Theses and dissertations (Applied Sciences)
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Item Characterisation and application of bambara protein-polysaccharide complex coacervates in encapsulation of bioactive compounds(2019) Busu, Nyasha M.; Amonsou, Eric OscarBambara groundnut (Vigna subterranea) is a leguminous crop that is indigenous to Africa. In South Africa, the legume is cultivated in KZN, Limpopo and Mpumalanga where it is considered a traditional food. Bambara groundnut is a good source of protein (15 – 28 %) and contains substantial amounts of starch. The legume thrives well in areas of low agricultural input. Despite its good protein content, bambara groundnut is mostly cultivated in rural areas for by subsistence farmers. In recent years, there has been increased interest in bambara groundnut protein as an alternative protein source. The purpose of this study is to investigate the complexation behavior of bambara protein with gum Arabic and test the application of the formed complexes in encapsulation and delivery of bioactive compounds. In the first part of this study, four protein fractions extracted at different pH including the salt-solubilisation method were complexed with gum Arabic. The protein content as well as physicochemical properties (SDS-PAGE, FTIR, Zeta potential, SEM) of the protein fractions and resulting bambara protein-gum Arabic (BPI-GA) complexes were then investigated. In subsequent parts of the study, bambara protein extracted by the salt-solubilisation method was complexed with gum Arabic. The influence of ionic strength and biopolymer mixing ratio on complex formation was investigated. Subsequently, the emulsification properties, foaming properties, encapsulation efficiency and release properties of the formed complexes were also investigated under simulated gastric and intestinal pH conditions. The salt-soluble fraction showed the highest protein content (82%) whilst the lowest protein content (76%) was recorded at pH 2. The FTIR analyses revealed an increase in β-sheet content with decrease in pH of extraction. Complexation of the protein fractions with GA resulted in the optimal pHs of interaction shifting towards acidic regions (pHopt: 4.8 to 2.9) as pH of protein extraction became more acidic. Upon complexation, protein fractions produced coacervate yields ranging between 41 - 68%, with the pH 2 fraction recording the lowest (41%) yield. Further, addition of gum arabic seemed to broaden the turbidity profiles. When assessed by SEM, the particles appeared as spherical and aggregated structures between 100-200 nm.Item Utilization of shrimp waste for the recovery of valuable bioactive compounds(2018) Dlamini, Nosihle; Permaul, KugenShrimp waste is a major by-product of crustacean processing and represents an interesting source of bioactive molecules. In addition, its use increases the sustainability of processing fishery products. The present study reports a process developed for recovering bioactive molecules from shrimp waste through the use of chemical methods. The samples of shrimp were confirmed to be from the species Haliporoides triarthrus. The recovery of chitin was 30% of the processing waste and 30-60% chitosan (CH) from chitin. CH was characterized by FTIR analysis and exhibited a degree of deacetylation (DDA) of 72%. From the demineralization extract, CaCO3 was extracted and confirmed by FTIR. Based on a kinetic study of acid hydrolysis, it was demonstrated that chitin can be quantitatively hydrolysed into glucosamine (GlN), N-acetyl glucosamine (GlcNAc) and their respective oligomers with 32% hydrochloric acid at 60oC and qualitatively from CH with 32% hydrochloric acid at 80oC. The oligomer mixed fractions were desalted by activated charcoal extraction and the components of each fraction were analysed by TLC and HPLC. Chitooligosaccharides (COS) and N-acetly chitooligosaccharides (NAcCOS) with degrees of polymerization (DP) ranging from 2 to 6 were obtained from CH and chitin, respectively. The antimicrobial activities of chitosan, COS and NAcCOS were investigated against five gram-negative bacteria and five gram-positive bacteria. Chitosan exhibited stronger bacteriostatic effects against gram- positive bacteria than gram-negative bacteria in the presence of 1% chitosan. The oligomers showed no bacteriostatic or bactericidal effects on all tested bacteria. A total 30.74± 0.078 µg.g-1 astaxanthin was extracted with 90% acetone from the species; Haliporoides triarthrus and TLC analysis indicated that the species contained both astaxanthin and its esters. Chitosan films were obtained by solution casting of blends of chitosan with glycerol, polyethylene glycol 200 (PEG-200) and polyethylene glycol 600 (PEG-600) as plasticizers. Films were characterized by FTIR, XRD diffraction, TGA, and SEM analysis. The tensile strength and elongation at break properties of the films were also evaluated. CH films and CH/GLY blended films were translucent in appearance and the CH/PEG 200 and CH/PEG 600 films were opaque. The CH films yielded mechanically resistant films without the use of a plasticizer. These data point to the feasibility of an integrated process for isolating highly bioactive molecules, such as oligosaccharides, with a broad spectrum of applications from shrimp processing waste.Item Fabrication of electrochemical biosensors for the determination of phenolic compounds by experimental and computational methods(2018) Kunene, Kwanele Winterose; Bisetty, Krishna; Kanchi, SuvardhanThe polyphenolic compounds of interest, bisphenol A (BPA) and its analogue bisphenol S (BPS) used in the plastic industry to manufacture baby bottles and beverage containers, were used in this study. They are generally used in the manufacture of polycarbonates, epoxy resins and unsaturated polystyrene resins. There is a growing concern in the public and scientific community about these organic compounds due to their endocrine disrupting activity and negative toxic impact on the wildlife. This has encouraged scientists to embark on research to find a sensitive and selective technique that will adequately determine these compounds even in trace amounts. The experimental research strategy adopted in this work was supported by computational methods. This work was conducted in two stages; Firstly, a sensitive EC biosensor was developed using a carbon screen printed electrode fabricated with the combination of silver doped zinc oxide nanoparticles with multiwalled carbon nanotubes (MWCNTs) and laccase enzyme. The EC behaviour of BPA towards the fabricated biosensor was investigated using cyclic voltammetry and differential pulse voltammetry under optimum experimental conditions. Secondly, a novel and selective PEC sensor was developed for the first time to detect BPS based on the vertically aligned ZnO nanorods (ZnO NRs) with a molecularly imprinted polypyrrole (PPy). Amperometric, cyclic voltammetry and impedance spectroscopy were used for the investigation of the photo induced electrochemical behaviour of BPS. Different characterisation techniques such as ultra-violet visible spectroscopy, fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, photoluminescence, Raman spectroscopy, grazing incidence X-ray diffraction and diffuse reflectance were used to characterize the synthesized nanostructures. Results revealed that the fabricated EC and PEC sensors exhibited good catalytic activity towards the determination of BPA and BPS respectively, in samples extracted from plastic water bottles. For the EC method, a low detection limit of 0.08 μM for BPA in a linear range 0.5 to 2.99 µM was detected. However, in the case of BPS, a highly selective PEC method was attained linearly ranging from 2.5 to 12.5 µM with a much higher limit of detection of 0.7 μM. Experimental results were further supported computationally for a better understanding of the optical properties of ZnO NRs-polypyrrole complex. Computational results were in good agreement with experimental results.Item Composition and functional bioactive properties of bambara groundnut protein and hydrolysates(2016) Arise, Abimbola Kemisola; Amonsou, Eric Oscar; Ijabadeniyi, Oluwatosin AdemolaBambara groundnut (Vigna substerranea) is an indigenous legume of African origin which is currently experiencing a low level utilisation. It is tolerant to drought and can grow under poor soil conditions in which other lucrative crops such as groundnut cannot grow. Bambara is a good source of protein comparable to that of cowpea and slightly lower than soya bean. In order to assess the potential use of bambara protein as a functional ingredient in food systems and as an important ingredient for the formulation of therapeutic product, the knowledge of its protein composition, structure and functionality becomes important. The main goal of this thesis was to determine the composition and bioactive properties of bambara protein and its hydrolysates. Specifically, a comparative study was carried out on the protein content, yield and functional properties of protein concentrates prepared from three different bambara landraces using different extraction methods (Salt solubilisation and Acid precipitation). There was no significant difference in protein content, yield and functional properties of the landraces. However, the method of extraction had an influence on their physicochemical and functional properties. Acid precipitation produced bambara protein concentrates with high protein content and yield (79% and 52% respectively) when compared to salt solubilisation (protein content - 57% and yield - 25%). Protein concentrates prepared through salt solubilisation method exhibited better functional properties in terms of water absorption capacity, oil absorption capacity, foaming capacity, foaming stability and emulsion activities when compared to concentrates obtained through acid precipitation. Furthermore, the composition of bambara proteins produced through isoelectric precipitation was determined. SDS PAGE revealed four major bands; a broad band at 55 kDa which was analysed to be vicilin, two medium bands at 62 kDa and 80 kDa and a high molecular weight (HMW) protein at 141 kDa. Further investigation of bambara protein revealed vicilin (55 kDa) with two sub units as the major protein in bambara and this was also confirmed by the proteomic map. The proteomic map revealed acidic amino acids as the major protein of bambara which is characteristic of vicilin, the map also showed that there were differences in the number of spots across the landraces with 77 spots matching each other. Circular dichroism spectroscopy exhibited reductions in α-helix, and β-pleated sheet conformations as pH varies. In addition, the tertiary structures as observed from the near-UV CD spectra were also influenced by shifts in pH conditions. Differential scanning calorimetry thermograms showed two endothermic peaks at around 67 and 81oC respectively. These can be attributed to thermal denaturation of vicilin and the HMW protein. Subsequent studies used isolates from red bambara since the composition of the landraces were similar. Bambara protein isolate was subjected to enzymatic hydrolysis using three proteases (alcalase, pepsin and trypsin) to produce various bambara protein hydrolysates (BPHs). BPHs were investigated for antioxidant and antihypertensive activities. The in vitro structural and functional characteristics of bambara protein and its enzymatic protein hydrolysate revealed that bambara groundnut possessed antioxidant properties against a variety of physiologically relevant free radicals. High surface hydrophobicity and the molecular size of the peptide seem to be important for scavenging of hydroxyl radicals, ferric reducing power and metal chelation. BPHs and peptide fractions were able to scavenge DPPH radicals with greater affinity for smaller size. Less than 1 and 1-3 kDa pepsin fraction was able to scavenge DPPH radical more than glutathione, BPHs and its fractions scavenge ABTS•+ three folds than the isolate. Scavenging of superoxide radicals was generally weak except for 5-10 kDa peptide fractions. All BPHs inhibited linolenic acid oxidation with greater affinity for the lower molecular size peptide. BPHs showed potential antihypertensive properties because of the in vitro inhibition of activities of angiotensin converting enzyme (ACE) and renin inhibition. The molecular size had significant effect on the ACE inhibitory properties with low molecular weight peptide (<1 kDa) fractions exhibiting significantly higher (p<0.05) inhibitory activities. However, enzyme type had synergistic effects on renin inhibition with alcalase hydrolysate showing highest inhibition at 59% when compared to other hydrolysates and their membrane fractions. The fractions with <1 and 1-3 kDa peptides showed a higher potential as antihypertensive and antioxidant peptides. Based on this study, incorporation of bambara protein isolate as an ingredient may be useful for the manufacture of high quality food products. Likewise, the bambara protein hydrolysates, especially the <1 kDa and 1-3 kDa fraction represent a potential source of bioactive peptides in formulating functional foods and nutraceuticals.