Thesis (MSc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: The importance of indigenous fruit wines is not well researched and documented. There is a need to develop and exploit these valuable food resources through improved production practices, storage, preservation and utilization technologies. The maruia fruit is beneficial in many ways, it can be used for making juice, jam, beer or can be eaten as a whole fruit. The highly nutritive nature of the fruit, its distinctive tropical flavor, its wild occurrence and demand by the local and international communities for the by-products of the fruit necessitated efforts to optimize the technological processes for the production of the possible by-products. This study focuses on the fermentation technology of the maruia fruit. The effect of enzymes prior to the fermentation process and post-fermentation was evaluated. For pre-fermentation processes we focused on the ability of commercial enzymes to increase juice yield, improve the clarification and filterability. For pre- and post-fermentation applications, aroma release was considered. The results indicated a significant increase in the yield depending on the enzyme used. An increase of at least 2% was recorded and a maximum of 12% yield increase was observed. The enzymes also had a phenomenal effect on the release of bound monoterpenes and hence enhancing the flavor of the juice. The panel of judges confirmed the results from the gas chromatography analyses by noting an increase in flavor intensity in the enzyme treated juice. The possibility of selecting a yeast strain that performs best during the fermentation of maruia pulp was also looked at. This study aimed at selecting a strain that produces wine and distillate with the typical maruia flavor complex. We showed the effect of the different yeast strains, in the wines and distillates, on the principal volatile compounds. We then correlated the performance of the different strains as perceived by the panel to the various volatile compounds. The effect of fermentation temperature on the performance of the different yeast strains was also considered. Fermenting the maruia pulp at different temperatures resulted in the production of wines and distillates with different volatile profiles for the different yeast strains. The wines and distillates fermented at a low temperature of 15°C were preferred to the wines and distillates fermented at 30°C. However, not all strains performed well at 15°C, strains like NT116 performed better at 30°C. The different commercial strains produced wines and distillates with significantly different flavor profiles. These differences in the flavor profiles were reflected in the sensory evaluation where, depending on the interaction of the volatile compounds some wines and distillates were preferred to others. The effect of the different commercial enzymes and yeast strains should thereof be further evaluated and optimized on a larger scale. This would greatly help prevent variation in quality of the fermented by-products of the maruia fruit. / AFRIKAANSE OPSOMMING: Die belang van inheemse vrugtewyne is nie goed nagevors en gedokumenteer nie. Daar is 'n behoefte om hierdie waardevolle voedselbronne te ontwikkel en te benut, deur verbeterde produksiepraktyke, storing, preservering en benuttingstegnologieë. Die maroelavrug is veelsydig op baie wyses, deurdat dit gebruik word vir die maak van sap, konfyt, bier, of as heel vrug geëet kan word. Die vrug is hoog in voedingswaarde, het In kenmerkende tropiese geur, kom wild voor, en is in aanvraag by plaaslike en internasionale gemeenskappe vir die by-produkte van die vrug. Dit maak dit essensieel om die tegnologiese prosesse vir die produksie van hierdie moontlike by-produkte te optimiseer. Hierdie studie fokus op die fermentasie-tegnologie van die maroelavrug. Die effek van ensieme voor en na die fermentasie-proses is geëvalueer. Vir prosesse wat voor fermentasie plaasvind, het ons gefokus op die vermoë van kommersiële ensieme om sapopbrengs te verhoog, asook om verheldering en filtrering te verbeter. Vir beide voor- en na-fermentasie toepassings is die vrystelling van aroma gemonitor. Die resultate dui op 'n betekenisvolle verhoging in die sapopbrengs, afhangende van die ensiem wat gebruik is. 'n Verhoging van ten minste 2% is opgeteken, en 'n maksimum van 12% opbrengsverhoging is waargeneem. Die ensieme het ook 'n geweldige effek op die vrystelling van gebonde monoterpene gehad, en dus die verhoging in die geur van die sap. Die proepaneel het die resultate bevestig van die gaschromatografie-analises, deur 'n verhoging in die geurintensiteit in die ensiembehandelde sap te bemerk. Daar is ook gekyk na die moontlikheid om 'n gisras te selekteer wat die beste presteer tydens die fermentasie van maroela-pulp. Hierdie studie het die doelstelling gehad om In gisras te selekteer wat wyn en distillaat produseer met In tipiese maroelageurkompleks. Ons het die effek van verskillende gisrasse aangedui in die wyne en distillate, op grond van van vlugtige komponente. Ons het dan die prestasie van die verskillende rasse, soos waargeneem deur die paneel, gekorrelleer met die verskeie vlugtige komponente. Die effek van fermentasie-temperatuur op die werkverrigting van die verskillende gisrasse is ook in ag geneem. Fermentasie van die maroela-pulp by verskillende temperature het gelei tot die produksie van wyne en distillate met verskillende vlugtige profiele vir die verskillende gisrasse. Die wyne en distillate wat by In laer temperatuur van 15°C gefermenteer is, is verkies bo die wyne en distillate wat by 30°C gefermenteer is. Alle rasse het egter nie baie goed presteer by 15°C nie, soos byvoorbeeld NT116 wat beter presteer het by 30°C. Die verskillende kommersiële rasse het wyne en distillate geproduseer met betekenisvol verskillende geurprofiele. Hierdie verskille in geurprofiele is gereflekteer in die sensoriese evaluering waar, afhangende van die interaksie van die vlugtige komponente, sommige wyne en distillate bo ander verkies is. Die effek van die verskillende kommersiële ensieme en gisrasse moet verkieslik verder op groter skaal geëvalueer en geoptimiseer word. Dit sal veral help om variasie in kwaliteit van die gefermenteerde by-produkte van die maroelavrug te voorkom.

Thesis (MSc. (Microbiology)) --University of Limpopo, 2018 / Refer to the document / National Research Foundation (NRF)

Thesis (M. Sc. (Microbiology) -- University of Limpopo, 2019 / Marula wine plays a fundamental role in the livelihoods of rural communities where it enhances social cohesion and also provides a reasonable income to the primary traders who often have no alternative source of income. Spontaneous fermentation will inevitably include microbes that produce undesirable metabolites, which lead to the spoilage and short shelf life of the wine. The aim of this study was to profile the microbial and chemical changes during fermentation of marula wine. Marula wines were collected from three areas in the Limpopo province namely: University of Limpopo, The Oaks village and Makhushane village. The bacterial species Gluconobacter oxydans, Acetobacter pasteurianus, Lactobacillus brevis, Lactobacillus nagelii, Lactobacillus parabuchneri and Lactobacillus plantarum species and yeast species Hanseniaspora guilliermondii, Pichia guilliermondii, Saccharomyces cerevisiae, Rhodotorula mucilaginosa and Meyerozyma caribbica were identified in marula wines at varying stages of fermentation. Non-fermenting yeast species such as H. guilliermondii together with lactic acid bacteria such as L. brevis and L. plantarum and the Enterobacteriaceae dominated the early stages of fermentation, whereas S. cerevisiae and Acetic acid bacteria dominated the late stages of fermentation. Chemical profiling of the marula juice and wine, which was achieved using both high performance liquid chromatography (HPLC) and gas chromatography (GC), revealed sucrose as the most abundant sugar in the marula juice with a range of 60.43 mg/mL to 73.20 mg/mL. Volatile organic compounds such as ethanol, ethyl acetate and isobutanol were observed during the fermentation process with none to very little of the volatile compounds detected in marula juice. Ethyl-acetate was the most abundant compound whereas ethanol concentration was observed to be high during the late stages of fermentation at a range of 1.16 g/L to 12.63 g/L. Common microbiota from different marula wines showed low intraspecific diversity indicating that the microorganisms responsible for the spontaneous fermentation are the same throughout the different areas that were selected for this study. The outcomes of the study provide empirical data to develop a wine with a long shelf life.

Thesis (Ph.D. (Microbiology)) -- University of Limpopo, 2022 / NRF and TIA

Sub-Saharan Africa is a treasure chest of natural materials remaining to be explored for commercial applications and as alternative foods to diversify and improve food sustainability. The Marula tree is available in abundance in South Africa and bears a fruit with a highly nutritious kernel containing high oil and protein content. The oil from the kernels has various applications from food to cosmetics. The accepted oil processing practice is required to be a green technology, producing no effluent or using toxic solvents. Therefore, the oil is extracted using an expeller. However, with average 55 wt. % oil in the kernel the extracted oil yield is far from optimal, typically ranging from as low as 7 wt. % to 47 wt. %. The latter is obtained only with proprietary modified expellers. Therefore, an alternative green technology which retains the native characteristics of the Marula oil is needed. Communication with local producers, South African and Namibian, confirmed the need for investigation of an alternative means of extraction of Marula oil from the seed kernels which can improve the yield and potentially the quality of the oilcake. The latter of which is typically adversely affected by the expelling process. A review of various processing technologies available for oil extraction was completed and supercritical fluid extraction utilizing carbon dioxide as the extraction solvent was identified as a potential solution. An overview on supercritical fluid extraction using carbon dioxide (SFE-CO2) of similar materials to the Marula kernels, such as hazelnuts, walnuts and pine kernels indicates that yields similar to that of solvent extraction and of the quality of the oils obtained by cold pressing can be obtained with the technique. The theory, practical applications, and how one can use the system to improve yield from various natural materials were reviewed. It was determined that the two main parameters one can manipulate on supercritical extraction systems to optimize the yield, were pressure and temperature. Subsequently kernels of the Sclerocarya birrea tree, common name Marula, cultivated in South Africa, were obtained for extraction with supercritical carbon dioxide. The effects of pressure and temperature on extraction yield were investigated. The total maximum yield of Marula kernel oil obtained was found to be 54 wt. %, compared to a solvent extracted yield of 52 wt. %, such that a 100 % oil recovery was obtained with SFE-CO2. The optimal conditions were found to be 450 bar and 60 °C as the yield per kg solvent initially was 41 g kg-1 CO2. Following the extractions, the oils were characterized for fatty acid composition using gas chromatography. Quality parameters of a cold pressed sample and a sample obtained at the optimal extraction conditions were determined and compared; and the results indicate that the two oils are of similar composition and quality. Supercritical fluid extraction using carbon dioxide was successfully verified as a potential processing method for the extraction of Marula oil from the kernels. The SFE-CO2 provided an improved yield compared to cold pressing and a quality of oil similar to cold pressed Marula oil. Additionally, after SFE-CO2 processing, the defatted Marula kernels contain high protein content, 69 wt. %, in the form of a pure white powder. Due to the favourable nutritional content the residue may be used for human consumption to create new products such as meat analogues, porridges, and shakes, or can be sold as a high protein powder. / MSc (Engineering Sciences in Chemical Engineering), North-West University, Potchefstroom Campus, 2015

Sub-Saharan Africa is a treasure chest of natural materials remaining to be explored for commercial applications and as alternative foods to diversify and improve food sustainability. The Marula tree is available in abundance in South Africa and bears a fruit with a highly nutritious kernel containing high oil and protein content. The oil from the kernels has various applications from food to cosmetics. The accepted oil processing practice is required to be a green technology, producing no effluent or using toxic solvents. Therefore, the oil is extracted using an expeller. However, with average 55 wt. % oil in the kernel the extracted oil yield is far from optimal, typically ranging from as low as 7 wt. % to 47 wt. %. The latter is obtained only with proprietary modified expellers. Therefore, an alternative green technology which retains the native characteristics of the Marula oil is needed. Communication with local producers, South African and Namibian, confirmed the need for investigation of an alternative means of extraction of Marula oil from the seed kernels which can improve the yield and potentially the quality of the oilcake. The latter of which is typically adversely affected by the expelling process. A review of various processing technologies available for oil extraction was completed and supercritical fluid extraction utilizing carbon dioxide as the extraction solvent was identified as a potential solution. An overview on supercritical fluid extraction using carbon dioxide (SFE-CO2) of similar materials to the Marula kernels, such as hazelnuts, walnuts and pine kernels indicates that yields similar to that of solvent extraction and of the quality of the oils obtained by cold pressing can be obtained with the technique. The theory, practical applications, and how one can use the system to improve yield from various natural materials were reviewed. It was determined that the two main parameters one can manipulate on supercritical extraction systems to optimize the yield, were pressure and temperature. Subsequently kernels of the Sclerocarya birrea tree, common name Marula, cultivated in South Africa, were obtained for extraction with supercritical carbon dioxide. The effects of pressure and temperature on extraction yield were investigated. The total maximum yield of Marula kernel oil obtained was found to be 54 wt. %, compared to a solvent extracted yield of 52 wt. %, such that a 100 % oil recovery was obtained with SFE-CO2. The optimal conditions were found to be 450 bar and 60 °C as the yield per kg solvent initially was 41 g kg-1 CO2. Following the extractions, the oils were characterized for fatty acid composition using gas chromatography. Quality parameters of a cold pressed sample and a sample obtained at the optimal extraction conditions were determined and compared; and the results indicate that the two oils are of similar composition and quality. Supercritical fluid extraction using carbon dioxide was successfully verified as a potential processing method for the extraction of Marula oil from the kernels. The SFE-CO2 provided an improved yield compared to cold pressing and a quality of oil similar to cold pressed Marula oil. Additionally, after SFE-CO2 processing, the defatted Marula kernels contain high protein content, 69 wt. %, in the form of a pure white powder. Due to the favourable nutritional content the residue may be used for human consumption to create new products such as meat analogues, porridges, and shakes, or can be sold as a high protein powder. / MSc (Engineering Sciences in Chemical Engineering), North-West University, Potchefstroom Campus, 2015

The marula tree has already become a very important alternative crop in South Africa with a great potential for further improvement. There are, however, no accurate records that have been reported on the fruit yield and quality of marula tree in Southern Africa. To utilise the fruit fully, to make proper selections from the wild trees and to develop new selections/cultivars, there is a need to have information on yield potential and fruit quality aspects. The overall objective was to determine the yield potential and to evaluate the fruit quality of different marula selections putting more emphasis on fruit quality parameters such as the fruit mass, size, total soluble solids (TSS), acidity and juice content. Four marula selections were identified during their peak production season(2004/2005/2006/2007) at Schoeman Boerdery (Orchard) between Marble-Hall and Groblersdal in Mpumalanga Province (South Africa). From each of the selections, namely; Pharulani, Toularula and Swarula, five trees were selected randomly and marked, while from the Wild marula only four trees were selected and marked. For the purpose of experimental design, the four selections were regarded as four treatments, and the marked trees per selection as the replicates. All selected trees were more or less the same in size and were in full production. The trees were growing in an orchard with planting distance of 9 m (interspacing) and 4.5 m (intraspacing). To determine yield potential, the number of fruit per square meter per tree was counted on pre-labelled branches on five positions around the tree canopy at three stages of fruit development. Collected data were converted to grams of fruit per square metre. Tree canopy size (CS), expressed as m2, was calculated by using the radius (r) of each canopy in the following formula: CS= (╥.r2) 4. For calculating the yield per tree, the average canopy size was multiplied by the number of fruit counted per m2. Twenty fruits from each labelled marula tree per selection were randomly selected; collected and taken to the Ecophysiology Laboratory at University of Pretoria for fruit quality traits, weight (mass), size, stone mass, peel mass, juice content, Total Soluble Solids (TSS) and Titratable acidity (TA). The yield efficiency/potential for both tree unit and hectare (ha) basis in 2006/2007 season, showed that the Swarula selection had the highest total yield as compared to other selections. Results showed that there was a positive relationship between fruit weight and size, that is, the bigger the weight, the bigger the size and the opposite. Generally, three selections, Swarula, Toularula and Pharulani had a higher fruit mass and size than “wild” marula. Pharulani had the highest seed/stone mass whereas “wild” marula had the lowest seed mass during the 2004/2005 and 2005/2006 fruiting seasons. Pharulani selection had the highest juice mass during both seasons whereas Swarula selection had the lowest juice mass. Results also showed that ‘wild’ marula had the highest TSS/TA ratio during both seasons whereas Pharulani had the lowest TSS/TA ratio. Optimal traits were therefore found in different selections and not in the same selections as envisaged. Copyright / Dissertation (MInst Agrar)--University of Pretoria, 2009. / Plant Production and Soil Science / unrestricted

STUDENT NUMBER: 0516082G Master of Science in Resource Conservation Biology Faculty of Science / The aim was to determine the availability of marula (Sclerocarya birrea) (A.Rich.) Hochst. Subsp.cafra (Sond.) Kokwaro trees for the harvesting of nut oils, through determining abundance, population structure and regeneration capacity, within the context of the socioeconomic and biophysical dimensions of villages in Zvishavane, Zimbabwe. Both arable and non arable lands in the study area were sampled within four randomly selected villages to measure species abundance, regeneration and population dynamics of the trees. The socioeconomic factors influencing marula commercialisation were determined through participatory rural appraisal (PRA) and household questionnaire survey techniques. Vegetation characteristics were assessed through the point centre quarter (PCQ) method by placement of transects across sampled villages. All households (100%) collected marula fruit for their household use, mainly for brewing marula beer, making jam and selling kernels for snacks or oil pressing. In 2005, the mass of marula harvest per household averaged 160 ± 18 (SD) kg, with a range of 50-800 kg within the study area. A linear regression analysis indicates no increase in mass of marula harvest with larger sizes of household (r2 = 0.0089, d.f = 50, p = 0.4048). In addition, household interviews revealed that there was no association between level of household wealth status and the household’s use of traditional medicine from marula trees (χ2 = 0.2233, d.f = 2, p = 0.8944). The vegetation survey indicated a elatively high density of marula within the study area of 8.03 ± 3.19 stems ha-1. There was a significant difference in marula densities between arable and non arable land uses (F1, 197 = 11.92, p=0.001). The arable land had 6.40 ± 5.29 stems ha-1 while non arable land had three times more at 19.63 ± 11.82 stems ha-1. However there was no significant difference in densities between the villages in the study area (F 3, 195 =1.063, p= 0.366). There was a significant difference between marula tree diameters between arable and non arable land (t 92, 107 = 1.69, p = 0.0401). The arable land had generally bigger tree diameters (31.2 ± 25.3 cm) than non arable land (26.5 ± 20.8 cm), suggesting a form of domestication through allowing marula to grow around homesteads and crop fields. The investigation of marula size class profiles shows a large proportion of smaller diameter trees and this indicates ongoing recruitment of trees into the population. A closer analysis of the smallest diameter class shows a higher proportion of saplings (> 6cm diameter) than of seedlings (< 4cm diameter). Fruit harvesting seems to have a low potential for any negative impact, compared to other uses of marula trees. However fruits have the highest economic return and therefore should be targeted for the commercialisation activities. The management of some destructive forms of marula tree use (such as harvesting for bark, firewood, and carving wood) however, do need to be monitored to limit negative impacts on the population. There is also a need to determine the annual quantity of harvestable marula fruits and also the sex ratio of marula trees (a dioecious species) on this particular site, as a prelude to developing sustainable harvesting quotas, so that harvesting rates do not exceed the capacity of populations to replace the individuals extracted. There are some non governmental organisations (NGOs) and local institutions that are working towards natural resources conservation in the area. Zvishavane water project (ZWP) and Phytotrade Africa are assisting the rural producers in marula commercialisation activities through providing information on processing, packaging and marketing. At a current value of US$1 kg-1of fruit, the 3200 ha study area is estimated to yield a total harvest of 1 120 000 kg of marula fruit per year, and this should translate to an approximate total value of $1 120 000 per year for the whole area. There is still a need to expand the supply of processed goods with added value to wider markets locally, nationally, and internationally. There is therefore an indication that marula products offer a promising economic alternative for the people in the rural areas of Zvishavane area and southern Africa as a whole. The cash injection earned from selling fresh marula products comes at a particularly crucial time of the year, when money is required for school fees, uniforms and books. However there is a need for long-term monitoring and evaluation of socio-economic and environmental impacts of marula commercialisation so as to achieve sustainable resource utilisation in the region.

Thesis ( PhD. (Biotechnology )) --University of Limpopo, 2005 / The marula tree (Sclerocarya birrea subsp. caffra), an indigenous, multipurpose, drought tolerant tree of Africa harbors great economic potential. Acceptance of marula-derived products internationally will directly increase the demand for marula resource. Rapid multiplication of marula trees of superior quality forms the basis of sustainable export growth. In vitro propagation and genetic improvement offer the opportunity for accelerated multiplication of selected tree material as well as to dramatically increase production, quality and efficiencies. The objectives of the study were therefore to develop a protocol for in vitro multiplication of marula and to determine the feasibility of Agrobacterium-mediated transformation of the marula tree. Nodal sections with axillary bud (s) were cultured on Murashige and Skoog (MS) medium supplemented with 4.8μM BA and 2.4μM KN and 0.1% polyvinylpyrrolidone (PVP) to obtain on average 2.5 microshoots per responding explant. The proliferated microshoots were elongated on MS medium supplemented with 1.2μM BA and 1.0μM KN. Elongated microshoots were rooted in MS medium at half salts strength supplemented with 10μM IBA and 0.3% activated charcoal (AC). On average 82% of the shoots rooted. Survival of acclimatized plantlets was 90%. RAPD analysis confirmed intraclonal genetic stability between parent plants and their clones within the limits of the technique.Nodal sections cocultivated with Agrobacterium tumefaciens for 3 days on MS multiplication medium supplemented with 100μM acetosyringone resulted on average in transient expression of 52.5% of the explants with 1.6 blue stained zones per explant. Cocultivated explants on MS selection medium containing 300mgl-1 kanamycin resulted in 1.5% chimeric putative transgenic shoots. This is the first report on the micropropagation and genetic transformation of marula, Sclerocarya birrea subsp caffra. / South Africa’s National Research Foundation Institutional Research Development Program (NRF-IRDP)

M. Tech. (Department of Chemical Engineering, Faculty of Engineering and Technology), Vaal University of Technology. / This study presents the use of sodium methoxide as a homogeneous catalyst and impregnated Perlite (potassium hydroxide/perlite) as heterogeneous catalyst for production of biodiesel using Baobab and Marula oil respectively. One factor at a time experimental design was used to study the effect of temperature, time, amount of catalyst and methanol to oil ratio on the transesterification of baobab oil using sodium methoxide as a catalyst. Response surface methodology was used to study the effect of temperature, time, amount of catalyst and methanol to oil ratio on the transesterification of marula oil using perlite as a catalyst. Biodiesel yield produced using sodium methoxide and baobab oil was 96% at 1 hr reaction time, 30 wt.% methanol to oil ratio, 1 gram of catalyst and 60°C reaction conditions. Biodiesel yield produced using perlite and marula oil was 91.38% at 3.55 hr reaction time, 29.86 wt.% methanol to oil ratio, 3.46 grams of catalyst and 70.41°C reaction conditions. Perlite catalyst was reusable for transesterification of marula oil while sodium methoxide was not reusable for transesterification of baobab oil. Baobab and Marula biodiesel fuel properties are comparable to American Society for Testing Materials standard (ASTM).