Master of Science / Department of Food, Nutrition, Dietetics, and Health / Brian L. Lindshield / Fortified-blended foods (FBFs), grain-legume porridges (most commonly corn and soy), are frequently used for food aid purposes. Sorghum and cowpea have been suggested as alternative FBF commodities because they are drought-tolerant, grown locally in food aid receiving countries, and are not genetically modified. The objective of this thesis was to determine the protein quality and iron bioavailability of newly formulated, extruded FBFs in broiler chickens, which have been suggested as a good model for assessing iron bioavailability. Five FBFs were formulated to contain whey or soy protein to compare protein quality, sugar, oil, and an improved micronutrient premix. These included three white sorghum-cowpea FBFs; two were extruded with either whey protein concentrate (WSC) or soy protein isolate (WSC+SPI) added, one was non-extruded (N-WSC). Two others were white sorghum-soy (WSS) and corn-soy (CSB14) FBFs. Two additional white-sorghum cowpea FBFs were reformulated and “over-processed” to contain no sugar, less whey (O-WSC) or soy protein (O-WSC+SPI), and less oil, thus producing a less expensive FBF. Two studies were performed using prepared (Prep) or dry (Dry) FBFs, along with the United States Agency for International Development (USAID) corn and soy blend FBF, CSB+, fed to chickens for 3 and 2 weeks, respectively; food intake, body weights, hemoglobin, and hepatic iron were assessed. In the Prep study, new FBFs significantly increased caloric and protein efficiency compared to CSB+, despite similar food intake and body weight gain. In the Dry study, CSB+ significantly decreased food intake and caloric efficiency, with the exception of O-WSC+SPI, and nonsignificantly reduced body weight gain and protein efficiency compared to new FBFs. CSB+ significantly reduced hepatic iron content compared to all FBFs in the Dry study, and was nonsignificantly decreased compared to new FBFs in the Prep study. In conclusion, sorghum and cowpea FBFs performed similarly to corn and soy FBFs, suggesting these commodities are suitable replacements for corn and soy. Soy protein isolate (WSC+SPI) was an effective alternative to whey protein concentrate (WSC), suggesting SPI can be a less expensive protein supplement in FBFs. Surprisingly, non-extruded sorghum and cowpea (N-WSC) was equally efficacious to extruded WSC. However, N-WSC did not meet viscosity requirements and is not precooked, which limits its viability as an FBF. O-WSC+SPI resulted in poorer outcomes compared to other FBFs, which suggests the protein quality of cowpea may be inferior and the inclusion of whey protein is needed in this formulation, as O-WSC with whey performed similarly to other FBFs. Overall, new FBFs, with the exception of O-WSC+SPI, resulted in improved food efficiency and hepatic iron outcomes compared to CSB+, suggesting they are of higher nutritional quality. However, further research is needed to refine and identify the best FBF formulations.

Membrane compartmentalisation allows eukaryotic cells to perform complex processes by combining dedicated sets of proteins in the same organelle. To achieve this, the cell must first target the appropriate proteins, primarily synthesised on cytosolic ribosomes, to the correct subcellular location. Components of the secretory pathway/endomembrane system begin this journey via their signal sequence-dependent delivery to the endoplasmic reticulum (ER). These ER targeting signals are hydrophobic, and typically function whilst the protein is being synthesised, via a so-called 'co-translational' pathway. However, some hydrophobic signals can also facilitate post-translational protein targeting to the ER, or initiate regulated protein degradation in the cytosol. Tail-anchored (TA) proteins are transmembrane proteins with a single C-terminal transmembrane domain that functions as both their subcellular targeting signal and membrane anchor. Recent evidence suggests that the canonical TRC40 pathway, through which mammalian TA proteins are delivered to the ER, may not be essential in vivo. In this thesis, I provide functional evidence for the existence of an orthologous SRP-independent (SND) pathway in mammalian cells and identify roles for both the signal recognition particle (SRP)-mediated pathway and presumptive mammalian SND pathway in the biogenesis of TA proteins. I conclude that although TRC40 normally plays a role in TA protein biogenesis, it is not essential, and speculate that these alternative pathways make a significant contribution to the apparent redundancy of the TRC40 pathway in vivo. The soluble components that act upstream of TRC40 during protein biogenesis also play an important role in the recognition and selective degradation of hydrophobic membrane and secretory proteins that mislocalise to the cytosol. I now provide preliminary evidence that TRC40 appears to exhibit dual functionality, having a non-essential role in TA protein delivery, whilst also contributing to protein quality control by acting as a putative holdase. My data suggest that both TRC40 and BAG6 can influence the proteasomal degradation of a novel class of substrates, which I have termed the aberrant short secretory proteins.

Most glyco-engineering approaches used to improve quality of recombinant glycoproteins involve the manipulation of glycosyltransferase and/or glycosidase expression. We investigated whether the over expression of nucleotide sugar transporters, particularly the CMP-sialic acid transporter (CMP-SAT), would be a means to improve the sialylation process in CHO cells. We hypothesized that increasing the expression of the CMP-SAT in the cells would increase the transport of the CMP-sialic acid in the Golgi lumen, hence increasing the intra-lumenal CMP-sialic acid pool, and resulting in a possible increase in sialylation extent of proteins being produced. We report the construction of a CMP-SAT expression vector which was used for transfection into CHO-IFNγ, a CHO cell line producing human IFNγ. This resulted in approximately 2 to 5 times increase in total CMP-SAT expression in some of the positive clones as compared to untransfected CHO-IFNγ, as determined using real-time PCR analysis. This in turn concurred with a 9.6% to 16.3% percent increase in site sialylation. This engineering approach has thus been identified as a novel means of improving sialylation in recombinant glycoprotein therapeutics. This strategy can be utilized feasibly on its own, or in combination with existing sialylation improvement strategies. It is believed that such multi-prong approaches are required to effectively manipulate the complex sialylation process, so as to bring us closer to the goal of producing recombinant glycoproteins of high and consistent sialylation from mammalian cells. / Singapore-MIT Alliance (SMA)

Wheat distillers dried grain with solubles (DDGS) is a co-product of ethanol production, primarily utilized as an animal feed ingredient. Reduced protein quality, a highly energy-intensive drying process, and product variability are some of the challenges that currently confront its production in western Canada. The main focus of this research undertaking was to examine, on a laboratory-scale, the effect of condensed distillers solubles (CDS) : wet distillers grain (WDG) blending ratio and drying conditions on the protein quality and physico-chemical characteristics of wheat DDGS. The potential of microwave-based drying methods in minimizing protein damage and energy consumption was investigated. An auxiliary case study was also conducted in the Philippines to apply the knowledge and skills acquired from the PhD research undertaking to a related problem situation in a developing country. It aimed to provide more information about brewers spent grain (BSG) supply and utilization in Misamis Oriental, Philippines and enhanced the efficiency and safety of BSG utilization as an animal feed ingredient. Laboratory-scale investigations used samples produced at three CDS:WDG blending ratios (by mass): 15:85 (15% CDS), 30:70 (30% CDS), and 45:55 (45% CDS) and dried under forced air convection (40-120C), and under microwave (420 – 805 W) and microwave convection (nominal settings of 130C-30% power to 190C-30% power) methods using a domestic microwave oven. Freeze-dried samples were used as standards in evaluating chemical composition and color of wheat DDGS. As CDS level in the blend was increased, protein and ash content of freeze-dried samples increased while fat, acid detergent fiber (ADF) and neutral detergent fiber (NDF) content decreased. These trends were attributed to proximate composition differences between CDS and WDG fractions. The CDS fraction had higher protein and ash and lower fat, ADF, and NDF contents compared to WDG. Variation in the CDS:WDG blending ratio employed in the source ethanol plant could be one of the factors contributing to the observed proximate composition differences between two ethanol plant-sourced wheat DDGS samples. This was verified through proximate analyses of: (i) plant-sourced wheat DDGS samples from two production batches; (ii) CDS and WDG samples obtained on the same production date; and (iii) laboratory-produced wheat DDGS samples at varying CDS:WDG blending ratios. Protein, ash, and NDF contents of forced-air convection-, microwave-, and microwave convection-dried samples also showed strong linear relationships with CDS level. Fat and ADF content, however, did not exhibit similar strong relationships with CDS level, indicating the influence of drying conditions. Maximum lysine and minimum acid detergent insoluble crude protein (ADICP) contents were achieved in blends with the highest CDS level (45% CDS) and dried under lower drying temperature (80C), microwave power (676 W), and microwave convection (150C-30% power) settings. Microwave- and microwave convection drying achieved desirable protein quality associated with lower temperature drying under much shorter times. Laboratory-scale drying of ethanol plant-sourced wet distillers grain with solubles (WDGS) under forced air convection produced DDGS samples with decreased lysine content as drying air temperature was increased. In terms of physical properties, dried samples with higher CDS level were significantly finer, denser, less flowable, less dispersible, have lower thermal diffusivity and higher internal friction coefficients, and produced denser and stronger pellets. Color parameters of freeze-dried samples were significantly affected by CDS level. As CDS level increased, lightness (Hunter L) decreased while redness (Hunter a) increased. The color parameters of forced-air convection-, microwave-, and microwave convection-dried DDGS samples did not, however, exhibit similar linear trends with CDS level as these were also affected by drying conditions, such as drying air temperature and microwave power level. Effective moisture diffusivity values, estimated from the drying data, were also significantly affected by drying conditions (drying air temperature and microwave power levels), CDS level, and interaction between drying air temperature and CDS level. Effective moisture diffusivity decreased as CDS level was increased. Physical properties of two commercial (ethanol plant-sourced) wheat DDGS samples, as affected by moisture content, were also assessed. Techno-economic evaluation results indicated that complete replacement of the conventional hot air drying with microwave drying technology was not yet economically feasible. Although energy consumption during drying was substantially reduced with the use of microwave energy, the cost of electricity to generate microwave energy was high. Incorporating microwave drying toward the end of the hot air drying process was seen as the more economically viable alternative. Drying of BSG was not commonly practiced in Misamis Oriental, Philippines. The material was typically stored in open concrete bins and commonly fed to dairy cattle in its wet form. A prototype batch dryer, powered either by solar or biomass energy, was developed to improve shelf life and safe use of BSG. Initial tests showed that drying spent grain using a biomass furnace was feasible. Operating the dryer using solar energy was not an attractive option because of the long drying time. Basic physical attributes, proximate composition, and moisture sorption characteristics of the dried BSG were also determined. Practical implications of the results of these two studies to their respective local contexts were discussed. Recommendations to further improve understanding of wheat DDGS protein quality and physico-chemical characteristics, BSG dryer performance, backyard farmers’ sustainable access and safe use of BSG were also presented.

Licentiatavhandling (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2006. / Härtill 2 uppsatser.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / Um dos problemas mundiais mais graves à a escassez de alimentos devido ao crescimento da populaÃÃo. InÃmeros estudos investigaram fontes nÃo convencionais de alimentos ricos em proteÃnas para atender Ãs necessidades de paÃses com situaÃÃo econÃmica desfavorÃvel. Bauhinia cheilantha, popularmente conhecida como pata-de-vaca ou unha-de-boi à uma leguminosa pertencente à subfamÃlia Caesalpinoideae de ocorrÃncia frequente nas Ãreas de mata de Caatinga. Poucos estudos com sementes do gÃnero Bauhinia sugerem seu potencial nutricional para alimentaÃÃo humana e/ou animal. Deste modo, o presente estudo objetivou avaliar as proteÃnas de B. cheilantha quanto ao seu valor nutricional usando ratos como modelo experimental. As sementes de B. cheilantha demonstraram bom potencial nutricional apresentando alto conteÃdo de proteÃnas (35,88  0,92%) e fibra alimentar (45,31  0,38%), enquanto os teores de cinzas, carboidrato e lipÃdio foram de 3,9%, 6,2 % e 8,71% de peso seco da farinha de B. cheilantha, respectivamente. O perfil de Ãcidos graxos presentes na semente mostrou elevado teor de Ãcido linolÃico (40,4%) e Ãcido olÃico (20,7%) que constituem importantes componentes para a manutenÃÃo da saÃde. A composiÃÃo de aminoÃcidos essenciais da farinha e do concentrado protÃico da semente apresentaram, perfil razoÃvel quando comparadas com recomendaÃÃes para crianÃas. Os fatores antinutricionais inibidores de tripsina (31,49 UI/gF), lectinas (793,6 UH/gF) e urease (32,89 U/gF) apresentaram-se iguais ou menores quando comparados a valores encontrados para soja e outras leguminosas, apesar da B. cheilantha ser uma espÃcie selvagem, enquanto taninos, flavononÃis, flavonas, flavanÃis ou xantonas, triterpenÃides, esterÃides e saponinas foram detectados em um teste qualitativo, podendo representar compostos antrinutricionais dependendo de suas quantidades. Para os testes biolÃgicos, ratos foram divididos em sete grupos que receberam dietas controles utilizando como fonte de proteÃna (caseÃna, como proteÃna de alto valor biolÃgico, soja como proteÃna de uma leguminosa com bom valor nutricional e uma dieta isenta de proteÃnas) e dietas experimentais utilizando como fonte de proteÃna (farinha da semente crua, farinha da semente previamente colocada em demolho, farinha da semente previamente colocada em demolho seguida de cozimento por 1 hora e com o concentrado protÃico da semente). Os animais expostos Ãs dietas com semente crua e em demolho e com concentrado protÃico apresentaram as menores taxas de ganho de peso (-17,51 g, -16,57 g e 10,40 g, respectivamente), enquanto a dieta com a semente tratada termicamente ofereceu melhora no ganho de peso (-2,50 g). A digestibilidade apresentada por todas as dietas foi considerada promissora variando de 70,36% a 90,96%. Os valores de NPU e valor biolÃgico, respectivamente para as dietas com B. cheilantha crua (-40,26% e -14,70%) e em demolho (-7,46% e -80,86%) foram melhorados com os processos de cozimento (25,63% e 35,37%) e extraÃÃo de proteÃnas solÃveis (26,62% e 29,34%). Os resultados de parÃmetros sÃricos e pesos secos relativos de ÃrgÃos nÃo demonstraram sinais de toxicidade. Assim, pode-se concluir que as sementes de B. cheilantha sÃo uma boa fonte de proteÃnas quando comparados seus parÃmetros com os de outras leguminosas utilizadas na alimentaÃÃo, apesar de nÃo oferecerem palatabilidade boa o suficiente para proporcionar crescimento adequado de ratos. / One of the most serious global problems is the shortage of food due to population growth. Numerous studies have investigated non-conventional sources of protein-rich food to meet the needs of countries with unfavorable economic situation. Bauhinia cheilantha, popularly known as cow-paw or ox-nail is a legume belonging to the subfamily Caesalpinoideae of frequent occurrence in forest areas of the Caatinga. Few studies with seeds of Bauhinia genus suggest its nutritional potential as food for humans and / or animals. Thus, this study aimed to evaluate the proteins of B. cheilantha regarding its nutritional value by using rats as experimental model. The seeds of B. cheilantha showed good nutritional potential with high protein content (35.88%) and dietary fiber (45.31%), while the ash content, carbohydrate and lipid levels were 3.9%, 6.2% and 8.71% of B. cheilantha flour dry weight, respectively. The fatty acids profile presented in the seed showed high content of linoleic acid (40.4%) and oleic acid (20.7%) which are important components to health maintenance. The essential amino acid composition of flour and protein concentrate from seed showed a reasonable profile when compared with recommendations for children. The antinutritional factors trypsin inhibitors (31.49 IU / gF), lectins (793.6 HU 0 / gF) and urease (32.89 U / gF) were equal to or smaller than those for soybean and other legumes, although B. cheilantha is a wild species, while tannins; flavononols, flavones, flavanols and xanthones, triterpenoids, steroids and saponins were detected in a qualitative test, which may represent antinutritional compounds depending on their amounts. For biological tests, rats were divided into seven groups receiving control diets using as protein source (casein as a source of high biological value, soy as a protein source from a legume with good nutritional value and a protein-free diet) and experimental diets using as protein source (raw seed flour, flour of the seed previously soaked, flour of the seed previously soaked followed by 1h boiling and with the protein concentrate from seed). The animals exposed to diets with raw, soaked and protein concentrate seeds had the lowest rate of weight gain (g -17.51, -16.57 g, 10.40 g) while the diet with the heat treated seed offered improvement in weight gain (-2.50 g). The digestibility of all diets were considered promising, ranging from 70.36 to 90.96%. The values of NPU and biological value for diets with raw seeds (-40.26% and -14.70%) and soaked seeds (-7.46% and -51.86%) were improved with the cooking processes (25.63% and 35.37%) and extraction of soluble proteins (26.62% and 29.34%). The results of serum parameters and organ dry weights showed no signs of toxicity. Thus, it can be concluded that the seeds of B. cheilantha are a good source of protein, when their parameters are compared with those of other legumes used as food, Nevertheless, palatability must be improved to provide adequate growth of rats.

Protein quality control is crucial to maintaining cellular function. A failure to clear misfolded, aggregation prone proteins can lead to the accumulation of toxic protein aggregates that interfere with cellular pathways and lead to cell death. In addition, the degradation of partially functional proteins can lead to loss of function diseases. Understanding proteins quality control mechanisms is therefore of fundamental importance to understanding these disease pathways. Systems that operate to monitor the structure of soluble protein domains are now relatively well understood. However, in addition to soluble domains, membrane proteins contain regions that span lipid bilayers, and a key question that remains is where and how these transmembrane domains (TMDs) that fail to assemble correctly or are otherwise aberrant are recognised within subcellular compartments. As such, in this study model chimeric proteins containing the luminal and cytoplasmic domain of the single-spanning membrane protein CD8 and exogenous TMDs derived from polytopic membrane proteins were used to investigate the handling of non-native TMDs in the secretory pathway. CD8 chimeras containing non-native TMDs were found to be recognised by endoplasmic reticulum (ER) quality control pathways. Importantly, ER-associated degradation of CD8 chimeras containing exogenous TMDs was reliant upon ubiquitination of cytoplasmic lysine residues prior to retrotranslocation and dislocation from the ER membrane. In contrast, CD8 containing the endogenous TMD but a misfolded luminal domain could be efficiently degraded when cytoplasmic lysines were removed, suggesting that the retrotranslocation mechanisms for these proteins are distinct and defined by the domain which is misfolded. A proportion of the CD8 chimeras containing non-native TMDs were able to exit the ER, and were retrieved to the ER from the Golgi. Golgi-to-ER retrieval was found to be at least partially mediated by Rer1. CD8 chimeras that escaped ER retrieval could also be retained in the Golgi and subsequently degraded in lysosomes, indicating the presence of an as yet undefined TMD-based Golgi quality control checkpoint in mammalian cells. Furthermore, in contrast to WT CD8 which was stable at the plasma membrane, CD8 chimeras containing non-native TMDs that trafficked to the cell surface were rapidly internalised and sorted to lysosomes. This process was largely independent of the cytoplasmic domain of CD8, suggesting signals within the TMD induced internalisation of these CD8 chimeras. The proportion of the CD8 chimeras that trafficked to the plasma membrane, and the stability of the protein at the cell surface, was dependent upon the presence of polar residues within the TMDs, indicating that exposed polar residues in non-native TMDs may alter the handling of proteins at the Golgi and cell surface. Together, these results further our understanding of the mechanisms by which proteins containing aberrant transmembrane domains are handled at multiple subcellular compartments.

Transgenic (TG) sorghum genotypes with altered kafirin synthesis were developed by the Africa Biofortified Sorghum Project, employing recombinant DNA technology, with the aim of improving the protein nutritional quality of the grain. In this project, the effects of altered kafirin synthesis on kafirin polymerisation, protein quality, protein body structure and endosperm texture in different TG lines were investigated. The first generation of TG lines were in a type II low-tannin sorghum background. Altered synthesis of different major kafirin sub-classes (α-, γ- and σ-kafirin) was targeted. Some TG lines had improved lysine content (3.17 g/100 g protein) and moderate (55%) to high (74%) cooked in vitro protein digestibility, compared to the parent (2.05 g/100 g protein; 47.4%, respectively). This is of significance as tannins reduce protein digestibility, by complexing with the proline-rich kafirins. Transmission electron microscopy revealed that the improved protein quality traits were associated with floury endosperm texture and irregular protein body structure. Irregular protein bodies were 2-3 ìm in diameter, with few to numerous invaginations, compared to normal protein bodies. The high digestibility TG line also had a unique dense protein matrix, with occasional thick dark-staining inclusions. It appears that reduced kafirin synthesis, specifically γ-kafirin, has a major effect on the protein body structure, which in turn results in changes in protein digestibility and endosperm structure. To further improve the protein quality and poor endosperm texture of the first generation of TG lines, improved non-tannin sorghums were transformed to suppress kafirin synthesis, or they were back-crossed into TG lines with improved protein quality. Co-suppression of the α-, γ- and σ-kafirin sub-classes and removal of the tannin trait, resulted in TG with high cooked protein digestibility (±80%), improved Amino Acid Score (0.8) and Protein Digestibility Corrected Amino Acid Score (0.7) compared to the non-TG null controls (±50%, 0.4 and 0.2, respectively). However, these high-protein quality lines still had a floury endosperm texture. They also had irregular shaped protein body structure, as described previously. When fewer kafirin sub-classes were suppressed (only γ- and σ-kafirin) the endosperm was corneous with normal protein body structure, but the improvement in cooked protein digestibility was less. Apparently, co-suppression of several kafirin sub-classes is required to obtain high-protein quality sorghum, but this seems to result in floury-type grain endosperm. Further work conducted on the high digestible TG line revealed that the proportion of kafirin-1, extracted with 60% tert-butanol alone, was greatly increased. However, the total amount of kafirin remained unchanged. Also, the kafirin was much less polymerised by disulphide bonding, and there was evidence of compensatory synthesis of other kafirin proteins. Hence, the mechanism for the increased protein digestibility of TG lines is probably related to their lower levels of disulphide-bonded kafirins, allowing better access of proteases. This work appears to confirm that disulphide bond formation in kafirin is responsible for the reduced protein digestibility of cooked sorghum. Since grain hardness is an important grain quality attribute, playing a major agronomic role, in sorghum processing and in the end-use quality of sorghum-based foods, further research should focus on transforming sorghum to have both improved protein nutritional quality and good grain endosperm texture. / Thesis (PhD)--University of Pretoria, 2012. / Food Science / unrestricted

Protein is essential in one's diet because it is an important component in many organs and tissues throughout the body. Athletes ingest protein in order to stimulate protein synthesis and increase lean muscle mass. In order to assist with obtaining adequate amounts of protein, athletes and bodybuilders purchase supplemental protein in the form of protein powders. Protein metabolism and digestion play key roles in this because if the protein is not metabolized or digested effectively, then those who are wishing to gain fat free mass will not be successful. A high quality protein will be digested, metabolized, and directed towards lean tissue accretion more efficiently than a lower quality protein. In order to be a high quality protein, it must contain the essential amino acids. Fortunately, whey protein is a high quality protein because it contains an abundant supply of the essential amino acids. Whey protein is a high quality protein; hence, many athletes and physically active individuals purchase whey protein supplements. Some individuals do not care about taste and overcome awful protein powder taste, while others value a good tasting whey protein. After extensive research, it appears that scientific taste tests on protein supplements are lacking. The purpose of this study was to test some of the most popular protein supplements (Muscle Milk, BSN, Nesquik Vanilla Milk and Optimum Nutrition) and discover which one tasted the best. In this study, there were 94 males and 68 females. The results showed that there was a difference in initial taste and after taste in protein supplements among a male and female population. The difference among the drinks was statistically significant. The findings showed that both genders thought BSN and Muscle Milk were close to "neither good nor bad" while Nesquick Milk was rated as "good" and Optimum was "bad." The initial taste ratings were BSN (mean=4.05; SD=1.7), Muscle Milk (mean=4.6; SD=1.8), Nesquick Milk (mean=5.4; SD=1.2), and Optimum Nutrition (mean=3.1; SD=1.6). This research study showed that there was a statistically significant difference in taste among protein drinks, but the results do not answer as to why that is. Future research would need to be conducted in order to find the answer as to why there is a difference in initial and after taste.

No description available.