Faculty of Applied Sciences
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Item The prebiotic effects of amadumbe (Colocasia Esculenta) and okra (Abelmoschus esculentus) mucilage(2023) Gajadhar, Sharmista; Amonsou, Eric Oscar; Mchunu, Nokuthula PeacePrebiotics have been shown to aid in the improvement and maintenance of human health through positive manipulation of gut microbiota. Diet-induced changes in gut microbial diversity has been recognized as a factor which contributes to the rising epidemics of chronic illnesses in both developed and developing countries. Traditional crops, amadumbe (Colocasia esculenta) and okra (Abelmoschus esculentus (L.) Moench) offer nutritional security to many communities in South Africa. These crops are rich in mucilage and are presumed prebiotics. Structural composition and functional properties of polysaccharides like mucilage are suggested to influence their fermentability by gut microbiota and potential health effects. The purpose of this study was to investigate the prebiotic effects of amadumbe and okra mucilages for potential application as dietary supplements. Mucilage was extracted from amadumbe and okra by cold water extraction. Purified mucilage was obtained by Sevag method, lipid removal and thereafter dialyzed. The composition and structure of crude and purified mucilage were analyzed using Fourier transform infrared spectroscopy (FT-IR), size exclusion chromatography (SEC) and high pressure liquid chromatography (HPLC). Functional properties including water and oil holding capacity, swelling and solubility were determined. The prebiotic potential of amadumbe and okra mucilage was carried out by in- vitro fermentation using human faecal sample. Glucose was the common monosaccharide present in both amadumbe and okra mucilage. Monosaccharides present in amadumbe mucilage were arabinose, mannose and xylose, while galactose, ribose and rhamnose were the main monosaccharides present in okra mucilage. The presence of β-glucan was found to be higher 0.20 g/100 g in amadumbe mucilage than in okra mucilage 0.07 g/100 g. The resistant starch content in amadumbe mucilage was higher 4 g/100 g than in okra mucilage 0.7 g/100 g. Asparagine, proline, glutamine, and threonine were the most common amino acids found in both amadumbe and okra mucilage samples. Purified amadumbe and okra mucilage displayed the same characteristic peaks as crude amadumbe and okra mucilage in the FT-IR spectrum but at a lower intensity suggesting that purification contributed to a more stable and uniform structure. The FT-IR spectrum indicated the presence of uronic acid and hydroxyl groups which confirm the existence of carbohydrate in both amadumbe and okra mucilage. The molecular weight of crude amadumbe and okra mucilages ranged between 219 and 224 kDa while molecular weight of purified amadumbe and okra mucilage ranged between 220 and 244 kDa. The purification process was seen to improve functional properties such as the water holding capacity, swelling and solubility of mucilages. In comparison to okra mucilage, crude and purified amadumbe mucilage showed low water holding capacity 5 and 9 g/100 g and high percentage solubility 61 and 73%. Amadumbe mucilage had a slightly higher oil holding capacity 11 g/100 g in comparison to okra mucilage 10 g/100 g. During in-vitro fermentation, inulin (positive control) rapidly decreased the pH of the fermentation medium from 7.0 to 6.5, in comparison to amadumbe (7.0 to 6.7) and okra (7.0 to 6.8) mucilage. At the end of fermentation inulin had maximum gas production of 233.19 mL, followed by amadumbe mucilage 158.98 mL and okra mucilage 113.98 mL. These results suggest inulin is more easily fermented by microbes compared to amadumbe and okra mucilage. Gut microbiota analysis at phylum level showed that amadumbe mucilage stimulated the proliferation of Actinobacteria and reduced the presence of Firmicutes in comparison to okra mucilage. At species level, okra mucilage promoted the growth of Bacteroidaceae bacteroidetes, Bacteroides ovatus and Bacteroides uniformis. These species are known to assist in protection of the gut and are capable of providing nutrients to other microbial species. This suggest that amadumbe and okra mucilages are fermented differently by gut microbiota possibly due to differences in their structure and composition. This study concluded that amadumbe and okra mucilages has potential to be utilized as an emerging prebiotic in food applications or as supplements.Item Enhancement of biohydrogen production from the aquatic weed Pistia stratiotes through a dark fermentation process(2019) Mthethwa, Nonsikelelo Precios; Pillai, Santhosh Kumar Kuttan; Bux, Faizal; Kiambi, Sammy LewisAquatic weeds are well known for their fast growth rate and high carbohydrate content that can be easily hydrolysed into fermentable sugars. This study was aimed at the utilization of an indigenous aquatic weed, Pistia stratiotes for biohydrogen production through the dark fermentation process. Characterization of the biomass, effect of pre–treatment methods on biomass hydrolysis, effect of reactor operational conditions and type of inoculum on enhancing hydrogen production potential of P. stratiotes was assessed. Physical and chemical pre–treatments were employed on P. stratiotes biomass to increase digestibility and to achieve high conversion rates of fermentable sugars. The highest sugar yield of 139± 0.8 mg/g was obtained when the oven dried biomass was subjected to H2SO4 (2.5%) pre– treatment followed by autoclaving at 121°C for 30 min. Biohydrogen production under different operational conditions was thereafter optimized using One–factor–at–a–time (OFAT) batch experiments in 120 mL serum bottles. A maximum hydrogen yield (HY) of 2.46 ± 0.14 mol-H2/mol-glucose (3.51 ± 0.20 mg-H2/g-dry weight) and 2.75 ± 0.07 mL h-1 hydrogen production rate was observed under optimized conditions (pH 5.5, Temp 35°C, S/X: 1.0 g-COD/g-VSS and HRT 8 h). The organic mass balance (92 – 96%) and electron– equivalent balance (92 – 98%) further indicated the reliability of the obtained fermentation data. Assessment of microbial activity was achieved using molecular techniques such as quantitative polymerase chain reaction (qPCR) targeting both 16s rRNA (of Clostridium spp., Bacillus spp., and Enterobacter spp.) and the functional hydrogenase gene (hydA). The highest gene activity of hydrogenase was noted at pH of 5.5 with 2.53×104 copies/ng-DNA compared to low pH: 4.5 (6.95 × 103 copies/ng-DNA) and high pH: 8.5 (7.77×103 copies/ng- DNA). A similar trend was also observed for the species containing a highly active hydrogenase (i.e. Clostridium spp., Bacillus spp., and Enterobacter spp.). During the optimum reactor conditions, three hydrogen producing bacterial strains Bacillus cereus and Enterobacter cloacae were successfully isolated. These isolates were used as inoculums for the pure culture studies and achieved HYs of 2.2, 1.10 and 1.97 mol-H2/mol-glucose respectively under optimized fermentation conditions. However, the thermally treated mixed culture displayed a marginally higher HY (2.46 mol-H2/mol-glucose) compared to the pure culture used alone. Furthermore, the cost estimation indicated a potential and economically feasible for biotransformation of P. stratiotes to hydrogen energy. In conclusion, the results from this study has revealed the potential of employing P. stratiotes biomass for biohydrogen production. The results also indicated the importance of employing suitable pre–treatment methods, operating conditions as well as inoculum types for enhanced hydrogen production using P. stratiotes.Item Microbial production of phytases for combating environmental phosphate pollution and other diverse applications(Taylor and Fancis Online, 2016) Kumar, Ashwani; Chanderman, Ashira; Makolomakwa, Melvin; Perumal, Kugen; Singh, SurenConcerns of phosphorus pollution and its impact on environments have driven the biotechnological development of phytases. Phosphoric acid, inositol phosphate, or inositols are produced after hydrolysis of phosphate from phytate, initiated by phytase. Research over the last two decades on microbial phytases has deepened our understanding of their production, optimization, and characterization. Despite the wide availability of phytase producing microorganisms, only a few have been commercially exploited. The current high cost of phytases, inability to withstand high temperatures (>85 C), a limited pH range, and poor storage stability are a major bottleneck in the commercialization of phytases. The development of novel phytases with optimal properties for various applications is a major research challenge. In this paper, recent advances in microbial phytase production, application of tools to optimize higher enzyme production, and characterization of phytases along with potential biotechnological applications are reviewed. Additionally the development of phytase assay methods and functions of phytate and phytate degradation products are discussed.Item Evaluation of biohydrogen production potential of sugarcane bagasse using activated sludge in a dark fermentation process(2016) Reddy, Karen; Bux, Faizal; Kuttun Pillai, Sheena Kumari; Gupta, Sanjay KumarAnaerobic dark fermentation is an efficient biological process to produce hydrogen from waste material. In South Africa, this technology has not been explored adequately to extract energy from biological wastes. Within the KwaZulu Natal region of South Africa, the sugar industry is a prominent venture that produces mass quantities of sugarcane bagasse amongst other waste products. This by-product can be an ideal source of substrate for biohydrogen generation. In this study, sugarcane bagasse was used as the main substrate for biohydrogen production by anaerobic fermentation using sewage sludge as the inoculum. Different pre-treatment methods were employed to maximize the release of fermentable sugars from the lignocellulosic biomass. Among the different pre-treatment methods employed, the maximum sugar yield (294.4 mg/g) was achieved with 0.25% H2SO4 for 60 minutes at 121°C. Prior to inoculation, the sewage sludge was also subjected to thermal pre-treatment to eliminate methanogens. Thermal pre-treatment of inoculum sludge for 30 min was effective in eliminating methanogens. Fluorescence in situ hybridization was used to positively identify the hydrogen producing bacteria present before and after treatment. The pre-treated substrate and inoculum was integrated into a dark fermentation process to further optimize the effect of pH, substrate to biomass, iron and magnetite nanoparticles on hydrogen production. The maximum hydrogen production (1.2 mol/mol glucose) was achieved at a pH range of 5-6, a substrate to biomass ratio of 3.5, and iron and magnetite nanoparticle concentration of 200 mg/L. Microbial analysis using quantitative polymerase chain reaction has confirmed the dominance of Clostridium spp. in the reactor. The highest hydrogenase gene activity (number of copies of hydrogenase gene expression/ng DNA) was recorded in the reactor supplemented with magnetite nanoparticles with lowest being in the raw sludge. There was a direct positive correlation between the hydrogenase gene copy number and the hydrogen yield obtained at different reactor conditions. Scanning electron microscopy was a useful to visually analyse the interaction of microorganisms with activated sludge. This study highlights the significance of anaerobic microorganisms from waste sludge being able to utilize agricultural waste material to produce biohydrogen which could be further scaled up for continuous hydrogen production. In addition, statistical tools used to predict the possible sugar (Design of experiments) and hydrogen yields (Gompertz model) produced would be helpful in saving time during full-scale operation of biohydrogen producing reactors.Item Improvement of d-lactate productivity in recombinant Escherichia coli by coupling production with growth(Springer Netherlands, 2012-06) Zhou, Li; Tian, Kang-Ming; Niu, Dan-Dan; Shen, Wei; Shi, Fui-Yang; Singh, Suren; Wang, Zheng-XiangCoupling lactate fermentation with cell growth was investigated in shake-flask and bioreactor cultivation systems by increasing aeration to improve lactate productivity in Escherichia coli CICIM B0013-070 (ackA pta pps pflB dld poxB adhE frdA). In shake-flasks, cells reached 1 g dry wt/l then, cultivated at 100 rpm and 42°C, achieved a twofold higher productivity of lactic acid compared to aerobic and O2-limited two-phase fermentation. The cells in the bioreactor yielded an overall volumetric productivity of 5.5 g/l h and a yield of 86 g lactic acid/100 g glucose which were 66% higher and the same level compared to that of the aerobic and O2-limited two-phase fermentation, respectively, using scaled-up conditions optimized from shake-flask experiments. These results have revealed an approach for improving production of fermentative products in E. coli.Item Improvement of ethanol production from sugarcane molasses through enhanced nutrient supplementation using Saccharomyces cerevisiae(Academic Journals, 2012-03) Nofemele, Zuko; Shukla, Pratyoosh; Trussler, Arthur; Permaul, Kugen; Singh, SurenSaccharomyces cerevisiae as a yeast cream was utilized for alcoholic fermentation using sugar cane molasses. In the present study, fermentation was optimized for urea and yeast hydrolysate (YH) dosage and the combined effect was evaluated. Total sugars as inverts (TSAI) composition of molasses were -1 determined by HPLC as 39% (m/v). Urea concentrations of 4, 2 and 3 gl showed optimal ethanol -1 production at 30, 35 and 40°C respectively. A YH concentration of 0.5 gl resulted in an ethanol yield of 8.7% (m/v) with a fermentation efficiency of 85.12%. Under optimized conditions (35°C) significant improvements were noticed with ethanol yield of 7.8% (m/v) and efficiency of 76.3%.Item Development of a starter culture for the production of Gari, a traditional African fermented food(2010) Edward, Vinodh Aroon; Singh, Suren; Franz, Charles M. A. P.; Van Zyl, Petrus J.Cassava, (Manihot esculenta Crantz), is used for the production of a variety of West African foods and ranks fourth in the list of major crops in developing countries after rice, wheat and maize. Gari is one of the most popular foods produced from cassava. Cassava may contain high levels of linamarin, a cyanogenic glucoside, which in its natural state is toxic to man. Therefore, some processing methods that can enhance the detoxification of cassava and lead to the improvement of the quality and hygienic safety of the food are vitally important for less toxic products to be obtained. Quality, safety and acceptability of traditional fermented foods may be improved through the use of starter cultures. There has been a trend recently to isolate wild-type strains from traditional products for use as starter cultures in food fermentation. A total of 74 bacterial strains and 21 yeast strains were isolated from a cassava mash fermentation process in a rural village in Benin, West Africa. These strains were assessed, together with 26 strains isolated at the CSIR from cassava samples sent from Benin previously, for phenotypic and technological properties. Twenty four presumptive lactic acid bacteria (LAB) were selected for further phenotypic, genotypic and technological characterization during a research visit to the BFE (now Max Rubner Institute of Nutrition and Food). After assessment, the strains VE 20, VE 36, VE 65b, VE 77 and VE 82 were chosen for further study as starter cultures. These L. plantarum strains were chosen on the basis of predominance and possession of suitable technological properties. The investigation of this study was complemented by further, similar studies on further Gari isolates in Germany by the BFE. That study was done independently from this study, but both studies served to select potential starter cultures for cassava fermentation for the production of Gari, as this was the common goal of the project. Thus, a wider final selection of potential starter cultures was decided on at the project level and this selection was further tested in fermentation experiments. A total of 17 strains were grown in optimized media in 2 L fermenters. These strains were freeze-dried and thereafter tested in lab-scale cassava mash fermentation trials. xiii The strains performed well in the small scale bucket fermentations. There was a rapid acidification evidenced by the increase in titratable acidity, ranging from 1.1 to 1.3 % at 24 hours, and 1.3 to 1.6 % at 48 hours. The effect of the starter was obvious in that it lowered the pH much faster and to lower levels than the control. It appeared that both the processing and starter culture addition played a role in the removal of cyanide during processing of the cassava into Gari. This was evident from the lower cyanide values obtained for fermentations that included starter cultures. The study also showed that especially the L. plantarum group strains could be produced as starter cultures at lower costs than compared to L. fermentum, W. paramesenteroides or L. mesenteroides strains. Overall the results of this study were crucial for the project in showing that a starter culture which is easy and economical to produce and which has the desired attributes is a feasible possibility for application in the field.