Theses and dissertations (Applied Sciences)
Permanent URI for this collectionhttp://ir-dev.dut.ac.za/handle/10321/6
Browse
11 results
Search Results
Item Development of electrochemical sensors for the detection of mycotoxins in food matrices using functionalised nanocomposites(2024-05) Naidoo, Lyndon; Bisetty, Krishna; Meier, Florian; Uwaya, Gloria EbubeThe analysis of pathogens in foods is of critical importance to ensure consumer safety and quality assurance, as contaminants pose serious risks to public health. Mycotoxins are naturally occurring carcinogenic toxins that arise from specific strains of fungi as they contaminate food. They are found in a wide variety of grains, cereals, and dairy products, causing cancer in both humans and animals. Thus, there is a growing demand for simple, sensitive and inexpensive sensors for mycotoxin detection in lieu of conventionally employed large-scale instrumentation. In this study, the development of electrochemical sensors for the detection of aflatoxin B1 (AFB1), zearalenone (ZEN) and ochratoxin A (OTA) in foods was investigated and presented as three case studies, respectively. In the first case study, an ultrasensitive aptasensor was developed for the indirect detection of AFB1 in the presence of a ferri/ferrocyanide ([Fe(CN)6]3-/4-) redox probe solution. The sensor was constructed by immobilizing an anti-AFB1 aptamer (Apt) to a carboxylated multiwalled carbon nanotube (cMWCNT) and iron oxide (Fe3O4) nanoparticle (NP) composite using a glassy carbon electrode (GCE). This resulted in the development of the GCE/cMWCNTsFe3O4 NP/Apt sensor. An electrochemical response was exhibited from AFB1 applying cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV), respectively, utilizing a [Fe(CN)6]3-/4- redox probe prepared in phosphatebuffered saline (PBS) solution with reference to the Ag/AgCl reference electrode under optimized conditions. DPV findings reported very low limits of detection (LOD) and quantification (LOQ) of 0.43 fg mL-1 and 1.44 fg mL-1 respectively in comparison to current literature, over a calibration range of 0.50 fg mL-1 to 5.00 fg mL-1. For real sample analysis, excellent spike recoveries from 95% to 105% were obtained for corn and rice flour. Density functional theory (DFT) was used to propose a reaction scheme by ascertaining the electronic properties of the redox-active functional groups of AFB1. This supported the experimental anodic response findings of DPV. The second case study focused on how PEGylated Fe3O4 NPs and cMWCNTs fabricated on a GCE could be used for the design of an electrochemical sensor for ZEN analysis. The qualitative and quantitative analyses of ZEN were completed using CV, EIS and DPV, respectively, under optimized conditions in a sodium phosphate buffer solution. The developed sensor reported significantly low LODs and LOQs of 0.34 fg mL-1 and 1.12 fg mL-1 respectively, over a calibration range of 1.00 fg mL-1 to 10.00 fg mL-1 by DPV. Excellent spike recoveries ranging from 92% to 106% were obtained for rice and corn flour. The Monte Carlo (MC) adsorption simulation studies predicted the strong interaction of ZEN with the constructed sensor. In the final case study, an OTA electrochemical sensor was designed using a nickel metalorganic framework (Ni-MOF) and carboxylated reduced graphene oxide (cRGO) on a GCE. The detection of OTA was achieved under optimized conditions in PBS solution with the developed GCE/Ni-MOF/cRGO electrode, employing CV, EIS and DPV as electrochemical tools. Applying DPV, the sensor reported very low LODs and LOQs of 3.29 fg mL-1 and 10.97 fg mL-1 respectively, over a calibration range of 10.00 fg mL-1 to 90.00 fg mL-1. Regarding real sample analysis, excellent spike recoveries from 95% to 105% were obtained for corn and rice flour. Molecular dynamics (MD) studies predicted that the Ni-MOF exhibited a strong electrostatic interaction with the OTA analyte, in agreement with the experimental findings. The synthesized nanomaterials (cMWCNTs-Fe3O4 NP, PEG-Fe3O4 NPs/cMWCNTs, and NiMOF/cRGO) utilized in this study were characterized by an array of techniques, including single particle inductively coupled plasma-mass spectrometry (spICP-MS), transmission electron microscopy (TEM), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDS), scanning electron microscopy (SEM), multidetector asymmetrical flow field-flow fractionation (AF4), and Fourier transform infrared spectroscopy (FTIR). Finally, computational modelling studies were undertaken to establish a synergy with the experimental approaches employed in each case study. These methodologies included DFT, docking studies, MC adsorption and MD simulations, which were aimed at predicting and assessing the atomic and molecular interactions between the mycotoxins and their respective electrode systems.Item Electrochemical aptasensor for the detection of mycotoxins in food samples by experimental and computational methods(2021) Kunene, Kwanele; Bisetty, Krishna; Kanchi, Suvardhan; Sabela, Myalowenkosi InnocentMycotoxins are secondary metabolites of fungi that are present in various foodstuff and feed commodities. A large number of mycotoxins exist, however only a limited number represent a major damages and toxic properties. Amongst them, the aflatoxins and ochratoxins are deemed to be the most poisonous and extensively circulated in the world and then, represent a real hazard to both human and animal. Depending on several factors like the consumption levels, exposure time, mechanisms of action, digestion and defense mechanisms, mycotoxins stimulate a wide spectrum of toxicological effects leading to both acute and chronic diseases, liver and kidney failure, skin rash, cancer, immune suppression, birth defects or even death. To address the harmful impact of mycotoxin contaminants in food and feed, health authorities in various countries world-wide have established guidelines in order to protect human and animal from the possible damages instigated by these toxins. Authorities such as the European Commission, US Food and Drug Administration (FDA), World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO) set up maximum level regulations for main mycotoxins in foods and feeds. To accomplish the expectations of these regulation levels, there is a great need for the development and validation of modern, uncomplicated, rapid, and detailed methodologies for the detection of toxins. In this study, various approaches for the rapid, inexpensive and ultrasensitive biosensors for the detection of two major mycotoxins were developed. The electrochemical-based aptasensor and immunosensor were developed for the determination of aflatoxin B1 (AFB1) and ochratoxin A (OTA) in different food products. The fabricated biosensors demonstrated good practical analytical feasibility for mycotoxins detection in real samples such as WeetBix, yoghurt, coffee and in wine samples with excellent recoveries and RSD values. To avoid fouling on the sensor surface by the constituents present in real samples, the carbon screen printed electrode (C-SPE) and carbon felt electrode (CFE) surfaces were modified with different nanomaterials such as silver nanoparticle (AgNPs), palladium nanoparticles (PdNPs), palladium doped boron nitride (PdNPs-BN) and titanium nanoparticles doped with boron nitride BN-TiO2. In addition, the aptamers and antibodies were immobilized on the modified electrode in order to enhance the selectivity of the sensor towards the detection of OTA and AFB1. The electrochemical aptasensor for OTA permitted for highly sensitive detection in Weet-Bix with a wide linear range (0.002 - 0.016 mg L-1) and limit of detection of 7×10-4 mg L-1. It is worth prominence that it is the first time that carbon screen printed electrode (C-SPE) modified with AgNPs was used, opening new pathways for highly precise analysis. Experimental results were further supported computationally for a better understanding of the interaction between the aptamer and the analytes. Computational results were in good agreement with experimental results. The same procedure was also established in voltammetric detection of AFB1 using CFE modified with BN-TiO2 (CF/BN-TiO2). A wide concentration range of 2.5 - 20 ng mL-1 with an excellent LOD of 0.002 ng mL-1 for AFB1 was obtained. For the case study of wine samples tested for AFB1 detection, a simple but very effective pretreatment method was effectively applied. The addition of acetonitrile to the wine reduces the non-specific interactions that might be accountable for inactivation of antibody and blocking of the sensor surface. Furthermore, the PdNPs-BN enhanced the electrical signal and the sensor sensitivity. Attained results allowed for AFB1 detection at concentrations range from 1.0 - 10 ng mL-1 with limit of detection of 0.832 ng mL-1 . In the case study of the electrochemical immunosensor for the detection of OTA in coffee, a linear detection range of 0.5 - 20 ng mL-1 was achieved with LOD of 0.096 ng mL-1 . The fabricated aptasensors and immunosensors in this study combines the most desirable characteristics of a good biosensor such as high sensitivity, inexpensive, rapid, and simple but portable method make proposed approaches an important and very promising tools for extensive biosensing applications.Item Incidence of mycotoxigenic fungi during processing and storage of bambara groundnut (Vigna subterranea) composite flour(2019) Olagunju, Omotola Folake; Ijabadeniyi, Oluwatosin Ademola; Mchunu, Nokuthula PeaceFungal contamination of food commodities is a global food security challenge that impacts negatively on the health of consumers. Mycotoxins are produced as secondary metabolites by some pathogenic fungi and may contaminate agricultural products while on the field or during harvesting and storage. Processing operations and storage conditions of temperature and relative humidity have marked effect on the ability of fungal pathogens to grow and produce mycotoxins in agricultural food commodities. The consumption of mycotoxin- contaminated foods, even at low doses over a prolonged period of time, may have deleterious effects on health of consumers. Bambara groundnut (Vigna subterranea (L.) Verdc) is an African legume gaining wide acceptance in various food applications due to its favourable nutritional composition, especially the high protein content. In several parts of Africa, it is used as a supplement in cereal-based foods, especially in weaning food for infants and young children. Bambara groundnut grows near or under the soil, which serves as inoculum of pathogenic fungi. Very little information is presently available on fungal and mycotoxin contamination of Bambara groundnut from Southern Africa. Hence, its safety for consumption from a mycological standpoint requires further studies. To establish the profiling of fungal contaminants in food commodities consumed in Durban, South Africa, 110 samples of regularly consumed food samples which included rice (23), spices (38), maize and maize-derived products (32), and Bambara groundnut (17) were randomly collected over a period of five months from retail stores and open markets. The food samples were screened for fungal contamination using conventional and molecular methods. Fungal isolates were characterized following DNA extraction, polymerase chain reaction and sequencing. Using a modified QuEChERS method, the detection and quantification of mycotoxins in Bambara groundnut was performed via Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS), and isolation and detection of the causative pathogen was carried out. The effect of processing operations of milling, a combination of roasting and milling, and spontaneous fermentation on the survival of the natural fungal population of Bambara groundnut, and aflatoxin production under simulated tropical conditions of storage was further studied. Processed Bambara groundnut flour samples were stored at temperature of 30±1 °C and 85±2% relative humidity for 30 days, vi and samples withdrawn at 5-day intervals for analyses, i.e., fungal counts, aflatoxin accumulation and changes in water activity during storage. Following the detection of aflatoxins in Bambara groundnut flour and the isolation of aflatoxigenic Aspergillus flavus in the seed, the effect of milling, roasting and milling, or lactic acid bacteria fermentation on the survival, growth and aflatoxin production of A. flavus in Bambara groundnut flour was studied. Irradiated seeds of Bambara groundnut were artificially inoculated with a 3-strain cocktail of A. flavus (2 x 106 spores/mL) and processed by milling, roasting at 140 °C for 20 min and milling. Slurries of irradiated Bambara groundnut flour were also inoculated with A. flavus spores and 1 x 108 CFU/mL inoculum of Lactobacillus fermentum or Lactobacillus plantarum. All inoculated samples were incubated at 25 °C for 96 h, samples withdrawn every 24 h were analyzed for viable A. flavus counts, changes in water activity during incubation, and aflatoxin production using Enzyme- linked Immunosorbent Assay (ELISA). Bambara groundnut flour samples fermented with lactic acid bacteria were further analyzed for pH, total titratable acidity, and viable lactic acid bacteria counts over the incubation period. The degradation of aflatoxin (AF) B1 by both lactic acid bacteria was also studied. Slurries of irradiated Bambara groundnut flour were spiked with 5 µg/kg of aflatoxin B1 (AFB1) and the percentage reduction over the incubation period was determined using HPLC. The survival, growth and aflatoxin production of A. flavus in Bambara groundnut and maize- composite flours as affected by milling, roasting and milling or lactic acid bacteria fermentation during storage was also studied. Processed and irradiated Bambara groundnut flour, maize flour and maize-bambara composite flour (70:30) were inoculated with 2 x 107 spores/ml of A. flavus and stored for up to 10 weeks at a temperature of 25±2 °C and relative humidity of 75±2%. Samples were withdrawn weekly and analyzed for viable populations of A. flavus, concentrations of aflatoxins B1, B2, G1 and G2, changes in pH and water activity over the storage period. The colonization of Bambara groundnut by A. flavus and the effects of fungal infection on the seed coat, storage cells and tissue structures were also studied. Irradiated Bambara groundnut seeds were artificially inoculated with spore suspension of aflatoxigenic A. flavus (2 x 106 spores/mL) and stored at a temperature of 25±2 °C and relative humidity of 75±2% for 14 days. Samples were withdrawn at 24 h intervals for 4 days, then at 7 and 14 days and examined using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Various fungal genera were isolated from the food samples under study with Aspergillus (52.5%) and Penicillium (31.8%) as the dominant genera. All the 110 food samples were contaminated with more than one fungal species. A. flavus and other Aspergilli, Penicillium citrinum and Fusarium oxysporum were isolated from Bambara groundnut seeds. Aflatoxigenic A. flavus was isolated from Bambara groundnut seed, with a co-occurrence of Aflatoxin (AF) B1 (0.13–6.90 µg/kg), AFB2 (0.14–2.90 µg/kg), AFG1 (1.38–4.60 µg/kg), and AFG2 (0.15–1.00 µg/kg) in the flour. The fungal counts of the samples during storage significantly (p≤0.05) increased, irrespective of the processing method from 6.3 Log10 CFU/g in Bambara groundnut flour to 6.55 Log10 CFU/g in fermented Bambara groundnut flour. Aflatoxin concentration was affected markedly by the processing methods in Bambara groundnut flour (0.13 µg/kg) and fermented Bambara groundnut flour (0.43 µg/kg), aflatoxin was not detected in roasted Bambara groundnut flour. The survival and growth of A. flavus was also markedly affected by lactic acid bacteria fermentation and roasting during incubation. Within 24 h of fermentation with L. fermentum, significant (p≤0.05) changes were recorded in viable population of A. flavus (6.30‒5.59 Log10 CFU/mL), lactic acid bacteria count (8.54‒13.03 Log10 CFU/mL), pH (6.19‒4.12), total titratable acidity (0.77‒1.87%) and a reduction by 89.2% in aflatoxin B1 concentration. Similar significant changes were recorded in Bambara groundnut flour fermented with L. plantarum. Aspergillus flavus in the artificially contaminated seeds were completely eliminated by roasting. Aflatoxin production was not detected in Bambara groundnut flour samples over the incubation period. During storage for 10 weeks, the population of A. flavus significantly (p≤0.05) decreased in roasted Bambara groundnut flour from 7.18 to 2.00 Log10 CFU/g. Similar significant (p≤0.05) decrease in A. flavus viable counts was recorded in fermented Bambara groundnut flour from 6.72 to 2.67 Log10 CFU/g, however after 7 weeks of storage and beyond, A. flavus was not detected. Significant (p≤0.05) decrease in aflatoxin B1 (0.36‒0.26 µg/kg) and aflatoxin G1 (0.15‒0.07 µg/kg) accumulation was also recorded in roasted Bambara groundnut flour. While A. flavus viable population significantly (p≤0.05) decreased in maize-Bambara composite flour from 6.90 to 6.72 Log10 CFU/g, aflatoxin B1 accumulation significantly (p≤0.05) increased from 1.17 to 2.05 µg/kg. Microscopy studies showed that the seed coat of Bambara groundnut was rapidly colonized by A. flavus within 24 h of inoculation. The infection of internal tissues of the cotyledon was through the ruptured seed coat, resulting in a disruption of the cellular architecture. Cell wall collapse, development of cavities in parenchymatous cells and ruptured storage cells resulted from A. flavus infection of the seed. This study reports a high prevalence of fungal contamination in some food commodities consumed in Durban, South Africa. The isolation of live mycotoxin-producing fungi from the food commodities necessitates the need for regular routine checks to ensure the mycological safety of agricultural products offered for sale to consumers. The detection of aflatoxigenic A. flavus and aflatoxins in Bambara groundnut flour at levels above the maximum tolerable limits raises health concerns on its utilization in food applications, and in supplementary feeding for infants and young children. Although roasting was effective in degradation of aflatoxins in Bambara groundnut seeds, elimination of fungal contaminants was not achievable which resulted in continued production of aflatoxin during storage. Fermentation using L. fermentum or L. plantarum is effective in eliminating A. flavus and degrading AFB1 in Bambara groundnut flour. Compositing Bambara groundnut with maize increased aflatoxin production in the flour. It is therefore necessary to implement legislation for aflatoxins in Bambara groundnut, and develop effective management practices during planting, harvesting and storage that will mitigate A. flavus infection in Bambara groundnut.Item Incidence and characterization of Fusarium species in crown rot of bananas(2002) Ramsunder, Kumindra Devrajh; Odhav, Bharti; Okole, Blessed N.Fusarium species produce toxic mycotoxins that are known to exert adverse health effects in humans and animals. No attempts have been made to establish mycotoxin-producing capabilities of isolates of Fusarium species from bananas exhibiting symptoms of crown rot. Crown rot is one of the most serious post harvest problems in banana and the disease is caused by different fungal species, principally Fusarium species. Banana, which is of great economic significance in growing countries (i.e. Costa Rica, Cameroon, Ecuador) is seriously affected by crown rot and is a major cause of fruit lossItem The isolation and characterization of phytoalexin and constitutive agents from plants for mycotoxin control(2000) Mohanlall, Viresh; Odhav, BhartiPlant medicine is an important area of commercial activity in South Africa. This is a rapidly expanding market, thus we are evaluating natural and stressinduced compounds (phytoalexins) from plants as agents that may be able to control mycotoxins. Natural compounds from Bridelia micrantha, Warburgia salutaris, Lippia javanica and Scenecio serratuloides and stress-induced compounds (phytoalexins) from Citrus sinensis cv Valencia were screened for antitunqal and antimycotoxic activity by bioautography against a test organism (Cladosporium cladosporoides) and mycotoxin producing fungi (Fusarium moniliforme and Aspergillus flavus).Item The potential of spice oils in the control of mycotoxin producing fungi(2000) Juglal, Sarla; Odhav, BhartiSpice oils are known to exhibit antifungal activity and therefore have the potential to control mycotoxin production. There is a need in the food industry to find measures to control mycotoxins that are frequently associated with grains that form the staple diet of the majority of the population in South Africa. Clove, cinnamon, oregano, tumeric, eucalyptus, neem, aniseed, mace and nutmeg oils were tested to determine their inhibitory potential against growth of Aspergillus parasiticus and Fusarium moniliforme using the agar overlay technique. Varying concentrations of the spice oils, ranging from 0.1 ppm to 2.0 ppm, were incorporated into broth cultures of A. parasiticus and maize patty cultures ofF. moniliforme. Levels of production of aflatoxins and fumonisin were determined using standard thin layer chromatography and highpressure liquid chromatography methods. In addition, the active component of the spice oils were isolated, characterised and tested. The inhibitory potential of these compounds for field use was tested by incorporating clove oil, whole cloves and ground cloves in sampItem Partial characterization of toxigenic Fusarium(2004) Govender, Leroosha; Govinden, R.Various methods have been developed for the analysis of Fusarium and its toxins. Advances in molecular biology can lead to efficient characterization of this group of fungi. This study was undertaken to examine random amplified polymorphic DNA, volatile compound production and hydrolytic enzyme production by 19 Fusarial isolates. These techniques were employed to assess their abilities in differentiating Fusarium species and F. verticillioides strains and extending the analysis to discriminate toxin producing capabilities amongst these fungiItem Concurrent analysis of the mycotoxins, cyclopiazonic acid, moniliformin and ochratoxin A using capillary zone electrophoresis(2000) Govender, Urishani; Odhav, BhartiMycotoxins are a group of natural poisons produced by certain strains of fungal species when they grow under favourable conditions on a wide variety of different substrates. These toxins have been implicated in a wide range of acute diseases in man and animals. Their toxic effects include oesophageal cancer and liver diseases in humans, and carcinogenic effects in experimental rats and poultry. Hence, there is a need to monitor toxin levels in food commodities.Item Modulating effects of Fumonisin B1 and Ochratoxin A on immune cells in human carcinoma(2005) Adam, Jamila Khatoon; Odhav, BhartiFumonisin B1 (FB1) and ochratoxin A (OTA) represent examples of mycotoxins of greatest public health and agro-economic significance. They exert adverse effects on humans, animals and crops that result in illnesses and economic losses. Fumonisin B1 are cancerpromoting metabolites of Fusarium proliferatum and F verticillioides, (formerly moniliforme), and are implicated in oesophageal cancer. Ochratoxins are metabolites of both Aspergillus and Penicillium species. These compounds are known for their nephrotoxic effects in all animal species and may promote tumours in humans. In man OTA exhibits unusual toxicokinetics, with a half-life in blood of 840 h (35 days) after oral ingestion. Although much is known regarding the toxicology of these toxins, little is known of the effects of these toxins on the immune system. The aim of this study was to determine and compare the immunornodulating effects of FB1 and OTA in human carcinoma. Initial experiments involved isolating lymphocytes and neutrophils from healthy volunteers. The isolated cells were exposed to either FB1 or OTA on a dose and time dependent level and LD50 of the toxins was determined. Thereafter, challenge tests were performed, whereby lymphocytes and neutrophils isolated from volunteers, oesophageal cancer patients and breast cancer patients were exposed to the LD50 dose of either FB1 or OTA for the appropriate time. The effect of the toxins was demonstrated by viability studies, light microscopy and electron microscopy. Cytokine receptors (CK, TNF and CSF) were evaluated by immuno-cytochemical methods and the levels of circulating cytokines (IL -1, IL-6, IL-8, IL-10 and TNF-a) were determined using ELISA kits.Item Molecular characterization of aflatoxigenic and non-aflatoxigenic aspergillus isolates(2007) Mngadi, Phakamile TruthFor decades the genus Aspergillus (of fungi) has been classified based on morphological and growth criteria. Members of the Aspergillus section Flavi are economically valuable and methods of differentiating them are thus very important. Several molecular methods have been developed to distinguish these strains. Also, a number of biochemical and genetic studies have been used in order to provide a better means of classification (Lee et al., 2004). Aflatoxins, the most frequently studied mycotoxins, are produced by certain Aspergillus species/strains/isolates of fungi. The aflatoxin biosynthetic pathway studies have led to a number of discoveries. Several structural and regulatory genes (and their enzymes) involved in the biosynthesis of aflatoxins have been discovered and purified (Trail et al., 1995). Aflatoxin production and contamination of agricultural crops are major causes of economic losses in agriculture. Thus, better methods of characterization/differentiation are required for both aflatoxigenic and non-aflatoxigenic isolates. Molecular biology is one of the current tools used to differentiate between these isolates. Polymerase Chain Reaction (PCR)-based randomly amplified polymorphic DNA (RAPD) analysis has been used successfully in the analysis of DNA relatedness of species of fungi, bacteria, plants and animals. Dendograms which evaluate/assess the likeness between different isolates has also been used (Martinez et al., 2001). Restriction fragment length polymorphism (RFLP) analysis has been applied to a number of studies to detect differences between fungi and to establish relationships between them. Therefore, the scope of this study was to investigate RAPD analysis (with dendograms) and detection of RFLPs by hybridization as molecular methods that can distinctly differentiate or characterize the aflatoxigenic and non-aflatoxigenic Aspergillus isolates.