Canola protein isolate (CPI) has tremendous potential as a protein alternative to soy within the global protein ingredient market. The overall goal of this thesis was to compare and contrast the gelling mechanism of CPI with a commercial soy protein isolate (SPI) ingredient. Specifically, the gelation properties of CPI and SPI were evaluated as a function of protein concentration (5.0–9.0%), destabilizing agent [0.1 – 5.0 M urea; 0.1 and 1.0% 2-mercaptoethanol], ionic strength (0.1, 0.5 M NaCl) and pH (3.0, 5.0, 7.0, 9.0). The fractal properties of CPI were evaluated as a function of protein concentration (5.0 – 9.0%) and pH (3.0, 5.0, 7.0, 9.0). In the first study, the gelling properties of CPI and SPI as a function of concentration were evaluated, along with the nature of the interactions within their respective gel networks. Overall, the magnitude of the storage modulus (G') of the gel was found to increase with increasing concentration at pH 7.0, whereas the gelling temperature (Tgel) remained constant at ~88ºC. As the NaCl level was increased from 0.1 to 0.5 M, the zeta potential was found to be reduced from ~-20 to -4 mV, but with little effect on Tgel or network strength. In the presence of 2-mercaptoethanol, networks became weaker, indicating the importance of disulfide bridging within the CPI network. Disulfide bridging, electrostatics and hydrogen bonding are all thought to have a role in CPI gelation. In the case of SPI, the magnitude of the storage modulus (G') and Tgel were found to increase and decrease (~80ºC to 73ºC), respectively, with increasing urea concentration at pH 7.0. Increases in NaCl from 0.1 to 0.5 M reduced the zeta potential from ~-44 to -13 mV and caused a shift in Tgel from ~84ºC to 67ºC, and increased G'. No gels were formed in the presence of 2-mercaptoethanol. In the second study, the effect of pH on the gelling properties of CPI and SPI was evaluated. Surface charge (i.e., zeta potential) measurements as a function of pH found CPI to be positively (+18.6 mV), neutral and negatively (-32 mV) charged at pH 3.0, ~5.6 and 9.0, respectively. On the other hand, SPI was observed to be positively (+35.4 mV), neutral and negatively (-51 mV) charged at pH 3.0, 5.0 and 9.0, respectively. An increases in NaCl concentration from 0 M to 0.1 M resulted in a reduction in surface charge at all pHs for both CPI and SPI. Differential scanning calorimetry was performed to determine the thermal properties of CPI. The gelation temperature was found to be above the onset temperature for denaturation. For CPI, the onset of denaturation was found to occur at ~68ºC and then increased to ~78-79ºC at pH 7.0-9.0. With respect to rheological properties, SPI did not gel at pH 9.0, and G' declined as pH increased from 3.0 to 7.0. CPI did not gel at pH 3.0, however the network formed at pH 5.0 became stronger (higher G') as pH increased. The SPI gelling temperature at pH 3.0, 5.0 and 7.0 was observed to be ~85.6, ~46 and ~81ºC, respectively. SPI gels formed at pH 5.0 earlier due to increased protein aggregation near its isoelectric point (pI). The gelation temperature for CPI at pH 5.0 and 7.0 were similar (~88ºC), then declined at pH 9.0 (~82ºC). Network structure of CPI as a function of pH also was investigated using confocal scanning light microscopy (CSLM). As the pH became more alkaline from pH 7.0 to pH 9.0, there was a decrease in lacunarity (~0.41->~0.25). However, the fractal dimension was found to increase (from ~1.54 to ~1.82) showing that increasing the pH resulted in a more compacted CPI network. In summary, protein-protein aggregation induced either by increasing concentration or changing the pH resulted in network formation for both CPI and SPI, where both networks were thought to be stabilized by disulfide bridging and hydrogen bonding. SPI underwent protein aggregation earlier than CPI near its pI value, whereas CPI gels formed the strongest networks away from its pI under alkaline conditions. In all cases, CPI grew in diffusion-limited cluster-cluster aggregation to from the gel network.

Doctor of Philosophy / Department of Biological and Agricultural Engineering / Donghai Wang / Sorghum distillers dried grains with solubles (DDGS), canola and camelina meals are the main co-products resulting from grain-based ethanol or oil production. The main objective of this research was to study physicochemical properties of proteins isolated from DDGS, canola and camelina meals and their adhesion performance. Acetic acid-extracted sorghum protein (PI) from DDGS had superior adhesion performance in terms of dry, wet and soak adhesion strength compared to acetic acid-extracted sorghum protein (PF) from sorghum flour and aqueous ethanol-extracted sorghum protein (PII) from DDGS. PI had a significantly higher wet strength (3.15 MPa) than PII (2.17 MPa), PF (2.59 MPa), and soy protein without modification (1.63 MPa). The high content of hydrophobic amino acids in PI (57%) was likely the key factor responsible for high water resistance. Canola protein was extracted from canola meal and modified with different concentrations of NaHSO3 (0 to 15 g/L) during protein isolation. Unmodified canola protein showed the highest wet shear strength of 3.97 MPa cured at 190 °C. Adhesion strength of canola protein fractions extracted at pH 5.5 and pH 3.5 (3.9-4.1 MPa) was higher than the fraction extracted at pH 7.0. NaHSO3 slightly weakened adhesion performance of canola protein; however, it improved handling and flow-ability due to breaking of disulfide bonds in proteins. Albumin, globulin, and glutelins were isolated from camelina meal. Adhesion performance of globulin fraction behaved better than glutelin fraction. The greatest wet shear strength of globulin was 3.3 MPa at curing a temperature of 190 °C. Glutelin had a more protein aggregation compared with globulin, as indicated by higher crystallinity and thermal stability, and dense protein aggregate. This compact structure of glutelins may partially contribute to lower adhesion strength as compared to globulin.

Biodegradable edible films are both economically and environmentally important to the food industry as packaging and coating materials, as the industry seeks to find a replacement to traditional petroleum-derived synthetic polymers. The overall goal of this thesis was to design a canola protein isolate (CPI)-based biodegradable and edible film that provides excellent mechanical, optical and water vapor barrier properties. A better understanding of the potential of CPI for use as a film-forming ingredient could lead to enhanced utilization and value of the protein for food and non-food applications. In study one, the mechanical, optical and water vapor barrier properties of CPI-based films were investigated as a function of protein (5.0% and 7.5% w/w) and glycerol (30%, 35%, 40%, 45%, and 50% w/w of CPI) concentrations. Overall, as the glycerol concentration increased for the 5.0% and 7.5% CPI-based films, mechanical strength and flexibility decreased and increased, respectively. Film strength was also found to increase at the higher protein concentration; however corresponding changes to film flexibility differed depending on the testing method used. For instance, puncture deformation testing indicated that film flexibility was reduced as the CPI concentration was raised, whereas tensile elongation testing indicated no change in extensibility between the two CPI concentrations. Film transparency was found to increase with increasing levels of glycerol and decreasing levels of CPI, whereas water vapor permeability was found to increase with increasing levels of both glycerol and protein. In study two, mechanical, optical and vapor barrier properties of CPI-based films were evaluated as a function of plasticizer-type (50% (w/w of CPI), glycerol, sorbitol, polyethylene glycol 400 (PEG-400)) and fixative condition (0% and 1% (w/w of CPI), genipin). CPI films prepared with sorbitol were significantly stronger than films with PEG-400, followed by films with glycerol, whereas the flexibility of CPI-based films with glycerol was higher than films with PEG-400, followed by films with sorbitol. In all cases, films prepared with genipin were stronger and less malleable than un-cross linked films. CPI films with glycerol were more transparent than films with sorbitol, followed by films with PEG-400, and the addition of genipin significantly increased the opacity of CPI films. CPI films prepared with glycerol also showed poorer water vapor barrier property than films with PEG-400, followed by films with sorbitol, however, no differences were observed in the presence and absence of genipin. In summary, as the plasticizer concentration increased or protein concentration decreased, CPI films became weaker, more flexible and clearer; however their water vapor barrier properties became poorer as both plasticizer and protein concentration increased. Moreover, CPI films with sorbitol and genipin were found to be stronger, less malleable and permeable to moisture than CPI films with or without genipin, and in the presence of glycerol or PEG-400. Overall, CPI could be considered as a potential material for the development of biodegradable edible packaging in the future.

Master of Science / Department of Biological and Agricultural Engineering / Donghai Wang / Currently, the majority of adhesives used for wood veneer, plywood, and composite applications are formaldehyde-based. Formaldehyde is derived from petroleum and natural gas, making it non-renewable and toxic. Therefore, extensive research has been conducted to develop bio-based adhesives to replace formaldehyde-based adhesives. Soy protein has shown great potential to partially replace formaldehyde adhesives, and canola protein has similar properties to soy protein. However, little research has been conducted on the feasibility of using canola protein for wood adhesive applications. The objective of this research was to study the adhesion performance of canola protein. Canola protein was modified with different chemical modifiers including sodium dodecyl sulfate (SDS), calcium carbonate (CaCO[subscript]3), zinc sulfate (ZnSO[subscript]4), calcium chloride (CaCl[subscript]2), and 2-octen-1-ylsuccinic anhydride (OSA) as well as combined chemical modifications. The wet, dry, and soak shear strengths of the adhesive formulations were determined. Viscosity testing, differential scanning calorimetry, and TEM and SEM imaging were used to characterize protein properties. Chemical modification with SDS (1%, 3%, and 5%), CaCO[subscript]3 (1%, 3%, and 5%), ZnSO[subscript]4 (1%), and OSA (2%, 3.5%, and 5%) improved the dry and soak shear strengths compared to unmodified canola protein. Canola protein modified with 3.5% OSA had improved wet, dry, and soak shear strengths. Combined chemical modification of canola protein did not show significant improvement on shear strength. Thermal modification of canola protein adhesives showed a trend of increasing shear strength with increasing press temperature. The data suggests that with further research, canola protein has potential to be used as a commercial adhesive or as an additive to formaldehyde-based adhesives to make them more environmentally-friendly.

The overall goal of this thesis is to better understand mechanisms governing associative phase separation within admixtures of plant proteins (e.g., pea and canola) and anionic polysaccharides (e.g., gum Arabic, alginate or é-carrageenan). The process involves the electrostatic attraction between two biopolymers of opposing charges, and typically results in the formation of both soluble and insoluble complexes during an acidic pH titration. If successful, polysaccharides could be triggered to coat the proteins surface to give novel, and hopefully improved functionality as ingredients for food and biomaterials.<p> In the first study, the effect of protein enrichment and pH on the formation of soluble and insoluble complexes in admixtures of pea legumin (Lg) and vicilin (Vn) isolates with gum Arabic (GA) was investigated by turbidimetric, surface charge and fluorometric measurements. The solubility of the protein isolates and mixed biopolymer systems was also studied as a function of pH. Enrichment of the crude Lg and Vn isolates by low pressure liquid chromatography led to a shift towards higher pHs at the onset of soluble complex formation in the presence of GA for both protein isolates, whereas the onset of insoluble complex formation was unaffected. Complexation of the Lg (or Vn) isolates with GA resulted in a shift in the pH where neutrality (zeta potential = 0 mV) occurred to lower pH values, relative to the Lg (or Vn) isolates alone. In the case of the enriched Vn isloate, changes to its tertiary structure were observed by fluorometry upon complexation with GA, whereas no changes were found for the enriched Lg isolate. Complexation of Lg and Vn isolates with GA also had little effect on their solubilities relative to protein alone solutions.<p> In the second study, the formation of soluble and insoluble complexes, and the nature of their interactions as determined by optical density analysis, were investigated in admixtures of canola protein isolate (CPI) and anionic polysaccharides (alginate and é-carrageenan) as a function of pH and biopolymer weight mixing ratio. The solubilities of formed complexes were also investigated versus protein alone. In both CPI-polysaccharide systems, critical pH associated with the onset of soluble and insoluble complexes shifted to higher pHs as the mixing ratios increased from 1:1 to 20:1 (CPI:polysaccharide), and then became constant. There complexes formed primarily through electrostatic attractive forces with secondary stabilization by hydrogen bonding. The solubilities of the CPI-alginate complexes were significantly enhanced relative to CPI alone or CPI-é-carrageenan, which were similar.

The overall goal of this thesis is to better understand mechanisms governing associative phase separation within admixtures of plant proteins (e.g., pea and canola) and anionic polysaccharides (e.g., gum Arabic, alginate or é-carrageenan). The process involves the electrostatic attraction between two biopolymers of opposing charges, and typically results in the formation of both soluble and insoluble complexes during an acidic pH titration. If successful, polysaccharides could be triggered to coat the proteins surface to give novel, and hopefully improved functionality as ingredients for food and biomaterials.<p> In the first study, the effect of protein enrichment and pH on the formation of soluble and insoluble complexes in admixtures of pea legumin (Lg) and vicilin (Vn) isolates with gum Arabic (GA) was investigated by turbidimetric, surface charge and fluorometric measurements. The solubility of the protein isolates and mixed biopolymer systems was also studied as a function of pH. Enrichment of the crude Lg and Vn isolates by low pressure liquid chromatography led to a shift towards higher pHs at the onset of soluble complex formation in the presence of GA for both protein isolates, whereas the onset of insoluble complex formation was unaffected. Complexation of the Lg (or Vn) isolates with GA resulted in a shift in the pH where neutrality (zeta potential = 0 mV) occurred to lower pH values, relative to the Lg (or Vn) isolates alone. In the case of the enriched Vn isloate, changes to its tertiary structure were observed by fluorometry upon complexation with GA, whereas no changes were found for the enriched Lg isolate. Complexation of Lg and Vn isolates with GA also had little effect on their solubilities relative to protein alone solutions.<p> In the second study, the formation of soluble and insoluble complexes, and the nature of their interactions as determined by optical density analysis, were investigated in admixtures of canola protein isolate (CPI) and anionic polysaccharides (alginate and é-carrageenan) as a function of pH and biopolymer weight mixing ratio. The solubilities of formed complexes were also investigated versus protein alone. In both CPI-polysaccharide systems, critical pH associated with the onset of soluble and insoluble complexes shifted to higher pHs as the mixing ratios increased from 1:1 to 20:1 (CPI:polysaccharide), and then became constant. There complexes formed primarily through electrostatic attractive forces with secondary stabilization by hydrogen bonding. The solubilities of the CPI-alginate complexes were significantly enhanced relative to CPI alone or CPI-é-carrageenan, which were similar.

Canola fibre-protein and can-sugar are the two by-products arising from a process for separating high quality protein fractions from canola meal. In the first trial chemical characteristics of fibre-protein and can-sugar were examined in comparison with commercial canola and soy meal. In the second trial in situ rumen degradability and kinetics of test feed was studied. Based on the findings of those two trials, available energy values were estimated based on NRC (2001) while protein contents potentially absorbable at small intestine were predicted using both NRC (2001) and DVE/OEB models. Subsequently a mixture of fibre-protein and can-sugar was used as an additive to dehydrated alfalfa pellet and two dairy cow trials were conducted to determine the palatability and examine effect on lactation performances of blended alfalfa pellet feeding in comparison with standard alfalfa pellet. Palatability difference was evaluated by Paterson -two choice alternating access method through a 7 day experimental period using 6 lactating Holstein cows. In the lactating performance trial, 6 cows were randomly assigned into two groups and two treatments were allocated over three experimental periods in a switchback design. <p>Can-sugar consisted of water soluble components (CP 15.6 %DM; SCP 96.2 %CP; NFC 99.9 %CHO) with non-protein nitrogen as the main CP fraction (NPN 96.2 %CP). Fibre-protein was a highly fibrous material (NDF: 55.6%; ADF: 46.3%; ADL: 24.1%) comparing to canola meal (NDF: 25.4%, ADF: 21.2%, ADL: 9.0%) due to presence of higher level of seed hulls in fibre-protein. Comparing to canola meal, fibre-protein contained 9% less CP and 1/4 of that consisted of undegradable ADIP. Rumen degradability of can-sugar was assumed as immediate and total as it was water soluble. Most of the ruminally undegradable nutrient components present in canola meal appeared to be concentrated into fibre protein during the manufacturing process and as a result fibre-protein has shown a consistently lower effective degradability of DM, OM, CP NDF and ADF comparing to both canola and soy meal. Available energy content in can-sugar was marginally higher than that of canola meal while fibre-protein contained only 2/3 that of canola meal. The predicted absorbable protein content at small intestine was about 1/2 that of canola meal. These results indicate that fibre-protein can be considered as a secondary source of protein in ruminant feed and a mixture of fibre-protein and can-sugar would nutritionally complement each other to formulate into a cheaper ingredient in ruminant ration. In the palatability study, there was no significant difference (P>0.05) in intake preference or finish time between the blended and standard alfalfa pellets. The results from the lactation study showed that there was no significant difference (P>0.05) in milk yield, dairy efficiency or milk composition between the blended and standard alfalfa pellets. The results from the two studies indicated that fibre-protein and can-sugar fractions could be used as an additive to alfalfa dehydrated pellet at 15% inclusion rate without compromising its palatability or the performance of dairy cows. <p>For future studies it is proposed to conduct feeding trials with varying levels of inclusions to alfalfa pellet to know the nutritional effect of fibre-protein and can-sugar while ascertain optimum inclusion rate.

Canola fibre-protein and can-sugar are the two by-products arising from a process for separating high quality protein fractions from canola meal. In the first trial chemical characteristics of fibre-protein and can-sugar were examined in comparison with commercial canola and soy meal. In the second trial in situ rumen degradability and kinetics of test feed was studied. Based on the findings of those two trials, available energy values were estimated based on NRC (2001) while protein contents potentially absorbable at small intestine were predicted using both NRC (2001) and DVE/OEB models. Subsequently a mixture of fibre-protein and can-sugar was used as an additive to dehydrated alfalfa pellet and two dairy cow trials were conducted to determine the palatability and examine effect on lactation performances of blended alfalfa pellet feeding in comparison with standard alfalfa pellet. Palatability difference was evaluated by Paterson -two choice alternating access method through a 7 day experimental period using 6 lactating Holstein cows. In the lactating performance trial, 6 cows were randomly assigned into two groups and two treatments were allocated over three experimental periods in a switchback design. <p>Can-sugar consisted of water soluble components (CP 15.6 %DM; SCP 96.2 %CP; NFC 99.9 %CHO) with non-protein nitrogen as the main CP fraction (NPN 96.2 %CP). Fibre-protein was a highly fibrous material (NDF: 55.6%; ADF: 46.3%; ADL: 24.1%) comparing to canola meal (NDF: 25.4%, ADF: 21.2%, ADL: 9.0%) due to presence of higher level of seed hulls in fibre-protein. Comparing to canola meal, fibre-protein contained 9% less CP and 1/4 of that consisted of undegradable ADIP. Rumen degradability of can-sugar was assumed as immediate and total as it was water soluble. Most of the ruminally undegradable nutrient components present in canola meal appeared to be concentrated into fibre protein during the manufacturing process and as a result fibre-protein has shown a consistently lower effective degradability of DM, OM, CP NDF and ADF comparing to both canola and soy meal. Available energy content in can-sugar was marginally higher than that of canola meal while fibre-protein contained only 2/3 that of canola meal. The predicted absorbable protein content at small intestine was about 1/2 that of canola meal. These results indicate that fibre-protein can be considered as a secondary source of protein in ruminant feed and a mixture of fibre-protein and can-sugar would nutritionally complement each other to formulate into a cheaper ingredient in ruminant ration. In the palatability study, there was no significant difference (P>0.05) in intake preference or finish time between the blended and standard alfalfa pellets. The results from the lactation study showed that there was no significant difference (P>0.05) in milk yield, dairy efficiency or milk composition between the blended and standard alfalfa pellets. The results from the two studies indicated that fibre-protein and can-sugar fractions could be used as an additive to alfalfa dehydrated pellet at 15% inclusion rate without compromising its palatability or the performance of dairy cows. <p>For future studies it is proposed to conduct feeding trials with varying levels of inclusions to alfalfa pellet to know the nutritional effect of fibre-protein and can-sugar while ascertain optimum inclusion rate.