The overall goal of this research was to develop a plant protein-based microcapsule capable of carrying, protecting and delivering flaxseed oil within the food and gastrointestinal environment. Specifically, the research aimed to: a) screen a variety of plant proteins and pre-treatment conditions based on their emulsifying properties for use as a wall material; b) develop and optimize encapsulation protocols for entrapping flaxseed oil; and c) study the oxidative stability and delivery of entrapped oils from capsules under different environmental and simulated gastrointestinal conditions. In Chapter 3 and 4, the emulsifying and physicochemical properties of legume and oilseed protein isolates, respectively produced from isoelectric precipitation and salt extraction were investigated. Findings in Chapter 3 indicated that both the legume source and method of production showed significant effects on the emulsifying and physicochemical properties of chickpea (ChPI), faba bean (FbPI), lentil (LPI), pea (PPI), and soy (SPI) protein isolates. The emulsion capacity (EC) values ranged between 476-542 g oil/g protein with LPI showing the highest capacity. Isoelectric-precipitated ChPI and LPI displayed higher emulsion activity index (EAI) (~46.2 m2/g), (emulsion stability index) ESI (~84.9 min) and (creaming stability) CS (98.6%), which were comparable to those of SPI. In Chapter 4, findings indicated that both protein source and method of production had significant effects on the physicochemical and emulsifying properties of canola (CaPI) and flaxseed protein isolates (FlPI). CaPI showed significantly higher EC (~515.6 g oil/g protein) than FlPI (~498.9 g oil/g protein). EAI for FlPI was found to be higher (~40.1 m2/g) than CaPI (~25.1 m2/g) however, ESI values of CaPI and FlPI were similar. Creaming stability of emulsions stabilized by CaPI and FlPI ranged between 86.1 and 96.6%. CaPI and FlPI were shown to have emulsion forming properties; however their stability was low. In Chapter 5, ChPI and LPI-stabilized emulsions were optimized based on pH, protein concentration and oil content for their ability to form and stabilize oil-in-water emulsions using response surface methodology. Droplet charge was shown to be only affected by pH, while droplet size and creaming index were affected by protein concentration, oil content and pH. Optimum conditions for minimal creaming (no serum separation after 24 h), small droplet size (<2 μm), and high net droplet charge (absolute zeta potential (ZP) value >40 mV) were identified as: 4.1% protein, 40.0% oil, and pH 3.0 or 8.0, regardless of the plant protein used for emulsion preparation. Flaxseed oil was microencapsulated by freeze (Chapter 6) or spray (Chapter 7) drying employing ChPI or LPI and maltodextrin. Effects of emulsion formulation (oil, protein and maltodextrin levels) and protein source (ChPI vs. LPI) on the physicochemical characteristics, oxidative stability, and release properties of the resulting capsules were investigated. Optimized capsule designs were found to have high encapsulation efficiencies, low surface oil, and afforded protection against oxidation over a 25 d room temperature storage study relative to free oil. Microcapsules were also able to deliver 84.2% of the encapsulated oil in the simulated gastrointestinal environments.

Through the implementation of in vivo feeding trials, the efficacy of three wheat gluten (WG) products, vital (Amytex®), hydrolysed (Merripro®) and soluble hydrolysed (Solpro®) wheat gluten as replacement of soy protein concentrate, and scFOS prebiotic (Profeed®) supplementation were analysed to assess their impacts on intestinal health and production of juvenile rainbow trout. Microbial community analysis in experiment one revealed a degree of diet based modulation with 7.5% and 15% inclusions of wheat gluten (WG) products. Bacterial species diversity was significantly reduced with 15% hydrolysed wheat gluten (HWG) inclusion compared to the plant protein control and 15% vital wheat gluten (VWG) treatments, with sequenced OTUs dominated by the phylum Firmicutes and possible promotion of probiotic species. No detrimental effects were observed on intestinal morphology. These findings led onto a longer duration feed trial with a more holistic, higher resolution approach. Experiment two revealed modulation of the allochthonous intestinal microbiota, with increased proportions of Enterococcus and Weissella in the 10% and 20% VWG treatments. Bacillus and Leuconostoc relative abundances were significantly increased with 10% HWG and soluble hydrolysed (Sol) wheat gluten inclusions. HSP 70 transcripts were significantly down-regulated in all WG treatments compared to the basal soy protein concentrate treatment (SPC) and increased intraepithelial leukocyte counts were observed with 10% VWG inclusion. Growth performance was unaffected by 10% dietary inclusions of WG, however, FCR’s were significantly improved in the 20% VWG treatment compared to the 10% HWG and Soluble treatments. This led to the investigation of increased inclusion levels of WG products in experiment three. All WG treatments in experiment three yielded significantly improved growth performance. Somatic indices were significantly increased with 30% blended WG inclusion compared to the SPC treatment. Modulation of allochthonous intestinal microbiota was observed to a lower degree than the previous experiments, with a dose response observed with increasing blended WG inclusion. In the final experiment two basal diets (SPC and 20% Blended) and two scFOS supplemented diets (SPC + FOS and 20% Blended + FOS) were investigated for the effect on growth performance, gut health and allochthonous microbial population. Growth performance was unaffected, however, modulation of the allochthonous microbial population was observed with an apparent synergistic effect of scFOS supplementation in WG diets. This synergistic trend was also observed in the transcription level expression of immune relevant genes. 20% WG inclusion with additional scFOS supplementation observed significant down regulation of the pro-inflammatory cytokine TNF-α, as well as HSP 70, CASP 3 and Glute ST compared to the 20% Blend treatment. The present research demonstrates dietary inclusions of WG products, solely or blended, at the expense of soy protein concentrate to modulate the allochthonous microbial population, potentially promoting probiotic species, whilst reducing the levels of intestinal stress in juvenile rainbow trout. Supplementation of the prebiotic scFOS modulated the microbial populations, enhancing the proportion of potential probiotic species, and combined with WG inclusions, reduce intestinal and oxidative stress and inflammation biomarkers, with no observed deleterious effects.

Of the organisms with sequenced genomes, plants appear to possess the most rhomboid protease-encoding genes. However, our knowledge of processes in plants that involve Regulated Intramembrane Proteolysis (RIP) and rhomboid proteases remains low. As expressed recently by other researchers, finding a natural substrate for a rhomboid protease represents the biggest experimental challenge. Using yeast mitochondria-based assays, a potential link between the plastid translocon component Tic40 and organellar rhomboid proteases was recently uncovered. In this particular link, rhomboid proteases appear capable of influencing the pattern of imported Tic40 in yeast mitochondria. Tic40 may thus represent a natural plant target of organellar rhomboid proteases. Here, we obtained further motif-oriented evidence supporting Tic40 as a natural plant rhomboid substrate. A comparative analysis of sequences revealed that Tic40 may also possess similar TMD motifs found in the model substrate, Spitz. Rhomboid proteases often require these motifs to cleave substrates within intramembrane environments. Using site-directed mutagenesis and yeast mitochondria assays, the impact of mutations occurring in the motifs ASISS, GV, QP, and GVGVG of Tic40 was assessed. In terms of cleavage and changing the pattern of imported Tic40, some of the mutations showed decreased activities and a few showed enhancements. More importantly, the overall observed pattern associated with select Tic40 mutations resembled the characteristics reported for the model substrates. In particular, mutations in the Tic40 GV motif produced similar results as that observed with Spitz, by drastically decreasing or increasing cleavage as a function of amino acid sequence. / Thesis (Master, Biology) -- Queen's University, 2010-05-30 10:22:07.72

Lipid droplets in plants (also known as oil bodies, lipid bodies, or oleosomes) are well characterized in seeds, and oleosins, the major proteins associated with their surface, were shown to be important for stabilizing lipid droplets during seed desiccation and rehydration. However, lipid droplets occur in essentially all plant cell types, many of which may not require oleosin-mediated stabilization. The proteins associated with the surface of nonseed lipid droplets, which are likely to influence the formation, stability, and turnover of this compartment, remain to be elucidated. Here, we have combined lipidomic, proteomic, and transcriptomic studies of avocado (Persea americana) mesocarp to identify two new lipid droplet-associated proteins, which we named LDAP1 and LDAP2. These proteins are highly similar to each other and also to the small rubber particle proteins that accumulate in rubber-producing plants. An Arabidopsis (Arabidopsis thaliana) homolog to LDAP1 and LDAP2, At3g05500, was localized to the surface of lipid droplets after transient expression in tobacco (Nicotiana tabacum) cells that were induced to accumulate triacylglycerols. We propose that small rubber particle protein-like proteins are involved in the general process of binding and perhaps the stabilization of lipid-rich particles in the cytosol of plant cells and that the avocado and Arabidopsis protein members reveal a new aspect of the cellular machinery that is involved in the packaging of triacylglycerols in plant tissues.

No description available.

With a rapidly growing world population and increased threats of climate change, Sub-Saharan Africa is most at risk for lower crop yields and facing hunger. Within Sub-Saharan Africa, Senegal has some of the highest levels of anemia and micronutrient deficiencies among women and children. Mungbean [Vigna radiata (L.) R. Wilczek] is a pulse crop that has recently been successfully introduced in Senegal to diversify a primarily cereal-based diet consisting of millet, maize, and rice. The potential for mungbean to be harvested as a leafy green as well as a pulse could allow for a more balanced and nutrient-rich diet. This diversification could help combat micronutrient deficiencies while earlier harvest of the leaves could help alleviate the pressures of the "hunger season." To understand the effects of leaf harvest on mungbean grain yield, yield components, and nutrition, a field trial was conducted in Blacksburg, Virginia for three consecutive years from 2020 to 2022. In a split-plot design, four frequencies of leaf harvest (0x, 1x, 2x, and 3x) on seven accessions of mungbean were tested in triplicate. The objective of this experiment was to determine if mungbean can be used as a dual-purpose crop as a leafy green vegetable without decreasing grain yield in Senegal. It was found that mungbeans could undergo up to two leaf harvest of immature leaves without reducing yield, total dry matter (TDM), or yield components. The harvested leaves were also found to be highly nutritious with 22.0% protein, 12.3% fiber and 8.5% ash with no significant differences between leaf harvest treatments. These results indicate that mungbean can be used as a dual-purpose crop for harvest as leafy greens and pulse in Senegal. Further, mungbeans were studied as a viable crop in southside Virginia. The objective was to evaluate the efficacy of mungbeans as an alternative crop to tobacco farmers in Virginia. Field trials were conducted on farmers' fields and at Virginia Tech's Southern Piedmont Agricultural Research and Extension Center in 2021 and 2022. A split-plot experimental design was used with early and late planting dates in the beginning and end of June as the whole plots and two commercially-available cultivars, Berken and OK 2000, as the sub-plots. Due to highly variable rain patterns in 2021 and 2022, as well as differing management practices, there were no consistent effects of genotype or planting date on yield, plant height, pods per plant, seeds per pod, or seed size. Yield ranged from 0.19 MT ha-1 to 1.18 MT ha-1 with an average yield of 0.84 MT ha-1 in 2021 and 0.38 MT ha-1 in 2022. Though there was variation in yield, across planting dates, cultivars, locations, and years, the highest yield was higher than global averages. It was concluded that while there is great potential with the growing mungbean market, more studies of breeding and supply chain issues and development of a production guide are needed for mungbean to be successful in Virginia. A final study compared soybean, edamame, and mungbean nutritional components and volatiles, two characteristics of importance to breeding objectives and food processing regarding plant alternative proteins. It was found that mungbean had significantly less protein (21.1%) than soy (36.2%) and edamame (38.3%). Mungbean also had lower fat (0.769%) compared to soy (13.5%) and edamame (14.0%). Analysis of aromatic compounds revealed that soybean, edamame, and mungbean each had unique profiles that could be advantageous to the production of specific plant protein foods. Overall, these studies demonstrate the growing importance and potential of mungbean in both Senegal and in the United States. / Master of Science / With a rapidly growing world population and increased threats of climate change, Sub-Saharan Africa is most at risk for lower crop yields and facing hunger. Within Sub-Saharan Africa, Senegal has some of the highest levels of anemia and micronutrient deficiencies among women and children. Mungbean [Vigna radiata (L.) R. Wilczek] is a pulse crop that has recently been successfully introduced in Senegal to diversify a primarily cereal-based diet consisting of millet, maize, and rice. The potential for mungbean to be harvested as a leafy green as well as a pulse could allow for a more balanced and nutrient-rich diet. This diversification could help combat micronutrient deficiencies while earlier harvest of the leaves could help alleviate the pressures of the "hunger season." To understand the effects of leaf harvest on mungbean grain yield, yield components, and nutrition, a field trial was conducted in Blacksburg, Virginia for three consecutive years from 2020 to 2022. In a split-plot design, four frequencies of leaf harvest (0x, 1x, 2x, and 3x) on seven accessions of mungbean were tested in triplicate. The objective of this experiment was to determine if mungbean can be used as a dual-purpose crop as a leafy green vegetable without decreasing grain yield in Senegal. It was found that mungbeans could undergo up to two leaf harvest of immature leaves without reducing yield, total dry matter (TDM), or yield components. The harvested leaves were also found to be highly nutritious with 22.0% protein, 12.3% fiber and 8.5% ash with no significant differences between leaf harvest treatments. These results indicate that mungbean can be used as a dual-purpose crop for harvest as leafy greens and pulse in Senegal.

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

Currently aquaculture is the largest growing food sector in the world, however, its future growth is limited by its heavy reliance on fishmeal (FM). Overfishing of wild marine fish stocks used for FM is putting too much pressure on the stocks, making FM unsustainable as a dominant protein source as aquaculture continues to grow. Plant proteins (PP) are an ideal alternative to FM because of their wide availability and relatively low cost. Soybean meal (SBM) is the most commonly used PP in aquaculture, but its inclusion in feeds is limited due to reduced digestibility and the presence of antinutritional factors ultimately leading to a reduced growth performance of fish that are fed with the SBM-based diet. Therefore, the goal of this thesis was to mitigate the negative effects of PP and improve the growth of fish fed with a PP-based diet, utilizing SBM as our PP in feeds. Three feeding trials were conducted to test the efficacy of 3 approaches towards improving the use of PP in fish. The first trial (Chapter 2), tested the effect of nutritional programming (NP) on the utilization of SBM in zebrafish (Danio rerio). NP is the theory that an organism can be ‘programmed’ to better utilize a dietary component by being exposed to that component in its early life stages. This study also tested the effect of NP through the broodstock by exposing the broodstock to SBM for 4 weeks prior to breeding. In addition, a combination of both programming techniques was also tested. The results found that neither of the programming techniques had a significant effect on the growth performance of the zebrafish. Among the two groups from the programmed broodstock, the group that also received early stage NP had a significantly higher expression of PepT1, a di- and tri- peptide transporter. Also, the dual programmed group had the highest length-to-width ratio of the distal villi among groups that were fed SBM, which signifies an increase in surface area for nutrient absorption in the intestine. The findings from this study suggest that early stage NP may increase the absorption of nutrient from PP-based feeds within the intestine. The second feeding trial (Chapter 3), utilized the supplementation of health-promoting dipeptides to improve the use of SBM-based feeds for zebrafish. The three dipeptides used in this study were alanyl-glutamine, carnosine, and anserine. The five groups in this study consisted of three groups receiving an SBM-based diet with one of the dipeptides supplemented into it, a (-) Control group receiving a non-supplemented SBM-based diet, and a (+) Control group receiving a FM-based diet. Both the alanyl-glutamine and carnosine supplemented groups experienced a significantly higher weight gain compared to the (-) Control group. In addition, the alanyl-glutamine supplemented group had a significantly higher length-to-width ratio of the intestinal villi and, had a numerically higher expression of both nutrient absorption genes measured, PepT1 and fabp2, compared to the (-) Control group. This finding suggests that the supplementation of alanyl-glutamine into SBM-based diets may improve the intestinal absorptive capacity of the fish fed with the SBM. The results from this study also support the use of both alanyl-glutamine and carnosine supplementation as a means of improving the growth performance of fish fed with a SBM-based diet. The third feeding trial (Chapter 4), was conducted on largemouth bass (LMB) (Micropterus salmoides). This study also focused on NP as a means of improving the utilization of dietary SBM, similar to Chapter 2. In this study, live feed was used as a vector to program the larval LMB to SBM. The programmed group in this study received Artemia nauplii that were enriched with a SBM solution, during the larval stage. The bass were then fed with a FM-diet for 7 weeks, before being reintroduced to SBM for the final 6 weeks of the study (PP-Challenge). The programmed LMB experienced a significantly higher weight gain compared to the non-programmed fish also undergoing the PP-Challenge, and achieved a weight gain similar to that of the LMB that were being fed with an FM-diet. In addition, the programmed LMB had significantly longer distal villi and a higher length-to-width ratio of the villi, compared to the non-programmed group. The findings from this study support the use of live feed as a vector for NP and improving the growth performance of a carnivorous aquaculture species fed with a SBM-based diet. The overall findings from these studies suggest that both NP and dipeptide supplementation are feasible means of improving the utilization of SBM in fish. The mechanism behind NP seems to lie in the intestine. In both zebrafish and LMB, NP was found to reduce the inflammatory impact on the intestine and increase the surface area for absorption of the intestinal villi. The supplementation of alanyl-glutamine had similar effects on the intestine as NP and improved the growth performance of zebrafish. The observations from these studies seem to point to mitigating the negative effects of SBM on the intestine as the key to improving the growth performance of fish fed with a SBM-based diet.

Plant-derived protein has gained popularity in recent years due to its health and environmental impact. Studies comparing the health benefits of animal and plant protein have mostly focused on soy as a plant protein representative due to its popularity. Demand for other protein sources such as peas and fava beans has been increasing; thus, the health impact of plant protein sources other than soy must be assessed. Evidence has shown that diet influences the gut microbiota and the metabolites in the body. Metabolites associated with amino acids are strong contributors to the metabolite distinction between dietary animal and plant foods. Certain gut bacteria are also known to be able to metabolize amino acids, thus influencing their survival in the gut. To discern the impact of plant and animal-based proteins, namely soy, pea, faba bean, beef, chicken, and pork, on the host gut microbiota as well as the metabolic profile, male C57BL/6J mice were fed with the proteins for 8 weeks. Results showed that each protein source influenced the gut microbiota and metabolic profile differently. However, these impacts were not caused by the amino acid profile alone. Other factors, such as myoglobin in meats and phenolic compounds in plant proteins, also play a role.

A bacteriose, ou mancha oleosa, doença causada por Xanthomonas axonopodis pv. passiflorae, é um sério problema em muitas áreas de produção de maracujá no Brasil, especialmente se associada à antracnose. A transformação genética é uma alternativa para obter plantas resistentes. Proteínas bactericidas, como as atacinas encontradas na hemolinfa de insetos, têm sido usadas para conferir resistência a espécies vegetais. Como as atacinas têm um peptídeo sinal que as direciona para o espaço extracelular em insetos, nós iniciamos este estudo investigando o direcionamento da atacina A em plantas. A seqüência do gene da atacina A (attA) com e sem o peptídeo sinal foi fusionada com os genes repórteres uidA e gfp e epidermes de cebola foram transformadas, via biobalística, com essas construções gênicas. A atacina A, de fato, é acumulada no apoplasto onde, justamente, bactérias fitopatogênicas se multiplicam antes de invadir as células vegetais. Visando obter plantas transgênicas resistentes à bacteriose, foram transformados tecidos foliares e hipocotiledonares com as linhagens LBA 4404 e EHA 105 de Agrobacterium tumefaciens contendo o gene attA. De um total de 313 explantes infectados, foram obtidos 31 brotos PCR+, o que representa uma eficiência de transformação da ordem de 10%. A expressão do transgene foi confirmada por RT-PCR e a resistência ao patógeno foi avaliada pela inoculação de X. axonopodis pv. passiflorae em folhas destacadas de plantas mantidas in vitro. Em dez plantas não houve formação de lesão foliar, indicando uma possível resistência ao patógeno. / Bacterial spot disease caused by Xanthomonas axonopodis pv. passiflorae is a serious problem in many passion fruit production areas in Brazil, especially if associated with anthracnose. Genetic transformation provides an alternative for obtaining resistant plants. Bactericide proteins such as attacins, found in the haemolymph of insects, have been used to confer resistance on plant species. As the attacins have a sign peptide that dispatches them to extracellular space in insects, we initiated our studies investigating the attacin A directing in plants. The attacin A gene (attA) sequence, with and without the sign peptide, was fused to uidA and gfp reporter genes, and onion epidermis were transformed using bioballistics with gene constructions. The protein did accumulate in the apoplast, where bacteria multiply before attacking plant cells. With the aim of obtaining transgenic plants of yellow passion fruit resistant to bacterial disease, leaf and hypocotyl-derived tissues were transformed with LBA 4404 and EHA 105 strains of Agrobacterium tumefaciens containing the attA gene. From a total of 313 infected explants, we obtained 31 PCR+ shoots, a transformation efficiency of 10%. Expression of the attA gene was confirmed by RT-PCR, and pathogen resistance evaluated by X. axonopodis pv. passiflorae inoculation in leaves obtained from in vitro plants. Leaf lesions were not observed in 10 shoots, suggesting a possible resistance to pathogen.