The main aim of this study was to find a plant extract or isolated compound that could be used to combat aspergillosis in animals. Aspergillus fumigatus is one of the most common pathogenic fungal species in humans and animals. A. fumigatus is also an economically important fungus in the poultry industry. Current treatment of the disease is hampered by drug resistance of the organism to conventional antifungals and also its widespread toxicity to the animals. Seven tree species that had good antifungal activity against Cryptococcus neoformans in the Phytomedicine Programme database were selected for further work. These tree species were: Combretum vendae A.E. van Wyk (Combretaceae), Commiphora harveyi (Engl.) Engl. (Burseraceae), Khaya anthotheca (Welm.) C.DC (Meliaceae), Kirkia wilmsii Engl. (Kirkiaceae), Loxostylis alata A. Spreng. ex Rchb. (Anacardiaceae), Ochna natalitia (Meisn.) Walp. (Ochnaceae) and Protorhus longifolia (Bernh. Ex C. Krauss) Engl. (Anacardiaceae). The antimicrobial activity of leaf extracts of the selected plant species were determined against four important nosocomial bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa) and five important animal fungi (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, Microsporum canis and Sporothrix schenckii) using a serial microplate dilution method. The minimal inhibitory concentrations (MIC), of an acetone extract of Loxostylis alata was the lowest against Aspergillus fumigatus with an MIC value of 0.05 mg/ml. The number of antifungal compounds in extracts was determined by bioautography. The acetone extract of L. alata had the most active zones (10). The antioxidant, antiplatelet and cytotoxic effects of the seven plant species were evaluated using established in vitro assays. All the extracts had comparably low toxicity except for the extract of C. harveyi that had high haemagluttination assay titre value, which indicates toxicity. The extracts of P. longifolia, K. wilmsii, O. natalitia, L. alata, C. harveyi and C. vendae contained antioxidant compounds in the qualitative assay using DPPH. In the quantification of antioxidation using ABTS, only the extracts of P. longifolia, L. alata, and C. vendae had substantial antioxidant activity with respective TEAC value of 1.39, 1.94 and 2.08. Similarly, in the quantitative DPPH assay, L. alata. (EC50, 3.58 ± 0.23 μg/ml) and K. wilmsii (EC50, 3.57 ± 0.41 μg/ml) did not differ significantly (p ≤ 0.05) from the positive control (L-ascorbic acid). K. anthotheca had a much lower antioxidant activity (EC<su>50 176.40 ± 26.56 μg/ml), and differed significantly (p ≤ 0.05) from all the other extracts and control. In addition, the extract of C. vendae and C. harveyi had significant (p ≤ 0.05) antiplatelet activity and did not differ from the control (aspirin) with EC50 of 0.06 ± 0.01 μg/ml, 0.19 ± 0.00 μg/ml, respectively. Lower EC50 values in the antioxidant and antiplatelet studies are indicative of superior activity of the plant extract against oxidation and platelet aggregation. Based on the results obtained L. alata was selected for further examination. To simplify the isolation of the antifungal compounds from the L. alata fractions the acetone extract was first separated into six different fractions based on polarity in a mild solvent-solvent fractionation process. The fractions were aqueous methanol, butanol, carbon tetrachloride, chloroform, hexane and water fractions. The antimicrobial activities of the fractions as well as other relevant pharmacological tests on the different fractions were carried out. The number of antimicrobial compounds present in the aqueous methanol (AM), butanol (BT), carbon tetrachloride (CCl4), chloroform (CC), hexane and water fractions was determined by bioautography. The CCl4 extract was active against six out of the 9 microbial strains used and was particularly active against S. aureus, E. faecalis, A. fumigatus, C. albicans, C. neoformans and M. canis with MIC of 0.04, 0.04, 0.1, 0.1, 0.06 and 0.03 mg/ml, respectively. Microsporum canis was the most sensitive organism with the lowest average MIC of 0.16 mg/ml. Qualitative antioxidation using DPPH and quantitative assay using both ABTS and DPPH radicals revealed the presence of several antioxidant compounds in the AM, BT and water fractions of Loxostylis alata. This supported the usefulness of L. alata in treating fungal diseases, as aspergillosis and most fungal infections are associated with immune depression of the host. Antioxidants may reverse several conditions associated with immune deficiencies, resulting in increased levels of interleukin-2, elevated numbers of total lymphocytes and T-cell subsets. Loxostylis alata is used in southern African traditional medicine to control labour pain and to boost the immune system. Extracts and compounds isolated from leaves of Loxostylis alata were therefore also evaluated for their in vitro antimicrobial, anti-inflammatory (cyclooxygenase-1 and -2) activities and evaluated for their potential toxic effects using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) and Salmonella typhimurium tester strains TA98 and TA100. Antimicrobial activity was evaluated using a serial microdilution assay. The bacterial strains used were Staphylococcus aureus (ATCC29213), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922). The fungal strains used were Cryptococcus neoformans, Sporothrix schenckii, Aspergillus fumigatus, Microsporum canis and Candida albicans. A bioassay guided fractionation of the crude extract yielded two antimicrobial compounds namely, Lupeol and μ-sitosterol Lupeol had the most pronounced zone of inhibition against S. aureus and A. fumigatus., When MICs of the 2 compounds were determined, only lupeol had relatively good activity with MICs values ≤ 100 μg/ml against 8 out of 10 of the tested pathogens. However, β-sitosterol had activity against only S. aureus and E. coli with MICs values of 90 and 110 μg/ml, respectively. In addition β-sitosterol had selective inhibition of COX-1 (IC50 = 55.3 ± 2) None of the compounds isolated were toxic in the Salmonella typhimurium/microsome assay and MTT cytotoxicity test. The isolation of these two compounds is reported for the first time from Loxostylis alata. It was disappointing that the two antifungal compounds isolated from L. alata had such a low activity against Aspergillus fumigatus. This inhibits the development of a single compound that can be used therapeutically. Because the crude extract had very good activity we decided to investigate the safety and potential use of this extract in target animal species. At a dose of 300 mg/kg, the chicks had some signs of intoxication, but not at a dose of 200 mg/kg. Aspergillosis was induced experimentally, in broiler chicks. The degree of infection was assessed by comparing degree and severity of clinical signs, lesion scores and fungal re-isolation from treated chicks with those from infected chicks not treated with the extract. The extract at a dose of 100 and 200 mg/kg reduced significantly (p ≤ 0.05) the lesions due to aspergillosis and the amount of Aspergillus fumigatus isolated from infected chicks in an excellent dose related response.. The crude extract of L. alata leaves was as active as the commercially used ketoconazole against avian aspergillosis. It appears likely that the crude acetone extract could be produced at a much lower cost than ketoconazole or other chemical antimicrobial products. If these results can be confirmed in larger studies and if the crude extract does not have a negative effect on the production of the poultry the crude extract of L. alata may prove to be a viable and cost effective alternative to using current antimicrobial products. This study proves that it may be worthwhile to invest human and financial resources in searching for plant related products than can increase animal health and productivity. Copyright / Thesis (PhD)--University of Pretoria, 2009. / Paraclinical Sciences / unrestricted

Thesis (PhD. (Microbiology)) --University of Limpopo, 2013 / The current study investigated four fungal species namely Aspergillus niger FGSC A733, Aspergillus versicolor EF23, Penicillium citrinum AZ01 and Trichoderma harzianum NCGR 0509 for their abilities to produce cellulases and xylanases in submerged and solid state fermentations. Five different substrates (carboxymethyl cellulose, xylan, common thatch grass, wheat bran and Jatropha curcas seed cake) were examined for their potential use as low cost feedstock for fermentation by the fungal species. Aspergillus niger FGSC A733 produced the highest titres of cellulase and xylanase in solid state fermentations using wheat bran as a substrate. However, because of the need to lower the cost of enzyme production, Jatropha seed cake a relatively underutilised oilseed cake was used. Supplementation of the Jatropha seedcake with 10% common thatch grass (Hyperrhenia sp) resulted in a fivefold increase in the levels of xylanase produced. Cellulase production was not affected by this supplementation. Addition of ammonium chloride increased production of xylanase while cellulase production was not affected nitrogen supplementation. Maximum xylanase was produced on Jatropha seed cake at 25 °C after 96 hours while cellulase was maximally produced at 40 °C after 96 hours of solid state fermentations. Peak production of xylanase was obtained at an initial pH of 3 whilst cellulase was maximally produced at an initial pH of 5. The crude xylanase was most active at pH 5 and cellulase at pH 4. The optimum temperature for cellulase activity was 65 °C and that of xylanase was 50 °C. Under optimized conditions, 6087 U/g and 3974 U/g of xylanase and cellulase per gram of substrate used were obtained respectively. The diversity of cellulases was investigated so as to determine the most appropriate enzyme mixture for saccharification of the common thatch grass. Proteins from the four species under investigation were partially purified by affinity chromatography on swollen Avicel. The proteins were analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis SDS-PAGE and zymography. Potential cellulase bands from SDS-PAGE were sequenced by mass spectrometry. The basic logical alignment tool (BLAST) and Clustal W were used for matching and identifying the sequences with closely related ones in the databases. The identified proteins from Penicillium citrinum AZ01 and Aspergillus versicolor EF23 were found to closely resemble a catalytic domain of cellobiohydrolase from Trichoderma sp. The three proteins obtained from Aspergillus niger showed resemblance to 1,4-beta glucan cellobiohydrolase A precursor from Aspergillus niger FGSC A733 was also found to have cellobiase and endoglucanase activity was determined using cellobiase and carboxymethyl cellulose as substrates. Cellulase and xylanase zymograms of proteins from A. niger FGSC A733 demonstrated six active bands ranging from 20 kDa to 43 kDa for cellulase and a 31 kDa active band for xylanase. The cellulase produced by Aspergillus niger FGSC A733 on Jatropha seed cake under optimised conditions was used for saccharification of 2% (w/v) common thatch grass (CTG) in combination with Celluclast™. Celluclast™ and Aspergillus niger cellulase were mixed at different ratios and the amount of glucose produced over time was monitored using high performance liquid chromatography (HPLC). A ratio of 2 volumes Celluclast™ to one volume Aspergillus niger cellulase was chosen for the saccharification process. The main enzymes in the mixture were identified using peptide mass fingerprinting as endoglucanases from the Celluclast™ and cellobiase from the Aspergillus niger cellulase. Concentration of the Celluclast™ tenfold times (164 FPU) improved the yield of glucose by 42.8 and 37.8% in acid and alkali pre-treated CTG, respectively. Concentrating Aspergillus niger cellulase (13.2 FPU) decreased the production of glucose by 4.8% in acid pre-treated CTG while in alkali pre-treated CTG, a 5% increase in glucose production was observed. Increasing the substrate loading of acid pre-treated CTG from 2% to 10% (w/v) resulted in a two and a half times increase in glucose production while an increase of 1.5 g/l glucose was obtained from 7% (w/v) alkali pre-treated CTG. Addition of xylanases from Aspergillus niger to the Celluclast™-Aspergillus niger cellulase mixture decreased glucose production by 16.3% on acid pre-treated CTG while there was an increase of 18.3% glucose in alkali pre-treated CTG. Addition of enzyme preparations from Aspergillus versicolor EF23, Penicillium citrium AZ01 and Trichoderma harzianum NCGR 0509 to the Celluclast™-Aspergillus niger cellulase mixture resulted in lower glucose production both in acid and alkali pre-treated CTG. Addition of Pentopan™ improved glucose production by 8 and 25% on 10% acid and 7.5% alkali loading of pre-treated CTG respectively. The optimal conditions for the production of the glucose rich hydrolysate in 10% (w/v) acid and 7% (w/v) alkali pre-treated CTG was found to be the use of Celluclast™-Aspergillus niger cellulase-Pentopan™ mixture (164 FPU Celluclast™ and 13 FPU Aspergillus niger cellulase, 7178 IU) Pentopan™ at 50 °C for 32 hours. The fermentability of the glucose in glucose-rich CTG hydrolysates to ethanol using Saccharomyces cerevisae WBSA 1386 and Candida shehatae CSIR Y-0492 was investigated. The highest yield of ethanol produced by S. cerevisae WBSA 1386 was 9.8 g/l in the alkali pre-treated CTG hydrolysate and 8.7 g/l in acid pre-treated CTG. C. shehatae CSIR Y-0492 produced 9 g/l of ethanol in alkali pre-treated CTG within 48 hours while acid pre-treated CTG hydrolysate produced 8.8 g/l of ethanol within 24 hours of the fermentation process. Addition of the nutrient supplement boosted the ethanol yield in the acid pre-treated hydrolysates. The consumption of glucose during fermentation by S. cerevisae WBSA 1386 and C. shehatae CSIR Y-0492 on average was 97%. The C. shehatae CSIR Y-0492 was expected to produce much higher ethanol yield than the Saccharomyces because of its ability to utilize xylose for ethanol production. This however was not observed in this investigation. The conclusion of this study is that it is possible to produce bioethanol from Hyperrhenia spp. (CTG) using a combination of fungal enzymes for the production of fermentable sugars.