This thesis is the first report linking differential distribution of cadmium (Cd) among tissues of hydroponically-grown soybean with Cd amendments – lower seed Cd-accumulating OAC Champion and higher seed Cd-accumulating OAC Bayfield – and the mechanisms responsible for these differences. OAC Champion retains 94% (64% for OAC Bayfield) of accumulated Cd in the root stock and Cd in its xylem sap is eleven-fold less concentrated than OAC Bayfield by seed fill. Though the movement of Cd to shoots is more restricted in OAC Champion, the concentration in some seed still approximates or exceeds 0.1 mg Cd • kg-1, indicating that in soils with elevated available Cd, there is potential for many cultivars of soybean to exceed the new suggested maximum for soybean, under an amendment to regulation (EC) No 1881/2006. Phytic acid was also assayed in seed tissue and OAC Bayfield was found to contain the highest concentrations.

Approaches for the evaluation of plant protein ingredients for salmonid feeds were investigated in a series of four trials. The first trial compared the apparent digestibility coefficients (ADC) of crude protein (CP) and amino acids (AAs) of two novel products - Indian mustard protein concentrate (IMC, 62% CP) and Indian mustard protein meal (IMM, 42% CP), to a commercially available soy protein concentrate (SPC, 57% CP) for two salmonid species, rainbow trout and Atlantic salmon. The second trial involved assessment of relative bioavailability of arginine (Arg) from IMC, IMM and SPC compared to that of a crystalline Arg (L-Arg) in rainbow trout using slope-ratio assay. In the third trial, the effects of phytic acid (PA) and lignin on nutrient utilization and partitioning in rainbow trout were assessed. Finally, a series of experiments was conducted in the final trial to establish the evaluation criteria for pellet quality assessment. The ADC of CP and most AAs in IMC and IMM were high (>90%). Differences in the ADCs of some AAs can be attributed to the high PA intake by fish fed 30%-IMC diet. The significantly higher (P<0.05) bioavailability of Arg from IMC (123 to 187%) and IMM (116 to 211%) relative to that of L-Arg, as determined by various regression approaches, reaffirmed the findings of the first trial that these ingredients are of excellent protein quality and can readily be used in compounded fish feeds. It can be inferred from the lack of effects of PA, lignin or PA plus lignin on most indices of physiology, performance, and nutrient utilization in the pair-fed fish, that like any other animal, controls feed intake when in the presence of one or more dietary ANF. It was also shown in the pellet quality assessment trial that minor changes in dietary composition can significantly alter physical properties of aquaculture feed. This study highlighted the importance of a comprehensive assessment for the effective evaluation of the nutritive value of plant protein ingredients for use in aquaculture feeds. / Ontario Ministry of Natural Resources; BIOEXX Specialty Proteins Ltd.; Martin Mills Inc.; MITACS

During seed development, myo-inositol (Ins) hexakisphosphate or phytic acid (PA) is stored in the form of phytin with mineral cations, and is mobilized following germination, releasing these nutrients that are required for seedling growth. Outside its role in seeds, PA and other phosphoylated Ins derivatives play critical roles in biological processes in many eukaryotes. PA also has negative influences on nutrition in both non-ruminant animals and humans due to its lack of digestibility. There have been two parallel PA biosynthetic pathways proposed, yet, the pathway is still poorly understood in terms of its regulation and enzymes involved. Here, genes encoding enzymes putatively implicated in castor bean PA biosynthetic pathways were identified in the genome and expression followed. Isolated castor bean embryos have the ability to resynthesize PA following germination if exogenous phosphate is available. It was found that the genes purported to act in PA synthesis were constitutively expressed in the embryos regardless of the availability of phosphate. Castor bean Ins 1,3,4,5,6-pentakisphosphate 2-kinase (RcIPK1), catalyzing the final reaction in PA biosynthesis, regardless of pathway, was chosen for further study. Even though only one copy of the RcIPK1 gene was present in the genome, numerous RNA variants were found, most likely due to alternative splicing events. These variants encoded six closely related protein isoforms based on in silico analysis. Functional analyses using yeast mutant strains lacking the IPK1 gene revealed that only three of the mRNA variants could rescue the temperature-sensitive growth phenotype, and it was demonstrated by HPLC analysis of Ins phosphates that their ability to complement the missing yeast IPK1 enzyme was associated with enzyme activity. Only these three isoforms possessed conserved motif III and motif IV important for IPK1 catalytic activity.

This study has assessed the effects of pulse type and processing (soaking and cooking) on antinutritional factors (α-amylase inhibitor, trypsin inhibitor, chymotrypsin inhibitor, lectins, phytic acid and oxalates) in a wide range of Canadian pulses, using soybean as a control. The contents of these antinutrients in Canadian pulses varied widely, but the levels were generally lower than those found in soybean. Analysis of variance indicated that both pulse type and processing had significant effects (P < 0.0001) on the investigated seeds. Soaking markedly decreased the contents of α-amylase inhibitor, trypsin inhibitor, chymotrypsin inhibitor, lectins and oxalates, but had no impact on phytic acid. Cooking of presoaked seeds appeared to be more effective; all proteinaceous antinutrients (α-amylase inhibitor, trypsin inhibitor, chymotrypsin inhibitor and lectins) were decreased by 80 – 100%, and significant reductions in phytic acid content (11 – 39%) were observed for all pulses, except common beans and soybean. / February 2016

Idag finns det flera hundra flamskyddsmedel på marknaden där en del är hälso- och miljöfarliga. Fytinsyra är en naturlig produkt från växtriket som är miljövänlig och icke-toxisk. Fytinsyra består till stor del av fosfor vilket gör att potential finns för ett grönt flamskyddsmedel vilket ska undersökas. Lösningar på 3 och 10 mass% fytinsyra har gjorts där sedan kalciumkloridhexahydrat, natriumhydroxid eller ammoniak med ett molförhållandet på 5 eller 12 har tillsatts. Tygbitarna, av bomull och polyester, doppades i lösningarna och torkades i 6 dagar för att sedan analyseras med ett ugnstest, förbränningstest, TGA och DSC. Bästa kombinationen för bomull blev när natrium tillsattes med ett molförhållande på 12 till 1. Denna lösning gav bäst resultat i förbränningstesten samt ger mest massa kvar enligt TGA analysen. Lösningen är även basisk vilket är bra då sura lösningar bryter ner bomullstyget. För bedömning av bästa kombinationen för polyester ligger DSC och TGA analyserna till grund. I DSC är det svårt att avgöra vilken lösning som är optimalast då natriumlösningarna höjer temperaturen för smältpunkten mycket medan effekten minskar som behövs för att smältningen ska äga rum har halverats i förhållande till obehandlad polyester. Däremot har effektförbrukningen för smältningen ökat för kalciumlösningarna medan temperaturen endast höjts någon grad. Därmed är det svårt att säga den optimala lösningen för polyester. / Today there are several hundred flame retardants on the market where some are hazardous for the health and environment. Phytic acid is a natural product from the plant kingdom, which is environmentally friendly and non-toxic. Phytic acid consists largely of phosphorus, which means that there is potential for a green flame retardant which will be investigated in this report. Solutions of 3 and 10% by weight of phytic acid were made where calcium chloride hexahydrate, sodium hydroxide or ammonia having a molar ratio of 5 or 12 were added. The cotton and polyester fabric pieces were dipped in the solutions and dried for 6 days, then analyzed by use of an oven test, a combustion test, TGA and DSC tests. The best combination for cotton was when sodium was added at a molar ratio of 12 to 1. This solution yielded the best results in the combustion test, and left the most bulk in accordance with the TGA analysis. The solution is also basic, which is good as acidic solutions break down the cotton fabric. To assess the best combination for polyester, the DSC and TGA analyzes were used. In DSC it is difficult to determine which solution is optimal as the sodium solutions raises the temperature where melting occurs by a lot, but the power needed for melting is halved relative to untreated polyester. On the other hand, the calcium solutions have increased the power needed for melting, but the melting point was raised by only some degree. Thus it is difficult to say which the optimal solution for polyester is.

Orientador: Ranulfo Monte-Alegre / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-20T07:41:57Z (GMT). No. of bitstreams: 1 Lima_MeiriBrum_M.pdf: 1014074 bytes, checksum: ae7eb9b355f6f79d9790f0d168228185 (MD5) Previous issue date: 2012 / Resumo: Processos biotecnológicos para produção de enzimas apresentam acentuado crescimento devido à necessidade de maximização dos processos biológicos industriais e à contribuição para reduzir impactos ambientais. Entretanto, a utilização e purificação das enzimas envolve inúmeras variáveis e custo de produção relativamente alto. A fitase hidrolisa o ácido fítico, ácido orgânico componente natural das sementes e oleaginosas, em inositol e fosfato inorgânico disponibilizando o fósforo antes não disponível e resultando melhor aproveitamento destes alimentos. O uso da fitase como aditivo em rações animais é obrigatório na Europa, Sudoeste Asiático, Coréia do Sul, Japão e Taiwan para reduzir o efeito ambiental provocado pelo excesso de fósforo excretado pelos animais. Nos útimos anos, além da adição em rações animais, a aplicação da fitase na dieta humana surgiu como alternativa potencial, pois estudos demonstram seus benefícios à saúde. O potencial de mercado mundial para fitase é de U$ 500 milhões, onde a China se destaca como o maior produtor chegando a U$ 200 milhões de acordo com estudo da Indústria de Rações da China (2008). Entretanto, essa enzima ainda não é produzida no Brasil. Este estudo tem por objetivo estudar o processo fermentativo para produção de fitase determinando parâmetros que auxiliarão na identificação da melhor condição de cultivo para sua produção. Propôs-se ainda a utilização de farelo de arroz como substrato a fim de agregar valor a esse subproduto da indústria de beneficiamento do arroz. Foram realizados ensaios de produção por Aspergillus niger e Aspergillus oryzae em frascos Erlenmeyer e usando de planejamento experimental para verificar a influência da adição de fontes de nitrogênio e sais minerais na fermentação. Foi feita a caracterização da atividade enzimática em função do pH e da temperatura, bem como a escolha da melhor cepa para produção de fitase / Abstract: Biotechnological processes for production of enzymes show strong growth due to the need for maximization of industrial and biological processes contributing to reducing environmental impacts. However, the use of enzymes and purification involves many variables and relatively hight cost of production. Phytase hydrolyzes phytic acid, an organic acid component of natural seeds and oilseeds, in inositol and inorganic phosphate available, resulting best use of foods. The use of phytase as an additive in animal feed is mandatory in Europe, Southeast Asia, South Korea, Japan and Taiwan to reduce the environmental effect caused by excess phosphorus excreted by animals. In recent years, plus the addition of animal feed, the application of phytase in the human diet has emerged as a potential alternative, because studies showed the heath benefits. The potential worldwide market for phytase is U$ 500 milion, where China stands as the largest producer coming to U$ 200 milion according to a study of China¿s Feed Industry (2008). However, this enzyme is not produced in Brazil. This study aimed to study the fermentation process for production of phytase determining parameters that will assist in indentifying the Best culture condition for their prodution. It was also proposed the use of Rice bran as substrate in order to add value to the industry by-product of rice processing. Were done tests of production by Aspergillus niger and Aspergillus oryzae in Erlenmeyer flasks and experimental design was used to verify the influence of the addition of nitrogen sources and mineral salts in fermentation. The characterization of the enzyme activity in function of pH na temperature and the choice of the best strain for the production of phytase was also done / Mestrado / Engenharia de Alimentos / Mestre em Engenharia de Alimentos

The giant freshwater prawn, Macrobrachium rosenbergii (De Man, 1879), is gaining popularity as a key aquaculture species; global production currently exceed 220,000 tonnes, however, industry expansion is limited by high operational costs, with the feed accounting for between 40 to 60% of these. Attention, therefore, has been devoted to increasing the inclusion of plant proteins into the formulation of aqua feeds as a consequence of the limited, unpredictable supply and increasing price of fishmeal. The concomitant introductions of anti-nutritional factors (ANFs), such as phytic acid (PA) with the plant protein fraction, however, are major impediments in the efforts toward the increased use of plant protein ingredients in aqua feeds. Phytic acid is an anti-nutrient that can curtail the development of this as PA has been reported to suppress growth impairing proper nutrient intake, diminishing the availability of minerals, and causing damage to the body tissues and organs which can result in mortality. Although the anti-nutritive effects of PA have been studied extensively in terrestrial agriculture farm species, as well as in a variety of fish species, there is almost no information regarding the effects of PA in crustaceans, including the freshwater prawn, M. rosenbergii. The aims of this present thesis were, therefore, to gain a greater understanding of dietary PA and microbial phytase and their effect on growth performance, feed utilisation, nutrient utilisation and digestibility, mineral availability and whole body proximate composition in juvenile M. rosenbergii. Specifically, the first major experiment set out to investigate the effect of including increasing amounts of PA in the diets presented to M. rosenbergii on growth. The dose-response relationships between PA and growth performance, feed utilisation, nutrient digestibility and utilisation and whole proximate composition were investigated. Four replicate groups of M. rosenbergii with a mean initial carapace length of 6.03 ± 0.30 mm and mean initial weight of 0.29 ± 0.02 g were fed graded levels of PA for 140 days. The basal diet, to which different levels of PA were added to obtain 0.26 (control), 6.48, 11.28, 16.53, 21.45 and 26.16 g PA kg-1, contained fishmeal, soy protein concentrate, wheat meal and corn starch. The results indicated that growth performance, feed utilisation and survival did not differ significantly between the groups receiving the different inclusions of PA within their diets. The apparent protein, lipid and energy utilisations responded negatively, decreasing significantly (p<0.05) with an increasing inclusion of PA, particularly within the groups of prawns fed the diet with the highest inclusions of PA, i.e. the 21.45–26.16 g PA kg-1 diets. The digestibility of protein and lipid were also reduced as the inclusion of PA increased. The whole body composition of protein (p<0.04), lipid (p<0.01) and gross energy (p<0.05) decreased significantly with an increasing supplementation of PA, while the ash content significantly increased (p<0.01), most notably in the groups of prawns receiving the highest levels of dietary PA. The second major experimental trial investigated the effect of microbial phytase on the growth of juvenile M. rosenbergii, when fed diets supplemented with various doses for a period of 80 days. The study set out to improve the growth performance, feed utilisation, nutrient digestibility and utilisation and body composition of M. rosenbergii when fed diets high in plant protein ingredients. To investigate this, four plant protein based diets, which included soybean meal, wheat gluten and wheat meal, were formulated and supplemented with microbial phytase at levels of 0, 500, 1000 and 2000 FTU kg-1 (one phytase unit per kg) and fed to sixty juvenile M. rosenbergii (mean initial carapace length of 8.51 ± 0.52 mm; mean initial weight of 0.40 ± 0.07 g) for 80 days. High levels of plant protein in the diets supplemented with 0–2000 FTU kg-1 did not result in any negative effect on growth performance, feed utilisation nor on the survival of M. rosenbergii. Noticeable moderate growth improvements in line with increasing microbial phytase supplement levels were observed and the highest growth performance was seen in the group fed 2000 FTU kg-1. Supplementation of the diets with 500–2000 FTU kg-1 were found to affect the nutrient utilisation, resulting in a significant (p<0.05) increase in the protein and lipid utilisation when compared to the prawns analysed from the control group. In addition to this latter finding, an increasing supplement of phytase in the diet also resulted in an increase in the dry matter fraction as well as improvements in the digestibility of protein and lipid. There were, however, no significant differences in moisture, protein, lipid, gross energy and the ash content of the whole body of the M. rosenbergii among the groups. This thesis, in a third major trial, explored the impact of other potential ANFs associated to PA, the binding effect of PA with mineral. The effects of graded levels of PA (i.e. 0.26–control, 6.48, 11.28, 16.53, 21.45 and 26.16 g PA kg-1) on the moult frequency and mineral availability in juvenile M. rosenbergii fed over a period of 140 days were determined. The levels of PA assessed in this feed trial had no major detrimental effects on moult frequency. Negative effects (p<0.05) of high PA levels (i.e. 21.45–26.16 g PA kg-1), however, were found on the whole body P concentration. An inverse trend was recorded for the Ca content in the whole body (p<0.005) and carapace (p<0.004) with increasing PA inclusion. These results are consistent with findings for marine shrimp species such as Marsupenaeus japonicus and Litopenaeus vannamei. The graded inclusion of PA in the experimental diets also resulted in a significant reduced (p<0.03) P content in the carapace. Significant changes (p<0.05) were observed in the carapace Zn, Cu, K and Na compositions, particularly in the prawns fed the diet containing 11.28 g PA kg-1, which suggests that the specific minerals were either selectively utilised or retained in the carapace. Accordingly, this thesis investigates the potential of adding dietary supplements of microbial phytase in order to improve mineral availability as proven in several fish species. To explore this, four experimental diets were formulated - three incorporating different levels of microbial phytase (i.e. 0 FTU kg-1, 1000 FTU kg-1 and 2000 FTU kg-1) and a fourth, a control. An aliquot of 15 g PA kg-1 was also added to each treatment. The mineral premix was omitted from the test group diets except the control diet was identical to the 0 FTU kg-1 diet but included a mineral premix and an aliquot of 8 g kg-1 monosodium phosphate which replaced an equal amount of wheat meal fraction. The trial found no significant differences in growth, feed utilisation and moult frequency with the microbial phytase level within the diet, however, survival was compromised. The proximate composition of the prawn whole body was in most cases unaffected by the level of phytase. Supplementation of the diets with microbial phytase did, however, result in significant higher (p<0.05) concentrations of minerals including: 1) Ca, Mg, K and Na in the whole body; 2) Ca and Zn in muscle tissue; and, 3) Ca and Mg in the carapace. The Zn content of the carapace, however, was negatively affected by the inclusion of microbial phytase suggesting the necessity of this mineral within the diet of M. rosenbergii. This thesis contributes to current understanding surrounding the inclusion of dietary PA and the benefits of microbial phytase within the experimental diets consumed by juvenile M. rosenbergii. The knowledge gained from this work provides a means to optimise the use of plant protein ingredients and with the potential to decrease the dependability of fishmeal without compromising M. rosenbergii production and profitability, thus ultimately promoting the sustainable expansion of M. rosenbergii aquaculture.

This diploma work discusses the chemical and physical characteristics of phytic acid, its positive and negative influence on people’s health, on the substances usability in the human organism and also its possible use in the food industry. Methods for analyzing phytic acid in the feed and food industry are also discussed. This diploma work also states the characteristics of analyzed raw materials and, for the analyzed raw material malt, it also covers the technological procedure for its production. For direct determination of phytic acid in barley caryopses and in malt, the isotachophoresis (ITP) method was used. This diploma work contains results of phytic acid determination in treated and untreated barley and malt variants by 6 different varieties in 4 test stations - specifically for harvests during the years 2006 and 2007. The final work contains comparisons of phytic acid content in different varieties of treated/untreated barley and malt variants. Comparisons are always within a single test station. Comparison are also included of phytic acid content for single varieties (across all examined test stations). This work furthermore, presents decreases in phytic acid content after malting. The ITP method was evaluated as quick and relatively simple. A method by using tests (from a commercial company) for obtaining phytic acid content was also used. This diploma work lists results of phytic acid content determination in untreated barley and malt variants at two test stations, for the year of harvest 2007. Further, this work contains a comparison of phytic acid content in different varieties for the given test station. The method by using tests was evaluated as a orientation method. This work states comparisons of phytic acid content obtained by measurements during the years 2006 and 2007. The diploma work also compares ITP with the method realized by using tests in two test stations for 5 untreated varieties of barley and malt for 2007 harvest.

Phytic acid (PA) accounts for up to 75% of the P in soybean (Glycine max L. Merr.) seeds, but it is indigestible by mono- and agastric animals resulting in economic and environmental detriment. Soybean lines with genetically reduced PA contents have been developed using three distinct mutant alleles at the MIPS1, LPA1, and LPA2 genes resulting in up to a 75% reduction in PA. Low PA (LPA) soymeal-based feeds have been tested on several agricultural species and shown to reduce the P in the animal effluent, but they have not been tested on any aquacultural species. However, LPA soybean lines often exhibit low field emergence making them commercially inviable. The cause of this phenomenon is widely debated with possibilities ranging from increased disease pressure to decreased seedling vigor. The objectives of this research were to 1) enhance field emergence of LPA soybean varieties through pre-planting seed treatments, 2) study the impact of the LPA mutant alleles on agronomic, quality, and seed composition traits, and 3) design a low-error method for studying the effect of LPA soymeal-based feeds on aquatic animals using Pacific White Shrimp (Litoenaeus vannamei). These results describe a variety of agronomic and genetic strategies with which the low field emergence of LPA soybeans can be addressed, reveal a heretofore not reported interaction between the mips1 and lpa2 alleles to further increase the digestibility of soymeal, and a possible method for studying LPA soymeal based feed on aquacultural animals. / Master of Science

The field of functional genomics aims to understand the complex relationship between genotype and phenotype by integrating genome-wide approaches, such as transcriptomics, proteomics, and metabolomics. Large-scale "-omics" research has been made widely possible by the advent of high-throughput techniques, such as next-generation sequencing and mass-spectrometry. The vast data generated from such studies provide a wealth of information on the biological dynamics underlying phenotypes. Though functional genomics approaches are used extensively in human disease research, their use also spans organisms as miniscule as mycoplasmas to as great as sperm whales. In particular, functional genomics is instrumental in agricultural advancements for the improvement of productivity and sustainability in crop and livestock production. Improvement in soybean production is especially imperative, as soybeans are a primary source of oil and protein for human and livestock consumption, respectively. The research presented here employs functional genomics approaches – transcriptomics and metabolomics – to discern the transcriptional regulation and metabolic events underlying two economically important agronomic traits in soybean: seed phytic acid content and Soybean mosaic virus resistance. At normal levels, seed phytic acid content inhibits mineral absorption in humans and livestock, acting as an antinutrient and contributing to phosphorus pollution; however, the development of low phytic acid soybeans has helped mitigate these issues, as their seeds increase nutrient bioavailability and reduce environmental impact. Despite these desirable qualities, low phytic acid soybeans exhibit poor seed performance, which negatively affects germination rates and yield and has prevented their large-scale commercial production. Thus, part of the focus of this research was investigating the effects of mutations conferring the low phytic acid phenotype on seed germination. Comparative studies between low and normal phytic acid soybean seeds were carried out and revealed distinct differences in metabolite profiles and in the transcriptional regulation of biological pathways that may be vital for successful seed germination. The final part of this research concerns Rsv3-mediated extreme resistance, a unique mode of resistance that is effective against the most virulent strains of Soybean mosaic virus. The molecular mechanisms governing this type of resistance are poorly characterized. Therefore, the research presented here attempts to elucidate the regulatory elements responsible for the induction of the Rsv3-mediated extreme resistance response. Utilizing a comparative transcriptomic time series approach on Soybean mosaic virus-inoculated Rsv3 (resistant) and rsv3 (susceptible) soybean lines, this final study provides gene candidates putatively functioning in the regulation of biological pathways demonstrated to be crucial for Rsv3-mediated resistance. / Doctor of Philosophy / Soybeans are a crop of great economic importance, being a primary source of oil and protein for human and livestock consumption, respectively. Increasing demand for soybean calls for improvement in its production. An emerging field that has had tremendous impact on this endeavor is the field of functional genomics. Functional genomics approaches generate large-scale biological data that can aid in discerning how specific processes are regulated and controlled in an organism. The research presented in this work utilizes functional genomics approaches to elucidate the biological mechanisms underlying two economically important traits in soybean: seed phytic acid content and Soybean mosaic virus resistance. Phytic acid is a compound found in soybean seeds that causes nutrient deficiencies and phosphorus pollution. Soybeans with reduced to phytic acid content have been developed to mitigate these problems; they have poor seed germination and emergence. The studies in this work employ functional genomics approaches to compare unique sets of low and normal phytic acid soybeans to help establish the relationship between seed phytic acid content and seed performance. These studies resulted in new and promising hypotheses for future studies on investigating the low phytic acid trait. The final focus of this work used a functional genomics approach to discern the molecular mechanisms underlying a unique mode of resistance to Soybean mosaic virus. The study identified genes in soybean that are potentially critical to resistance against Soybean mosaic virus.