility of PW-PG-PP was high and similar to the references. Purification of nPPI control also produced a highly soluble and thermally stable sample with good protein digestibility. HIC removed hydrophobic and polymerized proteins from nPPI, allowing for the fractionation and concentration of hydrophilic proteins in nPPI. This study proved the concept of “clean-label”, endogenous glycation of pea protein, utilizing endogenously produced maltodextrin and controlled Maillard reaction conditions. Additionally, both endogenous glycation coupled with HIC purification, as well and HIC purification alone, greatly improved the solubility and thermal stability of nPPI under acidic conditions and at a high-protein claim concentration (5% protein), with purified samples having nearly the solubility of whey protein, the gold standard for beverages. These processes also largely maintained the digestibility of PPI. Therefore, glycation and HIC purification created pea protein with potential value for application in high-protein, RTD beverages. Moreover, this work uncovered a PPI fractionation process that has the potential to increase specialty pea protein ingredient value, with the water soluble fraction suitable for beverage applications and hydrophobic fraction suitable for meat analogues. This work also provided foundational information, paving the way for future investigation and process optimization for scaled-up glycation and purification of pea protein.Item The extraction, characterization, modification, and texturization of novel pennycress (Thlaspi arvense) protein for food applications(2023-11) Mitacek, RachelThe food industry is actively seeking functional, nutritious, and sustainably produced crops as novel sources of plant protein ingredients to aid in feeding the growing population and address consumer demands. Pennycress (Thlaspi arvense) protein is an attractive alternative to market leading proteins, soy and wheat, as it is currently non-allergenic, non-GMO, and has vast environmental benefits. As a winter cover crop, pennycress provides soil stabilization, nutrient sequestration, and reduced nitrate leaching. Furthermore, pennycress oilseeds are high in protein and oil contents, which are attractive to valorize as two potential food ingredients. The oil from pennycress is currently utilized for industrial biofuels, leaving behind a proteinaceous meal as a by-product. Extracting protein from the meal will increase crop value, creating incentive for farmers to grow this sustainable crop, and will aid in addressing the growing consumer demands for alternative sources of plant proteins. Research on the utilization of pennycress oilseeds for food applications is limited due to antinutritional compounds, namely erucic acid and glucosinolates. Recent agricultural advancements have identified accessions of pennycress with no erucic acid, which are suitable for human consumption. In addition, glucosinolates, which are typically abundant in pennycress meal, are lost during protein isolation steps. Determining optimal, scalable protein extraction conditions that have a high yield of functional, nutritious protein isolates is crucial when evaluating novel crops for food applications. Furthermore, identifying differences in protein structural and functional characteristics among genetically diverse lines is an instrumental knowledge in the advancement of breeding efforts for pennycress. Many plant proteins are known to have inferior functionality compared to whey and soy protein, limiting their use in a variety of applications. Accordingly, this research was divided into two studies to evaluate protein extraction conditions and their impact on structural, functional, and nutritional characteristics of pennycress protein, and then to enhance inferior functional properties through targeted structural modification techniques. Therefore, the objectives of the first study were to 1) optimize protein extraction conditions to maximize yield and purity following two extraction methods, alkaline solubilization coupled with isoelectric precipitation and salt solubilization coupled with ultrafiltration and 2) characterize structural, functional, and nutritional properties of pennycress protein isolates as impacted by the extraction method, scaling up, and difference in genetic variety. Wild-type (W), and zero erucic acid (0EA) pennycress seeds harvested in 2017 were screw-pressed to expel the oil, milled to 60-mesh, and then residual oil was extracted using hexane to produce defatted pennycress meal (DPM). W-DPM was utilized for protein extraction following alkaline solubilization coupled with isoelectric precipitation and salt solubilization coupled with ultrafiltration. Pennycress protein isolate (PcPI) from alkaline extraction (W-PcPI-pH) had a dark, undesirable color, therefore, sodium sulfite was utilized during alkaline solubilization as a reducing agent to mitigate browning. Salt extracted pennycress protein isolate (W-PcPI-Salt) had superior color and functionality. Therefore, salt extraction was used for pilot plant scale up production of PcPI and for protein extraction from 0EA-DPM. Structural and functional characterization was performed on PcPI produced following selected alkaline (with and without sodium sulfite, W-PcPI-pH and W-PcPI-pH-S, respectively) and salt extraction conditions, scaled up salt extraction, and from 0EA seeds. Structural and functional properties of the PcPI samples were compared to native (nSPI) and commercial (cSPI) soy protein isolates. Furthermore, PcPI-salt and W-DPM were evaluated for in-vitro and in-vivo protein digestibility corrected amino acid score (PDCAAS). PcPI-pH, produced with and without the use of sodium sulfite, had relatively poor functionality overall as a consequence of excessive protein denaturation and aggregation and high surface hydrophobicity. On the other hand, W-PcPI-Salt had similar gel strength, three times higher solubility under acidic conditions, and 1.5 times higher emulsification capacity compared to cSPI. 0EA-PcPI-Salt had comparable functionality to that of W-PcPI-Salt. The scaling up process of W-PcPI-Salt resulted in partial denaturation and mild polymerization that contributed to enhanced surface hydrophilic/hydrophobic balance, water holding capacity (WHC), and gel strength compared to its bench scale counterpart. Protein profiling showed that PcPI contains primarily small molecular weight proteins compared to nSPI, contributing to inferior gelation and WHC. Finally, the in-vitro (0.87) and in-vivo (0.72) PDCAAS of PcPI-Salt was superior or comparable to other commercially available plant protein sources. Protein crosslinking and formation of soluble aggregates are required for the development of a strong 3-dimensional gel network that entraps water. Proteins of relatively large molecular weight are correlated with a high potential to form cohesive, strong gel networks. Crosslinking proteins in PcPI will increase gel strength and WHC for enhanced texturization potential, and incorporation into high-value meat analogue applications. Protein crosslinking can be induced by either transglutaminase (TG) or physical treatment with cold atmospheric plasma (CAP). Therefore, the objectives of the second study were to 1) evaluate the effect of CAP and TG modifications on the structural and functional characteristics of PcPI, and 2) to determine the texturization potential of the modified PcPI. CAP treatment with dielectric barrier discharge (DBD) was utilized to polymerize PcPI (PcPI-CP). The production of TG modified PcPI (PcPI-TG) was optimized for enzyme dose, the use of pre-treatment denaturation, and time based on lysine crosslinking and protein profile. PcPI-CP and PcPI-TG were evaluated for structural and functional properties compared to unmodified PcPI. Micro-compounding was utilized for bench scale texturization of unmodified, PcPI-CP, and PcPI-TG at 50% water content. The texturization potential was assessed through mechanical responses during micro-compounding, structural properties, and texture profile analysis. CAP treatment induced polymerization primarily through intermolecular disulfide interchange, whereas TG resulted in a relatively higher extent of polymerization that was induced through a combination of inter- and intramolecular disulfide linkages and other covalent interactions involving acidic subunits of cruciferin. Compared to unmodified PcPI, PcPI-CP and PcPI-TG had double and triple the gel strength, respectively. Furthermore, PcPI-TG had the highest WHC (almost 100%). Upon micro-compounding, unmodified PcPI did not form fibrous structures and instead was a soft mass with low resilience and cohesiveness. Micro-compounding of PcPI-CP resulted in hard, dense fibrous structures due to the low WHC. However, the high gel strength and WHC of PcPI-TG resulted in fibrous structures with more air incorporation upon micro-compounding. Results confirmed that polymerization, especially with TG, can enhance gelation properties and texturization potential of PcPI. This work was the first to optimize protein extraction conditions from pennycress and provide a comprehensive structural, functional, and nutritional comparison among the resulting isolates. Overall, this work demonstrated that PcPI can be successfully extracted from DPM with high protein purity and yield, and acceptable color. Furthermore, the characterization of PcPI from genetically diverse lines provided a benchmark of knowledge to progress pennycress breeding efforts. Results confirmed that salt extraction is scalable and can result in PcPI with favorable functional properties that are comparable, or in some cases superior to cSPI. The low gel strength of PcPI was overcome by inducing polymerization through CAP and TG treatment, ultimately enhancing texturization potential. In particular, TG modification increased the WHC of PcPI, which resulted in textural properties that are desirable for meat analogue applications. This research provided foundational knowledge for the processing, modification, and utilization of PcPI. The introduction of PcPI into the protein ingredients market provides a sustainable, nutritious, and highly functional protein source for use in a wide range of food applications.Item Extraction, Modification, and Chemical Characterization of Protein and Dietary Fiber from Camelina Sativa(2018-07) Boyle, ClaireCamelina sativa, a sustainable short-season cover crop, is an oilseed (35% oil) gaining interest due to the increasing global demand for sustainably sourced ingredients. Camelina provides numerous agricultural benefits—low production cost, low nitrogen requirements, drought resistance, cold weather tolerance, and short growing season—in addition to being high in protein (20%) and dietary fiber (30%), which are two of the fastest growing segments of the food ingredient market. In order to create functional, market-viable ingredients from camelina, the following need to be explored: efficient means of protein extraction, evaluation of protein functional properties, and chemical characterization of the dietary fiber constituents. The objectives of this study were as follows: (1) determine the impact of oil pressing conditions and protein extraction protocol on protein yield and content; (2) characterize structural differences in proteins extracted following salt precipitation and pH solubilization; (3) determine the impact of structure and enzymatic modification on the functionality of the different protein extracts; (4) isolate, quantify, and characterize the insoluble and soluble dietary fiber fractions of defatted camelina meal (DCM) prepared by two different oil pressing conditions. Protein extraction by pH solubilization and salt precipitation was tested and optimized. Camelina meal obtained from hot and cold press was further defatted by hexane and analyzed for protein content. Protein from DCM was extracted following degumming and pH solubilization at pH 12, separating non-protein material by centrifugation, acidifying the supernatant to pH 5 to precipitate out the protein, neutralizing and desalting. Protein from DCM was also extracted following salt precipitation, first by solubilizing the protein using 0.05 M phosphate buffer (pH 8, 1 M NaCl), followed by precipitation using 85% saturated ammonium sulfate solution, and desalting. To produce protein hydrolysates, extracted proteins were subjected to hydrolysis with Aspergillus oryzae protease by pH-stat methodology to a degree of hydrolysis less than 8%. Protein purity of the extracts was analyzed, and mass balances were tracked in order to evaluate extraction yields. The denaturation state, protein profile, and surface hydrophobicity of the protein extracts were determined using DSC, SDS-PAGE, and a fluorometric assay, respectively. Functionality was evaluated by determining protein solubility as well as emulsification, foaming, and gelation properties. Total dietary fiber (TDF) from DCM was determined following the AOAC method 2011.25, and three fractions —insoluble dietary fiber (IDF), soluble dietary fiber that precipitates in 78% ethanol (SDFP), and soluble dietary fiber that is soluble in 78% ethanol (SDFS) — were isolated preparatively. IDF and SDFP were analyzed spectrophotometrically for pectin content. The monomers of IDF and SDFP fractions were determined by alditol acetate formation and measured by GC-FID. Degree of pectin methylation (DM) of SDFP was determined by 1H NMR. The degree of polymerization (DP) of saccharides in the SDFS fraction (DP 2 – DP 7) was determined by liquid chromatography-ESI-mass spectrometry (LC-MS) using a ligand-exchange stationary phase and quantified by high performance anion exchange chromatography coupled with a pulsed amperometric detector (HPAEC-PAD). Disaccharides in DCM were differentiated and quantified spectrophotometrically following standard enzymatic assays. Compared to camelina protein concentrates (CPC) produced by alkaline pH extraction, CPC produced by salt extraction were less denatured and more functional. The functionality of the salt extracted CPC was comparable and sometimes better than that of soy protein isolate (SPI). Specifically, the solubility of the salt extracted CPC at pH 3.4 was significantly (P < 0.05) higher than that of SPI. Additionally, salt extracted CPC had significantly higher emulsification capacity and foaming capacity than SPI. On the other hand, the gelation property of CPC was inferior to that SPI, an observation attributed to the molecular size of camelina protein compared to SPI. Upon hydrolysis of CPC with Aspergillus oryzae protease, a limited benefit to solubility was noted at pH 7 post thermal treatment. TDF of DCM averaged 51.2% (45.3 – 49.1% IDF, 2.00 – 5.98% SDFP, 1.1 – 1.2% SDFS). The SDFS fraction was comprised mainly of stachyose and raffinose, which is in line with other Brassicaceae crops. The chief disaccharide present in DCM was verified to be sucrose (2.43 – 3.36%). Free glucose and fructose were also present in the SDFS fraction. Of the pectic polysaccharides measured in SDFP, low methoxyl pectin represented the major constituent, with a DM of 12.5 – 14.5%. Based on alditol acetate analysis, glucose was the main monomer in the IDF fraction. Other monosaccharides detected in the IDF fraction were xylose, arabinose, mannose, and galactose. The monosaccharide composition indicated the presence of cellulose, xyloglucans, galactomannans, and arabinoxylans in the IDF fraction. In SDFP, the monosaccharides rhamnose, arabinose, galactose, and mannose were evenly distributed. Monomer composition of the SDFP fraction indicated the presence of pectin and galactomannans. Results show that camelina meal contains a significant amount of protein and dietary fiber that can be isolated into functional ingredients. This is the first study to provide a comprehensive evaluation of protein and dietary fiber from camelina as potential alternatives to traditional ingredients. Further work is needed to understand how isolated camelina ingredients interact in various food matrices.Item Improvement of low fat Cheddar cheese texture using whey protein isolate aggregates(2015-02) Erickson, MollyTwo microparticulated whey protein fat mimetics were developed using the addition of lambda carrageenan to reduce whey protein aggregate size to approximately 2-10um. Interactions between the lamdba carrageenan and whey protein were analyzed using SDS-PAGE. Results suggest lambda carrageenan is not bound to the whey protein and that the aggregates were stabilized by disulfide bonding between the proteins. The fat mimetics were added to low fat Cheddar cheese at two addition rates and tested at one and two months of aging. Addition of fat mimetics produced a weaker cheese gel and produced no improvements in textural qualities as analyzed using rheological techniques. Low additions of fat mimetic produced a firmer texture. Though not significantly different than the low fat control, high addition rates of fat mimetic showed promise in improving texture, and confocal microscopy images suggest a disruption of protein structure with the high addition rates.Item Interrelationships between Soybean Seed Quality Characteristics(2016-10) Pfarr, MatthewThe quality of a protein for animal growth is partially determined by the relative abundance of essential amino acids. Those essential amino acids supplied in the lowest quantity relative to the animal’s requirement limit growth. Examination of soybean protein across genetic sources and environments has indicated that the abundance of potentially limiting amino acids within soybean protein may be influenced by the seed protein concentration. Our objective was to evaluate the effects of seed protein concentration on relative amino acid abundance under controlled environments in order to better understand the biological basis of this apparent relationship. This was accomplished through the use of source-sink treatments that altered seed protein concentration within environments. Increasing the source-to-sink ratio through partial pod removal and open environment treatments significantly increased seed protein; however, the resulting protein was disproportionately enriched in the amino acids glutamic acid and arginine at the expense of the limiting amino acids lysine, cysteine, methionine, threonine, and tryptophan. Defoliation treatments gave the opposite response to pod removal, resulting in a more favorable amino acid balance but with a lower seed protein concentration. Alternatively, the shade treatment increased protein concentration, but the relative concentration of the limiting amino acids was not reduced. This indicates that limiting amino acid abundance is not solely dependent on seed protein percentage and that limiting amino acids may be supplied by the vegetative tissue under C-limited conditions. The ultimate goal of soybean seed improvement is to increase yield while also increasing or maintaining seed protein concentration and the balance of the limiting amino acids. Meeting two of these goals was achieved through the current source-sink treatments as the open environment treatment increased seed yield and protein concentration while shade increased protein concentration and maintained limiting amino acid balance. Meeting all three goals concurrently for soybean improvement was not achieved in the current experiment and may be difficult.Item Optimization of Hemp (Cannabis sativa L.) Protein Extraction and Characterization of Protein Structure, Function, and Nutritional Quality across Different Cultivars(2022-01) Eckhardt, LauraThe global population is predicted to reach 9.7 billion people in 2050, presenting a significant challenge of producing enough nutritious food in a sustainable way. Specifically, the demand for protein is increasing, with marked increase in the demand for plant protein ingredients. Current plant protein sources (soy, wheat, pea) have limitations with respect to consumer perspective or from a functional or flavor perspective, necessitating exploration of novel sources.Hemp (Cannabis sativa L.), which has been cultivated for thousands of years, is an environmentally friendly crop. However, legal restrictions due to the presence of the psychotropic component delta-9 tetrahydrocannabinol (THC) have antagonized the hemp market for decades. In 2018, hulled hemp seeds, hemp seed protein powder, and hemp seed oil achieved “generally recognized as safe” GRAS status in the U.S. Hemp seeds contain high amounts of oil (~ 30%) and protein (~25%). Research on feasible production of hemp protein isolate (HPI) and on the functional properties for food applications is minimal. Determining optimal protein extraction procedures to produce HPI will be instrumental in the adoption as a desirable protein ingredient. Additionally, identifying differences in seed protein characteristics in different cultivars is needed to initiate breeding strategies to improve the prospects for food applications. Therefore, the objectives of this work were to 1) optimize protein extraction from hemp seed, following alkaline solubilization coupled with isoelectric precipitation and salt solubilization coupled with membrane filtration, to produce a protein isolate with acceptable color, purity, yield, structural and functional characteristics, and nutritional quality, and 2) evaluate HPI produced from four industrial cultivars for differences in color and protein structural, functional, and nutritional properties. Whole hemp seeds from one cultivar (CFX-2) harvested in 2016 were dehulled using an impact dehuller and further separated using sieves, an aerator, a gravity separator table, and manual separation. Dehulled seeds were pressed using a cold hydraulic press, ground, defatted with hexane, and milled to 50 mesh prior to protein extraction. Two methods of protein extraction were tested – alkaline solubilization coupled with isoelectric precipitation and salt solubilization coupled with membrane filtration. Optimal protein extraction conditions were determined by evaluating different parameters including solubilization pH, precipitation pH, salt (NaCl) solubilization concentration, and heating and assessing protein purity and yield. HPIs produced using both methods (pH-HPI and salt-HPI) were characterized in comparison to commercial soy protein (cSPI) and pea protein isolates (cPPI). Color was measured using a colorimeter. Structural analysis was performed using SDS-PAGE for protein profile, DSC for protein denaturation, a spectrophotometric method for surface hydrophobicity, zeta potential for surface charge, and Fourier transform infrared spectroscopy (ATR-FTIR) for protein secondary structure. Protein functionality including solubility, gel strength, water-holding capacity, and emulsification and foaming properties were evaluated. Structural and functional testing were performed in water and in 0.5 M NaCl due to sedimentation of salt-HPI in water. Nutritional quality was determined by calculating the protein digestibility-corrected amino acid score (PDCAAS) based on amino acid analysis and the pH drop in vitro protein digestibility assay. Whole hemp seeds from four industrial cultivars (CFX-2, Grandi, Joey, Picolo) harvested in 2019 were dehulled, separated, defatted, and milled, as described above. Four HPI samples were produced from the cultivars following the optimized pH extraction method. Color and protein structural, functional, and nutritional properties were characterized as described above. Structural and functional testing was only performed in water. Both the optimized pH-assisted (solubilization at pH 11, precipitation at pH 5) and salt-assisted (solubilization in 0.75 M NaCl at 50°C followed by ultrafiltration/diafiltration) protein extraction methods produced HPI with high protein purities (87 – 88% protein) and remarkable yields (> 80%). The use of dehulling prior to defatting and protein extraction resulted in HPIs with desirable light and bland colors. Both extraction methods produced isolates with similar protein profiles, but pH-HPI exhibited some protein polymerization and was partially denatured. These structural differences appeared to improve HPI properties. Specifically, salt-HPI needed to be dispersed in a dilute salt solution to form a gel, while pH-HPI formed a gel in water at relatively low protein concentration (10% protein). Overall, HPI was less functional than cSPI and cPPI, but had similar solubility to cSPI at acidic pH in water, and superior solubility (P < 0.05) and gel strength at neutral pH in 0.5 M NaCl. While the use of 0.5 M NaCl for solubilization improved HPI gel strength and solubility at neutral pH, it negatively impacted water holding capacity and foaming stability. The PDCAAS of HPI (0.58 for pH-HPI and 0.54 for salt-HPI) was within the range previously reported for whole hemp seeds, dehulled hemp seeds, and hemp seed meal (0.48 – 0.61). In general, alkaline solubilization coupled with isoelectric precipitation was determined to produce a more functional and nutritious HPI. Minimal structural differences among HPI extracted from the four cultivars were observed, which contributed to only slight differences in functionality and nutritional properties. All HPIs had similar solubility to cSPI at acidic pH, and three cultivars (Grandi, Joey, Picolo) produced significantly stronger gels (P < 0.05) than cSPI. There were no significant differences in in vitro digestibility among the four HPIs, but differences in amino acid profile led to significant yet minor differences in PDCAAS. This work demonstrated that protein can be successfully extracted from dehulled hemp seeds to produce an HPI with high protein purity, yield, and acceptable color. Protein extraction using pH produced an HPI with some promising functional attributes comparable or in some cases superior to cSPI and cPPI. This study was the first to optimize protein extraction parameters for hemp and to provide a comprehensive structural, functional, and nutritional comparison between pH-extracted and salt-extracted HPI. Additionally, this study was the first to examine the impact of cultivar on HPI properties. Since minimal differences among HPIs from four industrial cultivars were observed, more cultivars should be tested for differences that would warrant a hemp breeding program for improved functionality and nutritional quality. Future work is also needed to understand how HPI functions within a food matrix.Item Storage stability of a commercial spray dried hen egg yolk powder(2016-03) Guo, MufanDehydration is a good process approach for food preservation. However, dried food products may still suffer from deterioration if store in an abused environment such as high humidity (water activity (aw) > 0.6) and/or high temperature (> 45°C). These storage conditions can induce undesirable chemical reactions (disulfide bond interactions, Maillard reaction and/or lipid oxidation), resulting in a significant decrease in food quality. In this study, the storage stability of a commercial spray-dried egg yolk powder was evaluated. The dried egg yolk powder (DEY) was stored at three temperatures (room temperature, 35°C, and 45°C) and at six aw (0.05, 0.12, 0.37, 0.44, 0.54, 0.66) for at least two months, and several physicochemical changes and extent of protein aggregation were measured. The overall color change of DEY was that it became slightly darker (decrease of L* value), more red (increase of a* value), and less yellow (decrease of b* value) with increased storage time. The reaction kinetics of the L* value of DEY was also calculated using a first-order hyperbolic model. Its Q10 (rate increase with temperature increase at 10°C) was 2.9, which was more indicative of lipid oxidation, and the Ea (activation energy) was around 83 kJ/mole. The color change was mostly due to the browning pigments that were produced from the Maillard reaction and lipid oxidation. The glucose content went to zero after one-week during storage at 45°C at an aw of 0.66, confirming the occurrence of the Maillard reaction. The peroxide value of DEY storage at 45°C at aw of 0.66 was significantly increased compared to the control (vacuum packaged at -20°C), proving the occurrence of lipid oxidation. In addition, the Maillard reaction products and lipid oxidation products were both detected using the front face fluorescence spectrometer. After storage at an aw of 0.66 at 45°C for 8 weeks, protein solubility of DEY in TBS-SDS buffer [Tris-buffered saline (TBS: 20 mM Tris and 500 mM sodium chloride, pH 7.5) containing 1% sodium dodecyl sulfate (SDS, g/ml)] decreased to ~ 78% compared with that of the original DEY. Formations of buffer-soluble and –insoluble protein aggregates were discovered using SDS-PAGE. The protein aggregates were mainly formed through unfolded intermediates and unfolded states as well as direct chemical linkages. The proteins in DEY were all denatured after storage at an aw of 0.66 at 45°C for 8 weeks, resulting in numerous unfolded intermediates and states that could interact with each other to form aggregates. The spray drying process during the manufacturing of DEY also caused denaturation of protein, which explained the detection of buffer-insoluble protein aggregates in the original sample. Increases of disulfide bond links and protein-lipid interaction during storage were also found using techniques such as Raman spectrometry, fourier transform infrared spectroscopy, and front-face fluorescence spectrometry, indicating that some of the protein aggregates were induced by chemical reactions. The high molecular weight protein aggregates (HMWPAs) were further evaluated. Results showed that 32 proteins were involved with formation of buffer-soluble and -insoluble HMWPAs. They were products of natural egg yolk proteins and egg white proteins including serum albumin, vitellogenin, apovitellenin, as well as ovotansferrin, ovalbumin, lysozyme, ovomucoid, and ovastatin. Most of them contain disulfide bonds and some of them contain ligand and fatty acid binding sites, which corresponded with the theory of the direct chemical linkages induced protein aggregates. Overall, physicochemical changes and protein aggregates were found during the storage of DEY and it is mostly due to three undesirable chemical reactions, i.e., disulfide bond interactions, the Maillard reaction and/or lipid oxidation. Therefore, most effective approaches to reduce and/or inhibit the occurrence of those reactions include adjusting storage temperature and humidity as well as vacuum packaging after drying.