Thesis (Ph. D. (Plant Production)) -- University of Limpopo,2016 / Refer to document / University of Limpopo, The Technology Innovation Agency (TIA), The Land Bank Chair of Agriculture ─ University of Limpopo, The Flemish Interuniversity Council (VLIR) and, The Agricultural Research Council - University Collaboration Centre

A thermophilic fungus was isolated from composted horse manure. The organism was as a Chaetomium sp. by sequencing the highly conserved ITS region of the fungus and comparing to known regions in a genomic database and was referred to as TM-417. TM-417 was found to have an optimal growth temperature of 45 oC and an optimal pH of 7.0. An extracellular DNase and RNase was found to be produced by the isolate and were purified 145.58-fold and 127.6-fold respectively using a combination of size exclusion chromatography and a novel affinity membrane purification system. The extent of purification was determined electrophoretically using 4-15% gradient polyacrylamide gels. Both DNase and RNase were dependent on metal co-factors for activity. The metal ion Mg2+ was the preferred ion for the DNase, whereas for the RNase, Zn2+ and Mn2+ yielded an increase in enzyme activity over that with Mg2+. The purified DNase demonstrated maximum activity at pH 6.0 with no activity at pH 2.0 or 10.0. The RNase exhibited two peaks of maximum activity, on at pH 3.0 and the other at pH 7.0 with no activity at pH 2.0 or 10.0. The optimal temperature for the purified DNase was 65oC. The optimal temperature for the RNase was 70oC. The molecular of the DNase and RNase were determined to be 56 kDa and 69kDa respectively using a Sephadex G-75 column. A standard curve was generated using several standard proteins of known molecular weight.

DEVELOPMENT AND VALIDATION OF A LIGHT WEIGHT, ENERGY DENSE, READY TO EAT (RTE) BAR Jacob Wilhelm-Maria Heick Providing additional calories in the form of an RTE bar to endurance athletes will increase performance and muscle re-synthesis, reduce muscle breakdown, and shorten recovery time. An RTE bar containing a blend of dairy proteins and carbohydrates will create a product with superior functionality, including bioactive and immunity enhancing properties from dairy derived ingredients. The protein will provide benefits in the form of easily digestible calories, essential amino acids and physical satiate. A formulation was developed and optimized, resulting in a final product that meets the required nutritional profile: 400kcal, 25grams protein per 100 gram serving size. The desired physical characteristics were achieved through processing by both conventional baking and freeze drying. The latter method improves the stability and functionality of the RTE bar. In order to meet the protein requirements of the RTE bar without compromising sensory properties, a unique protein source was developed. Using high concentrations of conventional protein sources like Whey Protein Concentrate (WPC) resulted in stale off-flavors and unappealing textures. Milk Protein Precipitate (MPP) was developed for this formulation. MPP is a curd-like ingredient created through the combined heat and acid precipitation of dairy proteins. MPP can be used effectively in high concentrations provides a subtle dairy flavor. MPP delivers a balance of casein and whey, similar to that found in milk. The effectiveness of the RTE bar formulation as a post exercise recovery food was evaluated in a human studies experiment conducted on the Cal Poly campus. The human subjects study utilized 34 Cal Poly students in a single-blind cross-over design experiment. The study compared the effects of this high protein RTE bar against a calorically equal carbohydrate bar. The bars were administered after subjects completed the pre-assigned hikes on three consecutive days. Following the cross-over design, subjects received the alternate bar in the second period of the experiment. Several blood markers involved in metabolism and inflammation were measured before and after the two treatment periods. No blood marker showed a statistically significant difference between bars, but several trends were observed. Body weight and fat percent were also unaffected by bar composition.

Epigallocatechin gallate (EGCG), also known as epigallocatechin-3-gallate, is a polyphenol that is most abundant in tea. It has been shown from many studies that consumption of EGCG can contribute to weight loss, however, the underlying mechanism is not fully understood. To determine how EGCG acts to reduce fat, an organism model Caenorhabditis elegans (C. elegans) is introduced, which is a useful animal system in exploring crucial biological mechanisms that are readily applicable to humans. In this study, different strains were raised for two days on a diet with or without 100µM and 200µM EGCG treatment: N2 (i.e., wild type) and mutants (i.e., knockdown of fat metabolism related genes). EGCG’s effect on fat reduction was characterized by triglyceride content, food consumption and physiological behaviors. Our results showed that 100 and 200 µM EGCG significantly reduced the triglyceride content of wild type worms by 10% and 20%, respectively, without affecting its food intake and physiological behaviors. Additionally, EGCG could effectively reduce fat accumulation in C. elegans dependent on acs-2 and atgl-1.

A flavonol specific glucosyltransferase cloned from Citrus paradisi has strict substrate and regio-specificity (Cp3OGT). The amino acid sequence of Cp3OGT was aligned with sequences of an anthocyanidin UDP- dependant glucosyltransferase (UGT) from Clitorea ternatea and a UGT from Vitis vinifera that can glucosylate both flavonols and anthocyanidins. Using homology modeling to identify candidate regions followed by site directed mutagenesis, three double mutations were constructed and biochemically characterized. S20G+T21S mutant protein retained activity with flavonols similar to the wildtype Cp3OGT but the mutant had optimum activity at 60°C and broadened substrate acceptance to include the flavanone naringenin. S290C+S319A mutant protein retained 40% activity with quercetin relative to WT, and had an optimum pH shift. H154Y+Q87I mutant protein was only 10% active with quercetin relative to WT. Docking analysis revealed that H154, Q87 and S20 could be involved in orienting the acceptor molecules within the acceptor binding site whereas S319 and S290 residues are involved in maintaining the active site conformation.

Amidos nativos apresentam algumas desvantagens tecnológicas que impulsionam pesquisas voltadas à modificação, visando melhorar as propriedades físico-químicas e promover propriedades funcionais específicas. Modificações como dextrinização e esterificação estão entre as mais utilizadas, porém, apesar dos avanços recentes na área de amidos modificados, ainda há pouca informação sobre as propriedades de dextrinas esterificadas. Assim, o presente estudo teve como objetivos principais a obtenção e caracterização de dextrinas esterificadas com ácido málico a partir dos amidos de mandioca e milho. A modificação dos amidos foi realizada com ácido málico (2,5; 5,0; 7,5 % m/m) durante o processamento térmico (150 °C/ 4 horas) com baixa umidade (≤ 3 %). Para avaliar o efeito de um catalisador ácido, foram realizados ensaios com a adição de HCl (0,05 % m/m), sob as mesmas condições. A termoconversão do amido na presença de ácido málico produz dextrinas esterificadas com baixo grau de substituição (GS) e características aniônicas, além de alta claridade de pasta, solubilidade a frio e baixa viscosidade em elevados teores de sólidos (RVA). A degradação macromolecular reduz o teor de amilose dos amidos após a modificação, assim como a viscosidade intrínseca. Porém, as dextrinas com maior GS apresentaram maior viscosidade intrínseca em solvente DMSO, o que sugere a ocorrência de ligações cruzadas nas dextrinas com maior concentração de ácido málico. Os resultados de dextrose equivalente (DE) também foram dependentes da concentração de ácido málico e os valores variaram de 6,89 a 11,33% nas dextrinas de mandioca e de 8,56 a 12,38 % nas dextrinas de milho. A variação de cor identificou maior tendência a tonalidades amareladas, característica típica de dextrinas termoconvertidas. Na análise de DRX, foi observado que a modificação alterou o padrão de difração das amostras de amido de mandioca e diminuiu a cristalinidade relativa (CR) dos grânulos dos amidos de ambas as fontes botânicas estudadas. A morfologia dos grânulos, analisada por MEV, também foi alterada em ambas as fontes de amido estudadas, assim como suas propriedades térmicas, de maneira que não foi observada transição de fase referente à gelatinização em análise de DSC. / Native starches have some technological disadvantages which stimulate researches for modification, aimed improve physicochemical properties and promote specific functional properties. Modifications as dextrinization and esterification are widely used, however, despite recent advances about modified starches, there is still little information about the properties of esterified dextrins. The aim of this study was to prepare and characterize esterified dextrins from cassava and maize starches using malic acid. The modification of starches was performed by heating a dried mixture (≤ 3%) of starch and malic acid (2.5; 5.0; 7.5 % w/w). To evaluate the effect of an acid catalyst, tests were prepared with addition of HCl (0.05% w/w) under the same conditions. The starch pyrodextrinization in presence of malic acid produces esterified dextrins with low degree of substitution (DS) and anionic properties, as well as high clarity of paste, cold solubility and low viscosity at high solids contents. The macromolecular degradation decreases the amylose content values after modification, as well as the intrinsic viscosity values. However, the dextrins with high GS showed higher intrinsic viscosity in DMSO, which suggests the occurrence of crosslinking in the dextrins with a higher concentration of malic acid. The results of dextrose equivalent (DE) were dependent of malic acid concentration and values ranged from 6.69 to 11.33% in dextrins from cassava starch, and 8.56 to 12.38% in dextrins from maize starch. Color variations indicated its direct dependence on pyrodextrinization conditions and showed trends to yellow tones, typical feature of pyrodextrin. In X-ray diffraction, was observed that modification changed x-ray pattern in dextrins from cassava starch and decreases the relative crystallinity (RC) of the granules from cassava and maize starches, and its granular morphology (SEM) and thermal properties were affected, it was not observed phase transition related to the gelatinization in DSC analysis.

Amidos nativos apresentam algumas desvantagens tecnológicas que impulsionam pesquisas voltadas à modificação, visando melhorar as propriedades físico-químicas e promover propriedades funcionais específicas. Modificações como dextrinização e esterificação estão entre as mais utilizadas, porém, apesar dos avanços recentes na área de amidos modificados, ainda há pouca informação sobre as propriedades de dextrinas esterificadas. Assim, o presente estudo teve como objetivos principais a obtenção e caracterização de dextrinas esterificadas com ácido málico a partir dos amidos de mandioca e milho. A modificação dos amidos foi realizada com ácido málico (2,5; 5,0; 7,5 % m/m) durante o processamento térmico (150 °C/ 4 horas) com baixa umidade (≤ 3 %). Para avaliar o efeito de um catalisador ácido, foram realizados ensaios com a adição de HCl (0,05 % m/m), sob as mesmas condições. A termoconversão do amido na presença de ácido málico produz dextrinas esterificadas com baixo grau de substituição (GS) e características aniônicas, além de alta claridade de pasta, solubilidade a frio e baixa viscosidade em elevados teores de sólidos (RVA). A degradação macromolecular reduz o teor de amilose dos amidos após a modificação, assim como a viscosidade intrínseca. Porém, as dextrinas com maior GS apresentaram maior viscosidade intrínseca em solvente DMSO, o que sugere a ocorrência de ligações cruzadas nas dextrinas com maior concentração de ácido málico. Os resultados de dextrose equivalente (DE) também foram dependentes da concentração de ácido málico e os valores variaram de 6,89 a 11,33% nas dextrinas de mandioca e de 8,56 a 12,38 % nas dextrinas de milho. A variação de cor identificou maior tendência a tonalidades amareladas, característica típica de dextrinas termoconvertidas. Na análise de DRX, foi observado que a modificação alterou o padrão de difração das amostras de amido de mandioca e diminuiu a cristalinidade relativa (CR) dos grânulos dos amidos de ambas as fontes botânicas estudadas. A morfologia dos grânulos, analisada por MEV, também foi alterada em ambas as fontes de amido estudadas, assim como suas propriedades térmicas, de maneira que não foi observada transição de fase referente à gelatinização em análise de DSC. / Native starches have some technological disadvantages which stimulate researches for modification, aimed improve physicochemical properties and promote specific functional properties. Modifications as dextrinization and esterification are widely used, however, despite recent advances about modified starches, there is still little information about the properties of esterified dextrins. The aim of this study was to prepare and characterize esterified dextrins from cassava and maize starches using malic acid. The modification of starches was performed by heating a dried mixture (≤ 3%) of starch and malic acid (2.5; 5.0; 7.5 % w/w). To evaluate the effect of an acid catalyst, tests were prepared with addition of HCl (0.05% w/w) under the same conditions. The starch pyrodextrinization in presence of malic acid produces esterified dextrins with low degree of substitution (DS) and anionic properties, as well as high clarity of paste, cold solubility and low viscosity at high solids contents. The macromolecular degradation decreases the amylose content values after modification, as well as the intrinsic viscosity values. However, the dextrins with high GS showed higher intrinsic viscosity in DMSO, which suggests the occurrence of crosslinking in the dextrins with a higher concentration of malic acid. The results of dextrose equivalent (DE) were dependent of malic acid concentration and values ranged from 6.69 to 11.33% in dextrins from cassava starch, and 8.56 to 12.38% in dextrins from maize starch. Color variations indicated its direct dependence on pyrodextrinization conditions and showed trends to yellow tones, typical feature of pyrodextrin. In X-ray diffraction, was observed that modification changed x-ray pattern in dextrins from cassava starch and decreases the relative crystallinity (RC) of the granules from cassava and maize starches, and its granular morphology (SEM) and thermal properties were affected, it was not observed phase transition related to the gelatinization in DSC analysis.

The increasing market demand for fresh produce promotes a keen interest in developing a rapid, sensitive and reliable method for monitoring plant health and determining the shelf-life of postharvest produce. The objective of this study is to explore the capability of Surface-enhanced Raman spectroscopy (SERS) in these applications. SERS integrates Raman spectroscopy which measures molecular vibrations and nanotechnology which enhances the weak Raman signals. Herein, we developed two SERS methods based on a surface detection approach using nanoparticles solution and a headspace detection approach using gold nanoparticles (AuNPs) fibers, to detect biochemical changes during postharvest storage of arugula leaves. Compared with surface detection, the headspace detection revealed significant spectral changes during the storage, particularly in the shifts around 500, 950 and 1030 cm-1. These changes analyzed using principal component analysis (PCA) to establish a prediction model for shelf-life determination. Through analyzing reference standard compounds, we identified the dimethyl disulfide (DMDS), 1-propanethiol and methanethiol (MT) were most likely to account for the signature spectra of headspace arugula at the late storage period due to the activities of spoilage bacteria. The headspace detection method was also applied to monitor the stress responses of living basil to abiotic stresses (pesticide/salinity). However, the volatile analysis of the basil plants response to abiotic stresses (pesticide/salinity) showed indistinctive results. In conclusion, the headspace detection based on SERS provides a new strategy for quality monitoring of fresh produce in the food industry.

Cholesterol is an important biological molecule with many important functions. However, high serum cholesterol is a health hazard. Thus analysis of cholesterol is important and many analytical techniques have been developed. The objectives of the proposed research are to develop an economical, rapid method for the determination of total cholesterol with good selectivity and enhanced sensitivity. For evaluation of proposed flow injection analysis with peroxyoxalate chemiluminescence (FIA-POCL) method, figures of merit such as accuracy, precision, and linear dynamic range will be assessed. The proposed procedure was then applied to the determination of total cholesterol in foods. The procedure was linear for cholesterol from 0.01 to 0.120 mg/mL. The relative standard deviation was 2.57%. The recoveries were 97.5-103.3% for commercial standard cholesterol sample, and 101.5-108.0% for butter. The proposed method was applied to analysis of cholesterol in food and the results were consistent with expected values.

Abstract There are eight foods that contribute to food allergies in the western world and peanut is the most common. Currently, there are no medical treatments that can cure an individual of food allergy, so avoidance of the allergic food is the only option. In the United States, there are three immunodominant allergic proteins accountable for patient sensitization to peanut, Arachis hypogea 1, 2, and 3 (Ara h 1, Ara h 2, Ara h 3). Therefore, research into why peanuts are more allergic than other foods that have homologous proteins is critical and may be obtained by studying the structural and allergenic properties of individual allergens and the changes that occur due to food processing. In this study, the basic and acidic subunits of Ara h 3 were cloned, expressed, and purified, and compared with each other and with the native Ara h 3 purified from peanut for differences in binding to IgE from peanut allergic individuals. Also, an in vitro Maillard reaction was performed on purified native raw Ara h 3 and patient serum IgE western blots were performed. This study concluded that an in vitro Maillard reaction enhanced IgE binding to Ara h 3, IgE binding to native Ara h 3 was in most cases higher than to the recombinant Ara h 3 subunits, and recognition of the acidic subunit was much higher than the and basic subunits in both the recombinant and native forms of the protein were investigated. Keywords: