Thesis (M.Tech.: Biotechnology)- Dept. of Biotechnology & Food Technology, Durban University of Technology, 2007 xv, 102 leaves / For 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.

Thesis (M.Tech.: Food Technology)-Dept. of Biotechnology, Durban University of Technology, 2007 xv, 122 leaves / Several herbs are well known for their nutritional applications. The reported study tested some herbs for their efficacy as antioxidants. The herbs and combinations thereof were analyzed for antioxidant activity using two comparative methods: the beta-carotene bleaching method and through measurement by the Rancimat apparatus. The efficacy of the antioxidants was also tested in a cosmetic base cream. The concentration of the natural antioxidant vitamins, such as vitamin C, vitamin E and beta-carotene was determined by analytical methods. The methods used for analysis provided adequate results for interpretation. All herbs exhibited antioxidant activity, at comparable levels. The two methods used for analysis showed variable results, as previous researchers have shown. In terms of antioxidant potency, the herbs were ranked as follows: fenugreek > coriander > oregano > sage (beta-carotene bleaching method), oregano > sage > coriander > fenugreek (Rancimat method). Oregano and sage were good synergists, when combinations of herbs were used. Fenugreek, unlike the other herbs under investigation, is a prooxidant when used at higher concentrations. A direct relationship was observed between the concentration of herbs and antioxidant potency for sage, oregano and coriander. Fenugreek which is prooxidant at higher concentrations, should be used as an antioxidant independently rather than a synergist. This was the most important finding in the reported study. Similar antioxidant activities of the herbs were observed in the base cosmetic formulation. The reported study has provided an adequate base for further quantitative research into the innovative topic of antioxidants.

Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 xxi, 117 leaves / The aromatic compounds Polychlorinated Biphenyls (PCBs) are one of the largest groups of environmental pollutants. The greatest concern is the release of PCBs in the water systems by industrial effluent, accidental spillages or leaks. PCBs are able to bioaccumulate in the fatty tissues of animals, fish and humans. The impact on human health due to PCBs has prompted interest in their degradation. The application of microbial degradation of PCBs can transform many PCB metabolites. There are a wide variety of microorganisms that can degrade PCBs or utilise them as sole carbon sources. This study focused on isolating microrganisms from industrial wastewater capable of aerobic degradation of PCBs. The degradation potential of the selected isolates were investigated by using different analytical techniques viz. ultra violet or visible spectrophotometer (UV/Vis), thin layer chromatography (TLC) and gas chromatography electron capture detector (GC-ECD).

Thesis (M.Tech.: Food Technology)-Dept. of Biotechnology, Durban University of Technology, 2007 xv, 122 leaves / Several herbs are well known for their nutritional applications. The reported study tested some herbs for their efficacy as antioxidants. The herbs and combinations thereof were analyzed for antioxidant activity using two comparative methods: the beta-carotene bleaching method and through measurement by the Rancimat apparatus. The efficacy of the antioxidants was also tested in a cosmetic base cream. The concentration of the natural antioxidant vitamins, such as vitamin C, vitamin E and beta-carotene was determined by analytical methods. The methods used for analysis provided adequate results for interpretation. All herbs exhibited antioxidant activity, at comparable levels. The two methods used for analysis showed variable results, as previous researchers have shown. In terms of antioxidant potency, the herbs were ranked as follows: fenugreek > coriander > oregano > sage (beta-carotene bleaching method), oregano > sage > coriander > fenugreek (Rancimat method). Oregano and sage were good synergists, when combinations of herbs were used. Fenugreek, unlike the other herbs under investigation, is a prooxidant when used at higher concentrations. A direct relationship was observed between the concentration of herbs and antioxidant potency for sage, oregano and coriander. Fenugreek which is prooxidant at higher concentrations, should be used as an antioxidant independently rather than a synergist. This was the most important finding in the reported study. Similar antioxidant activities of the herbs were observed in the base cosmetic formulation. The reported study has provided an adequate base for further quantitative research into the innovative topic of antioxidants.

Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnolgy, Durban University of Technology, 2007 xviii, 143 leaves / Activated sludge flocs are responsible for flocculation, settling and dewaterability. It is important to maintain the growth off loc-forming bacteria for efficient sludge settleability and compaction for good quality effluent. Filamentous bacteria on the other hand are believed to provide rigid support network or backbone upon which floc-forming bacteria adhere to form stable activated sludge flocs (Wilderer et al., 2002; Ramothokang et al., 2003). Filamentous bacteria can also be detrimental to the process when they outgrow floc-forming bacteria. Morphologically filamentous bacteria are at an advantage as they have higher outward growth velocity and can extend freely to bulk liquid substrate. Proliferation of filamentous bacteria causes foaming and bulking (Martins et al., 2004). Although chemical alleviation measures to circumvent bulking are present, they are symptomatic (Chang et al., 2004). Eikelboom (1975) developed the first identification keys for the classification of filamentous bacteria that is primarily based on morphological characteristics and microscopic examination. Although very useful, this type of identification has its limitations. For instance some filamentous bacteria can change morphology in response to changes in the environment and although some of them can be morphologically similar they may vary considerably in their physiology and taxonomy (Martins et al., 2004). A vast number of filamentous bacteria are still very poorly understood which could be due to the problems of cultivation due to their slow growing nature and maintenance of cultures (Rossetti et al., 2006). This limitation necessitates a molecular approach to resolve the taxonomy of filamentous bacteria as it is a culture-independent technique which is highly accurate. This project was undertaken to verify the identity of pure cultures of filamentous bacteria isolated previously through the application of molecular techniques. The 16S rDNA are conserved regions in bacterial cells and they can be extracted and specific nucleic acid fragments amplified. Denaturation gradient gel electrophoresis enabled the separation of fragments of identical length but different size and served as an indication of purity (Muyzer et al., 1993).

Thesis (M.Tech.: Food Technology)-Dept. of Biotechnology, Durban University of Technology, 2007 xiii, 100 leaves / There are many ways for milk and dairy products to develop flavour defects. Sensory evaluation, has been the traditional approach to characterize off flavours. The need for odour sensing devices becomes greater when volatile and semi-volatile organic compounds are present in the product in parts per billion or even in the parts per trillion concentration range that cause off flavours. Today, sophisticated, sensitive instrumental tests such as electronic nose technology coupled with gas chromatography are capable of detecting, identifying and quantifying the specific chemical agents responsible for off flavours. This study focused on the use of the electronic nose as a novel technology for the detection and monitoring of milk quality by testing the effects of heat treatment at 63˚C and shelf life. Microbiological testing, sensory evaluation and gas chromatographic analysis were carried out together with aroma profiling using the electronic nose to determine milk quality.

Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnolgy, Durban University of Technology, 2007 xviii, 143 leaves / Activated sludge flocs are responsible for flocculation, settling and dewaterability. It is important to maintain the growth off loc-forming bacteria for efficient sludge settleability and compaction for good quality effluent. Filamentous bacteria on the other hand are believed to provide rigid support network or backbone upon which floc-forming bacteria adhere to form stable activated sludge flocs (Wilderer et al., 2002; Ramothokang et al., 2003). Filamentous bacteria can also be detrimental to the process when they outgrow floc-forming bacteria. Morphologically filamentous bacteria are at an advantage as they have higher outward growth velocity and can extend freely to bulk liquid substrate. Proliferation of filamentous bacteria causes foaming and bulking (Martins et al., 2004). Although chemical alleviation measures to circumvent bulking are present, they are symptomatic (Chang et al., 2004). Eikelboom (1975) developed the first identification keys for the classification of filamentous bacteria that is primarily based on morphological characteristics and microscopic examination. Although very useful, this type of identification has its limitations. For instance some filamentous bacteria can change morphology in response to changes in the environment and although some of them can be morphologically similar they may vary considerably in their physiology and taxonomy (Martins et al., 2004). A vast number of filamentous bacteria are still very poorly understood which could be due to the problems of cultivation due to their slow growing nature and maintenance of cultures (Rossetti et al., 2006). This limitation necessitates a molecular approach to resolve the taxonomy of filamentous bacteria as it is a culture-independent technique which is highly accurate. This project was undertaken to verify the identity of pure cultures of filamentous bacteria isolated previously through the application of molecular techniques. The 16S rDNA are conserved regions in bacterial cells and they can be extracted and specific nucleic acid fragments amplified. Denaturation gradient gel electrophoresis enabled the separation of fragments of identical length but different size and served as an indication of purity (Muyzer et al., 1993).

Thesis (M.Tech.: Food Technology)-Dept. of Biotechnology, Durban University of Technology, 2007 xiii, 100 leaves / There are many ways for milk and dairy products to develop flavour defects. Sensory evaluation, has been the traditional approach to characterize off flavours. The need for odour sensing devices becomes greater when volatile and semi-volatile organic compounds are present in the product in parts per billion or even in the parts per trillion concentration range that cause off flavours. Today, sophisticated, sensitive instrumental tests such as electronic nose technology coupled with gas chromatography are capable of detecting, identifying and quantifying the specific chemical agents responsible for off flavours. This study focused on the use of the electronic nose as a novel technology for the detection and monitoring of milk quality by testing the effects of heat treatment at 63˚C and shelf life. Microbiological testing, sensory evaluation and gas chromatographic analysis were carried out together with aroma profiling using the electronic nose to determine milk quality.

Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 xi, 114 leaves / Eighteen traditionally leafy vegetables consumed as food or medicinal compounds by a majority of people in the KwaZulu Natal province of South Africa were analysed for the presence of potentially harmful chemicals (antinutrients) and for their toxicity and mutagenicity. The purpose of the study was to determine whether leafy vegetables were safe for human consumption. Chemical analysis showed that none of the vegetables contained cyanogenic glycosides, however all the vegetables contained oxalic acid ranging from 24.1 mg/ml to 798.2 mg/ml with Solanum nigrum, Portulaca oleracea and Mormodica balsamina showing the highest concentrations. Most of the vegetables contained negligible amounts of phytic acid and saponins, except for Momordica balsamina (3.01 mg/ml and 1.83 mg/ml, respectively). Fourteen of the plants contained alkaloids with Portulaca oleracea having the highest content (1.53 g total alkaloids/5 g leaf material). Eight of the plants were found to inhibit trypsin activity. These chemical analyses were carried out in duplicate and the mean and standard deviation were used. The Ames test revealed that none of the leafy vegetables produced a mutagenic frequency above 1, except 10 000 µg/ml organic extract of Senna occidentalis (mutagenecity considered at mutagenic frequency above 2), thus none were considered mutagenic. All 18 organic extracts did not kill off more than 50% brine shrimp and were thus considered non-toxic. On the other hand the aqueous extracts of seven vegetables, namely, Physalis viscosa, Amaranthus dubius, Justicia flava, Bidens pilosa, Senna occidentalis, Chenopodium album and Ceratotheca triloba, killed more than 50% of the shrimp and are thus considered toxic above 100 µg/ml. The MTT assay carried out on the organic extracts indicated that 17 vegetables did not kill off more than 50% of HepG2 cells and were thus considered non-cytotoxic. The aqueous extracts of four vegetables, namely, Justicia flava, Asystasia gangetica, Momordica balsamin and Senna occidentalis, however killed more than 50% of the shrimp and were thus considered cytotoxic above 1 000 µg/ml. It may be concluded from the antinutrient analyses and the bioassays on the 18 vegetables that caution needs to be maintained with the consumption of certain leafy vegetables included in this study, especially Senna occidentalis.

Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 xi, 114 leaves / Eighteen traditionally leafy vegetables consumed as food or medicinal compounds by a majority of people in the KwaZulu Natal province of South Africa were analysed for the presence of potentially harmful chemicals (antinutrients) and for their toxicity and mutagenicity. The purpose of the study was to determine whether leafy vegetables were safe for human consumption. Chemical analysis showed that none of the vegetables contained cyanogenic glycosides, however all the vegetables contained oxalic acid ranging from 24.1 mg/ml to 798.2 mg/ml with Solanum nigrum, Portulaca oleracea and Mormodica balsamina showing the highest concentrations. Most of the vegetables contained negligible amounts of phytic acid and saponins, except for Momordica balsamina (3.01 mg/ml and 1.83 mg/ml, respectively). Fourteen of the plants contained alkaloids with Portulaca oleracea having the highest content (1.53 g total alkaloids/5 g leaf material). Eight of the plants were found to inhibit trypsin activity. These chemical analyses were carried out in duplicate and the mean and standard deviation were used. The Ames test revealed that none of the leafy vegetables produced a mutagenic frequency above 1, except 10 000 µg/ml organic extract of Senna occidentalis (mutagenecity considered at mutagenic frequency above 2), thus none were considered mutagenic. All 18 organic extracts did not kill off more than 50% brine shrimp and were thus considered non-toxic. On the other hand the aqueous extracts of seven vegetables, namely, Physalis viscosa, Amaranthus dubius, Justicia flava, Bidens pilosa, Senna occidentalis, Chenopodium album and Ceratotheca triloba, killed more than 50% of the shrimp and are thus considered toxic above 100 µg/ml. The MTT assay carried out on the organic extracts indicated that 17 vegetables did not kill off more than 50% of HepG2 cells and were thus considered non-cytotoxic. The aqueous extracts of four vegetables, namely, Justicia flava, Asystasia gangetica, Momordica balsamin and Senna occidentalis, however killed more than 50% of the shrimp and were thus considered cytotoxic above 1 000 µg/ml. It may be concluded from the antinutrient analyses and the bioassays on the 18 vegetables that caution needs to be maintained with the consumption of certain leafy vegetables included in this study, especially Senna occidentalis.