<p>Pectinases are a group of enzymes produced by bacteria, fungi, higher plants and animals. Pectinases can modify and degrade pectins, a class of heterogeneous and multifunctional polysaccharides present in middle lamellae and primary cell walls of plants. Pectins have been showed to play diverse roles in cell physiology, growth, adhesion and separation. Pectinases are used technically in the processing of fiber production and fruit juice or wine making. We have studied the mechanisms and applications of pectinases, especially in retting, a microbiological process where bast fibers in flax and other bast fiber cultivars are released from each other and from the woody core.</p><p>A strong correlation was found between the ability to perform retting and the degradation of sparsely esterified pectin, a substrate of polygalacturonase. This led to the conclusion that polygalacturonase plays a key role in the enzymatic retting of flax. We purified and characterized an extracellular polygalacturonase produced by Rhizopus oryzae, a very potent retting organism. The purified enzyme which appeared to be the single active component in retting, has non-methylated polygalacturonan as its preferred substrate. Peptide sequences indicate that the enzyme, like another polygalacturonase (EC. 3.2.1.15), belongs to glycosyl hydrolase family 28. It contains, however, an N-terminal sequence absent from other fungal pectinases, but present in an enzyme from the phytopathogenic bacterium, Ralstonia solanacearum.</p><p>Our finding that removal of calcium ions from the plant material by pre-incubation in dilute acid in enzymatic retting could reduce enzyme consumption by several orders of magnitude, improves the economical feasibility of the enzymatic retting process. Comparisons with different acids showed that the action was mainly pH dependent.</p><p>Pectinases were employed as analytical tools in a study of stored wood discoloration and, together with cellulases, in a mechanical process for making pulp from flax and hemp in paper production. </p>

Thesis (Ph.D. (Environmental Science)--North-West University, Potchefstroom Campus, 2007.

BACKGROUND: There is an increased need to replace materials derived from fossil sources by renewables. Sugar-cane derived carbohydrates are very abundant in Brazil and are the cheapest sugars available in the market, with more than 400 million tons of sugarcane processed in the year 2007. The objective of this work was to study the preparation of sugar acrylates from free sugars and free acrylic acid, thus avoiding the previous preparation of protected sugar derivatives, such as glycosides, or activated acrylates, such as vinyl acrylate. RESULTS: Lipase catalyzed esterification of three mono- and two disaccharides with acrylic acid, in the presence or absence of molecular sieves was investigated. The reactions were monitored by high-performance liquid chromatography (HPLC) and the products were analyzed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. The main products are mono- and diacrylates, while higher esters are formed as minor products. The highest conversion to sugar acrylates was observed for the D-glucose and D-fructose, followed by D-xylose and D-maltose. Molecular sieves had no pronounced effect on the conversion CONCLUSIONS: A feasible method is described to produce and to characterize sugar acrylates, including those containing more than two acrylate groups. The process for production of these higher esters could potentially be optimized further to produce molecules for cross-linking in acrylate polymerization and other applications. The direct enzymatic esterification of free carbohydrates with acrylic acid is unprecedented.

Pectinases are a group of enzymes produced by bacteria, fungi, higher plants and animals. Pectinases can modify and degrade pectins, a class of heterogeneous and multifunctional polysaccharides present in middle lamellae and primary cell walls of plants. Pectins have been showed to play diverse roles in cell physiology, growth, adhesion and separation. Pectinases are used technically in the processing of fiber production and fruit juice or wine making. We have studied the mechanisms and applications of pectinases, especially in retting, a microbiological process where bast fibers in flax and other bast fiber cultivars are released from each other and from the woody core. A strong correlation was found between the ability to perform retting and the degradation of sparsely esterified pectin, a substrate of polygalacturonase. This led to the conclusion that polygalacturonase plays a key role in the enzymatic retting of flax. We purified and characterized an extracellular polygalacturonase produced by Rhizopus oryzae, a very potent retting organism. The purified enzyme which appeared to be the single active component in retting, has non-methylated polygalacturonan as its preferred substrate. Peptide sequences indicate that the enzyme, like another polygalacturonase (EC. 3.2.1.15), belongs to glycosyl hydrolase family 28. It contains, however, an N-terminal sequence absent from other fungal pectinases, but present in an enzyme from the phytopathogenic bacterium, Ralstonia solanacearum. Our finding that removal of calcium ions from the plant material by pre-incubation in dilute acid in enzymatic retting could reduce enzyme consumption by several orders of magnitude, improves the economical feasibility of the enzymatic retting process. Comparisons with different acids showed that the action was mainly pH dependent. Pectinases were employed as analytical tools in a study of stored wood discoloration and, together with cellulases, in a mechanical process for making pulp from flax and hemp in paper production.

The overarching goal of this research was to formulate an encapsulated powder using a modified lentil protein isolate-maltodextrin mixture to encapsulate flaxseed oil by freeze drying. The primary objectives were: a) to examine the physicochemical and emulsifying properties of lentil protein isolates with different degrees of hydrolysis; b) to design and test the physicochemical properties of encapsulated flaxseed oil using a wall material with native, heat treated and partially hydrolyzed lentil proteins in combination with maltodextrin; and c) test the oxidative stability of encapsulated flaxseed oil with the capsule design with the lowest surface oil and highest encapsulation efficiency versus free oil. During the first study, the physicochemical and emulsifying properties of lentil protein isolates (LPI) were investigated as a function of their degree of hydrolysis (DH of 4, 9 and 20%) following exposure to trypsin/heat. Interfacial tension, surface characteristics (charge and hydrophobicity) and intrinsic fluorescence were determined and related to changes in the emulsification activity (EAI) and stability indices (ESI) of unhydrolyzed (u-LPI) and hydrolyzed LPI (h-LPI) in a flaxseed oil-water emulsion. Most importantly surface hydrophobicity declined from ~30 to ~24 for the u-LPI and h-LPI (DH 4-20%), respectively. The changes in physicochemical properties induced by hydrolysis had a detrimental effect on EAI and ESI values, which declined from ~51 to ~47 m2 g-1 and ~12 to ~ 11 min for u-LPI and h-LPI (DH 4-20%), respectively. In the second study, the physicochemical properties of encapsulated flaxseed oil within lentil protein-based maltodextrin microcapsules were investigated using native (n-LPI), pre-treated (heated, un-hydrolyzed (u-LPI); and heated, hydrolyzed (h-LPI)) lentil protein isolates and as a function of oil load (10.0, 20.0 and 30.0% of total solids). The moisture, water activity, surface oil and encapsulation efficiency (EE) were assessed, along with droplet size and emulsion morphology. Light microscopy imaging of the emulsions, showed that the h-LPI had slightly larger oil droplets than the n-LPI and u-LPI, which both appeared similar. Microcapsules prepared from h-LPI showed significantly higher surface oil and lower EE than both the n-LPI and u-LPI materials. The microcapsules prepared using n-LPI with 10.0% oil loading were found to have the lowest surface oil content (~3.7%) and highest EE (~62.8%) for all formulations, and were subjected to an oxidative storage stability test over a 30 d period vs. free oil. The encapsulation process however induced autooxidation leading the production of a greater amount of primary oxidative products than free oil. Findings indicate that future studies are necessary to enhance the stability of the flaxseed oil through the encapsulation process.

Grain quality in chickpea (<i>Cicer arietinum</i> L.) is a major factor affecting its consumption for human nutrition and health benefits. Some of the major factors affecting chickpea grain quality are: seed weight, size, colour, protein, starch and amylose concentration, and amylopectin structure. The objectives of this study were to: 1) determine variation, repeatability and genotype by environment interaction on thousand seed weight, starch, amylose and protein concentration of chickpea cultivars adapted to western Canada; 2) assess variations in global chickpea germplasm for thousand seed weight, seed size, protein, starch and amylose concentrations; and 3) characterize the desi and kabuli type chickpea for starch concentration, composition, and amylopectin structure to study their effect on starch enzymatic hydrolysis. Limited variation was observed in seed composition of chickpea cultivars adapted to the western Canadian prairies. Significant genotype by environment interaction occurred for starch, amylose, and protein (except for kabuli) concentrations, seed yield and thousand seed weight indicating that testing over a wide range of environments is needed to identify genotypes for grain quality improvement. Repeatability of starch, amylose, and protein concentrations was low and inconsistent across chickpea market classes. Broad sense heritability was higher than repeatability across all traits for all market classes implying that repeatability estimates do not set upper limits to heritability if significant genotype by environment interaction is present. The negative relationship between seed constituents and yield indicates that selection for chickpea cultivars with desired seed composition may require compromise with yield and indirect selection. All the mini core accessions that had above average seed diameter score in both desi and kabuli also had above average score for thousand seed weight. Selecting mini core with promising intrinsic and extrinsic quality characteristics may reduce yield. Slowly digestible starch was negatively correlated with hydrolysis index in both pure starch and meal starch of desi and kabuli. Amylose had a strong relationship with resistant starch but not with rate of starch hydrolysis. Genotypes with a significantly higher rate of starch hydrolysis had significantly lower 60-80 µm starch granule size volume. Amylopectin B2 chains were related to slowly digestible starch of meal (except kabuli) and extracted starch. Resistant starch positively correlated with B1 fraction of amylopectin chain length in both desi and kabuli meal starch. Our results suggest that there is no major difference between starch composition in the two chickpea market classes, although only three genotypes of each class were tested. The meal components affect the starch hydrolytic properties and the effect is genotype specific. The results also show that amylopectin structure influences starch hydrolytic properties. These observations emphasize that complete characterization of seed components is needed to obtain meaningful results regarding the desired nutritional and health benefits attributed to any grain.

This study aims efficient conversion of cotton stalks to cellulosic ethanol through ionic liquid pretreatment and enhanced enzymatic hydrolysis. Among several ionic liquids, EMIMAc exhibited the most striking impact on cotton stalks with respect to the changes in biomass structure and digestibility. Cotton stalks, which were subjected to EMIMAc pretreatment at 10% (w cotton stalks/w EMIMAc) of biomass loading and 150&deg / C for 30 minutes, were found to be 9 times more digestible than untreated cotton stalks. Besides, glucose and ethanol yields, which were based on the cellulose content of untreated cotton stalks, were found as 67% and 66%, respectively. These yields were insufficient regarding efficient conversion of the cellulosic portion of cotton stalks to glucose and ethanol which is linked to the superior solvation capability of EMIMAc towards biomass. In order to enhance aforementioned yields, EMIMAc pretreatment was conducted at 30% of biomass loading. Though lignin extracted was much lower, higher yields were obtained compared to the former case since 96% of cellulose was recovered upon EMIMAc pretreatment and reduced crystallinity was observed for pretreated biomass. Glucose yield was achieved as 84% even at a substrate loading of 15% (w/v). Additionally, 76% of ethanol yield and 3% (v/v) of ethanol titer were obtained upon fermentation. Accordingly, reduction in biomass crystallinity was satisfactory to improve enzymatic accessibility of the biomass. Besides, EMIMAc maintained its effectiveness as a pretreatment agent upon recycling since no change in terms of hydrolysis of pretreated samples was observed upon EMIMAc recycling for three times.

Bioethanol from lignocellulose is expected to be the most likely fuel alternative in the near future. SEKAB E-Technology in Örnsköldsvik, Sweden develops the technology of the 2nd generation ethanol production; to produce ethanol from lignocellulosic raw material. The objective of this master’s thesis was to achieve a better knowledge of the potential and limitations of separate hydrolysis and fermentation (SHF) as a process concept for the 2nd generation ethanol production. The effects of enzyme concentration, temperature and pH on the glucose concentration in the enzymatic hydrolysis were investigated for pretreated spruce at 10% DM using a multiple factor design. Enzyme concentration and temperature showed significant effects on the glucose concentration, while pH had no significant effect on the concentration in the tested interval of pH 4.5-5.5. To obtain the maximum glucose concentration (46.4 g/l) for a residence time of 48 h, the optimal settings within the studied parameter window are a temperature of 45.7⁰C and enzyme concentration of 15 FPU/g substrate. However, a higher enzyme concentration would probably further increase the glucose concentration. If enzymatic hydrolysis should be performed for very short residence times, e.g. 6 h, the temperature should be 48.1⁰C to obtain maximum glucose concentration. The efficiency of the enzymes was inhibited when additional glucose was supplied to the slurry prior to enzymatic hydrolysis. It could be concluded that end product inhibition by glucose occurs and results in a distinct decrease in glucose conversion. No clear conclusions could be drawn according to different techniques for slurry and enzymes, i.e. batch and fed-batch, in the enzymatic hydrolysis process. Investigations of the fermentability of the hydrolysate revealed that the fermentation step in SHF is problematic. Inhibition of the yeast decrease the fermentation efficiency and it is therefore difficult to achieve the 4% ethanol limit. Residence time for enzymatic hydrolysis (48 h) and fermentation (24 h) need to be prolonged to achieve a sufficient SHF process. However, short processing times are a key parameter to an economically viable industrial process and to prolong the residence times should therefore not be seen as a desirable alternative. SHF as a process alternative in an industrial bioethanol plant has both potential and limitations. The main advantage is the possibility to separately optimize the process steps, especially to be able to run the enzymatic hydrolysis at an optimal temperature. Although, it is important to include all the process steps in the optimization work. The fermentation difficulties together with the end product inhibition are two limitations of the SHF process that have to be improved before SHF is a preferable alternative in a large scale bioethanol plant.

Mechanical pressing of soybean is highly desirable as it provides, at low cost, non-contaminated, protein-rich, low-fat soyflour which can be further processed into nutritious edible foods. Unfortunately, mechanical pressing of this low-fat oilseed ($<$20%) yields only 50-70% of the available oil, in contrast to the solvent extraction method which recovers over 98% of the oil. The main focus of the study was to maximize the oil recovery from soybean using mechanical oil expression by applying two pretreatments, enzymatic hydrolysis and extrusion cooking of soybeans, and by varying the pressing conditions including three applied pressures (20, 40 and 60 MPa), three pressing temperatures (22, 60 and 90°C) and two sample sizes (10 and 20 g). To characterize the material properties affecting mechanical oil expression from soybean a mathematical simulation of uniaxial compression was developed which incorporated the time dependent variation of soybean properties. The mathematical simulation was based on Terznaghi's theory of consolidation for soils and was solved using measured values of the coefficients of permeability, volume change and consolidation. A compression-permeability test cell was specifically developed for these measurements. For validation of the model, in addition to extruded soy, sunflower seeds (oil content ca. 45%) were also compressed under the same pressing conditions. Improvements in oil recovery due to enzymatic pretreatment of soybean were small, while the extrusion pretreatment increased the oil recovery from only a trace for raw soybean to 90.6%. Such oil recovery using mechanical pressing of soybean has not been reported in the past. The measured values of oil recovery, coefficients of permeability, volume change and consolidation for soybean and sunflower seeds were found to vary significantly $(P<0.05)$ with time of pressing, applied pressure, pressing temperature and the size of the sample. For extruded soy samples, the developed model predicted the values of oil recovery versus pressing time with an average error of 15%, while for sunflower seed samples the average prediction error was 40%. The high error values were attributed to the presence of hulls in the sunflower seed samples, as well as error during measurement of the coefficient of permeability. The coefficient of consolidation was found to have the greatest influence on oil recovery. The incorporation of time dependent material properties in the developed simulation was demonstrated to give more accurate and consistent prediction in trends of oil recovery as compared to using constant material properties. The correlationship developed between the oilseed material properties and the oil recovery obtained from uniaxially compressed oilseeds would help researchers and designers to better evaluate the mechanical oil expression equipment and systems. To the extent that the developed model adequately predicted oil recoveries from both sunflower and soybean oilseeds, the model is expected to be applicable to other oilseeds as well.

Grain quality in chickpea (<i>Cicer arietinum</i> L.) is a major factor affecting its consumption for human nutrition and health benefits. Some of the major factors affecting chickpea grain quality are: seed weight, size, colour, protein, starch and amylose concentration, and amylopectin structure. The objectives of this study were to: 1) determine variation, repeatability and genotype by environment interaction on thousand seed weight, starch, amylose and protein concentration of chickpea cultivars adapted to western Canada; 2) assess variations in global chickpea germplasm for thousand seed weight, seed size, protein, starch and amylose concentrations; and 3) characterize the desi and kabuli type chickpea for starch concentration, composition, and amylopectin structure to study their effect on starch enzymatic hydrolysis. Limited variation was observed in seed composition of chickpea cultivars adapted to the western Canadian prairies. Significant genotype by environment interaction occurred for starch, amylose, and protein (except for kabuli) concentrations, seed yield and thousand seed weight indicating that testing over a wide range of environments is needed to identify genotypes for grain quality improvement. Repeatability of starch, amylose, and protein concentrations was low and inconsistent across chickpea market classes. Broad sense heritability was higher than repeatability across all traits for all market classes implying that repeatability estimates do not set upper limits to heritability if significant genotype by environment interaction is present. The negative relationship between seed constituents and yield indicates that selection for chickpea cultivars with desired seed composition may require compromise with yield and indirect selection. All the mini core accessions that had above average seed diameter score in both desi and kabuli also had above average score for thousand seed weight. Selecting mini core with promising intrinsic and extrinsic quality characteristics may reduce yield. Slowly digestible starch was negatively correlated with hydrolysis index in both pure starch and meal starch of desi and kabuli. Amylose had a strong relationship with resistant starch but not with rate of starch hydrolysis. Genotypes with a significantly higher rate of starch hydrolysis had significantly lower 60-80 µm starch granule size volume. Amylopectin B2 chains were related to slowly digestible starch of meal (except kabuli) and extracted starch. Resistant starch positively correlated with B1 fraction of amylopectin chain length in both desi and kabuli meal starch. Our results suggest that there is no major difference between starch composition in the two chickpea market classes, although only three genotypes of each class were tested. The meal components affect the starch hydrolytic properties and the effect is genotype specific. The results also show that amylopectin structure influences starch hydrolytic properties. These observations emphasize that complete characterization of seed components is needed to obtain meaningful results regarding the desired nutritional and health benefits attributed to any grain.