Biotechnology

Occurrence of epibiont attachment on filamentous bacteria is a common phenomenon in activated sludge. In this study, an attempt has been made to elucidate the intrinsic nature of the attachment between the epibionts and filamentous bacteria based on microscopic observations. Characterization of the epiflora based on fluorescence in situ hybridization using group level probes revealed that the epibionts colonizing these filamentous bacteria largely belongs to the class Alphaproteobacteria, followed by Beta and Gammaproteobacteria. The ultrastructural examination using transmission electron microscopy pointed to the existence of a possible cell-to-cell interaction between epibionts and the selected filaments. Common bacterial appendages such as pili and fimbria were absent at the interface and further noted was the presence of cell membrane extensions on epibiont bacteria protruding towards the targeted filamentous cell. Fibrillar structures resembling amyloid-like proteins were observed within the filament cells targeted by the epibionts. An interaction was apparent between amyloid such as proteins and epibionts with regards to the direction of fibrillar structures and the distance of approaching epibiont bacteria. Due to the lack of visual evidence in support of penetration, the role of these amyloid-like fibrils as potential attachment sites for the epibionts was taken into consideration, and required further validation using conformational antibodies.

Occurrence of epibiont attachment on filamentous bacteria is a common phenomenon in activated sludge. In this study, an attempt has been made to elucidate the intrinsic nature of the attachment between the epibionts and filamentous bacteria based on microscopic observations. Characterization of the epiflora based on fluorescence in situ hybridization using group level probes revealed that the epibionts colonizing these filamentous bacteria largely belongs to the class Alphaproteobacteria, followed by Beta and Gammaproteobacteria. The ultrastructural examination using transmission electron microscopy pointed to the existence of a possible cell-to-cell interaction between epibionts and the selected filaments. Common bacterial appendages such as pili and fimbria were absent at the interface and further noted was the presence of cell membrane extensions on epibiont bacteria protruding towards the targeted filamentous cell. Fibrillar structures resembling amyloid-like proteins were observed within the filament cells targeted by the epibionts. An interaction was apparent between amyloid such as proteins and epibionts with regards to the direction of fibrillar structures and the distance of approaching epibiont bacteria. Due to the lack of visual evidence in support of penetration, the role of these amyloid-like fibrils as potential attachment sites for the epibionts was taken into consideration, and required further validation using conformational antibodies.

Optimization of multiple objectives and constraints of fermentation process problems have been extensively studied in recent years. High-performance, robust, cost-effective and reliable computing models that provide innovative solutions to fermentation problems have been developed. This paper reviews the problems of the optimal design of batch fermentation technology and the development of computer-based solving problems using evolutionary algorithms (EAs) to generate optimal or nearoptimal solutions to the problems in the fermentation industries. In this review, the latest developments of evolutionary algorithm techniques are focused and optimization of fermentation processes improvement by the techniques is presented.

Evolutionary algorithms are widely used in single and multi-objective optimization. They are easy to use and provide solution(s) in one simulation run. They are used in food processing industries for decision making. Food processing presents constrained and unconstrained optimization problems. This paper reviews the development of evolutionary algorithm techniques as used in the food processing industries. Some evolutionary algorithms like genetic algorithm, differential evolution, artificial neural networks and fuzzy logic were studied with reference to their applications in food processing. Several processes involved in food processing which include thermal processing, food quality, process design, drying, fermentation and hydrogenation processes are discussed with reference to evolutionary optimization techniques. We compared the performances of different types of evolutionary algorithm techniques and suggested further areas of application of the techniques in food processing optimization.

Lactic acid bacteria (LAB) were isolated from raw and fermented milk samples. They were evaluated for hydrogen peroxide (H 2 O 2 ) production. Sixty-three strains of LAB belonging to the genera Lactobacillus, Leuconostoc, Lactococcus and Streptococcus were isolated and all the isolates produce H 2 O 2 with Lactococcus lactis having the highest yield of 0.4279 mg/L in de Mann Rogosa Sharpe (MRS) broth. Among the high-level H 2 O 2 -producing strains are Lactobacillus bulgaricus, Lactobacillus casei, L. lactis and Streptococcus thermophiles. They were subsequently selected and their culture supernatants were evaluated at different temperatures, pH values and using different carbon and nitrogen sources. The highest quantity of H 2 O 2 (0.6517 mg/L) was produced at 37°C by S. thermophilus, while L. casei produced the lowest quantity of 0.1132 mg/L at 40°C. S. thermophilus produced the highest quantity of 0.5912 mg/L at pH 5.5, while the lowest quantity of 0.1042 mg/L was produced at pH 7. L. bulgaricus produced the highest concentration of0.6512 mg/L when galactose was used as carbon source and lowest quantity of 0.0210 mg/L was produced by L. lactis in basal medium containing sorbitol as carbon source. L. casei produced both the highest and lowest quantities of 0.1895 and 0.0207 mg/L of H 2 O 2 in medium supplemented with yeast extract and ammonium sulphate as nitrogen sources respectively. The antimicrobial activity of the culture supernatants against Escherichia coli K12, Staphylococcus aureus and Candida albicans using agar well diffusion assay was evaluated. Cell-free supernatant by L. bulgaricus had the highest inhibitory activity against E. coli K12 with 21 mm zone of inhibition and against S. aureus with 15 mm zone of inhibition but showed no antagonistic activity against C. albicans. The study revealed that lactic acid bacteria isolated from raw and fermented milk in South-West Nigeria are capable of producing hydrogen peroxide which has antagonistic effect on pathogenic organisms, thus, may be promising sources of preservative that may in future be applied to food.

The aim of the study was to investigate the use of dark fermented paperboard mill wastewater (PMW) containing volatile fatty acids for polyhydroxyalkanoates (PHA) production. Six sequencing batch reactors (SBRs) were initially fed with synthetic feed containing acetate and operated at different organic loading rates (OLRs) of 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 gCOD l -1 day -1 for PHA-storing bacteria enrichment. The maximum PHA content and yield (Y PHA/S ) were 67.44 ± 5.88 % and 0.45 ± 0.39 C-mol Cmol -1 at OLR of 1.5 gCOD l -1 day -1 , respectively. The synthetic feed was replaced by dark fermented PMW effluent rich in acetate, butyrate, propionate, and lactate at the accumulation stage resulting in a PHA content and yield of 58.57 ± 4.02 % and 0.46 ± 0.09 C-mol C-mol -1 , respectively. The maximum specific PHA production rate (q PHA max ) amounted to 0.29 ± 0.1 C-mol C-mol -1 X -1 h -1 . Illumina MiSeq sequencing of bacterial 16S rRNA gene showed that Proteobacteria and Bacteroidetes increased from 37.4 to 77.6 % and from 2.49 to 17.66 % at enrichment and accumulation stages, respectively. Actinobacteria (15.44 %), Chloroflexi (8.15 %), Planctomycetes (7.46 %), and Acidobacteria (6.0 %) were detected at the enrichment SBRs.

Lactic acid bacteria (LAB) were isolated from raw and fermented milk samples. They were evaluated for hydrogen peroxide (H 2 O 2 ) production. Sixty-three strains of LAB belonging to the genera Lactobacillus, Leuconostoc, Lactococcus and Streptococcus were isolated and all the isolates produce H 2 O 2 with Lactococcus lactis having the highest yield of 0.4279 mg/L in de Mann Rogosa Sharpe (MRS) broth. Among the high-level H 2 O 2 -producing strains are Lactobacillus bulgaricus, Lactobacillus casei, L. lactis and Streptococcus thermophiles. They were subsequently selected and their culture supernatants were evaluated at different temperatures, pH values and using different carbon and nitrogen sources. The highest quantity of H 2 O 2 (0.6517 mg/L) was produced at 37°C by S. thermophilus, while L. casei produced the lowest quantity of 0.1132 mg/L at 40°C. S. thermophilus produced the highest quantity of 0.5912 mg/L at pH 5.5, while the lowest quantity of 0.1042 mg/L was produced at pH 7. L. bulgaricus produced the highest concentration of0.6512 mg/L when galactose was used as carbon source and lowest quantity of 0.0210 mg/L was produced by L. lactis in basal medium containing sorbitol as carbon source. L. casei produced both the highest and lowest quantities of 0.1895 and 0.0207 mg/L of H 2 O 2 in medium supplemented with yeast extract and ammonium sulphate as nitrogen sources respectively. The antimicrobial activity of the culture supernatants against Escherichia coli K12, Staphylococcus aureus and Candida albicans using agar well diffusion assay was evaluated. Cell-free supernatant by L. bulgaricus had the highest inhibitory activity against E. coli K12 with 21 mm zone of inhibition and against S. aureus with 15 mm zone of inhibition but showed no antagonistic activity against C. albicans. The study revealed that lactic acid bacteria isolated from raw and fermented milk in South-West Nigeria are capable of producing hydrogen peroxide which has antagonistic effect on pathogenic organisms, thus, may be promising sources of preservative that may in future be applied to food.

Optimization of multiple objectives and constraints of fermentation process problems have been extensively studied in recent years. High-performance, robust, cost-effective and reliable computing models that provide innovative solutions to fermentation problems have been developed. This paper reviews the problems of the optimal design of batch fermentation technology and the development of computer-based solving problems using evolutionary algorithms (EAs) to generate optimal or nearoptimal solutions to the problems in the fermentation industries. In this review, the latest developments of evolutionary algorithm techniques are focused and optimization of fermentation processes improvement by the techniques is presented.

Evolutionary algorithms are widely used in single and multi-objective optimization. They are easy to use and provide solution(s) in one simulation run. They are used in food processing industries for decision making. Food processing presents constrained and unconstrained optimization problems. This paper reviews the development of evolutionary algorithm techniques as used in the food processing industries. Some evolutionary algorithms like genetic algorithm, differential evolution, artificial neural networks and fuzzy logic were studied with reference to their applications in food processing. Several processes involved in food processing which include thermal processing, food quality, process design, drying, fermentation and hydrogenation processes are discussed with reference to evolutionary optimization techniques. We compared the performances of different types of evolutionary algorithm techniques and suggested further areas of application of the techniques in food processing optimization.

In this study, a novel halophilic cyanobacterium was isolated and identified as Euhalothece sp. KZN. This fast-growing strain had the ability to synthesise high yields (12 mg g −1) of C-phycocyanin (C-PC), a highly fluorescent blue light-harvesting pigment with numerous potential uses in the biotechnology and commercial sectors. This study elucidated the individual and interactive role of different nutrients in BG11 growth medium for enhancing C-PC production in Euhalothece sp. KZN. Nine components of BG11 medium were screened for their effects via fractional factorial design (FFD). The results revealed a significant influence of nutrients, viz. MgSO 4 , NaNO 3 and minor nutrients (citric acid, EDTA-iron citrate, CaCl 2 and Na 2 CO 3) on C-PC yield. These three components were further explored for their optimum concentration for enhancing C-PC production using a central composite design. The optimum values for these essential nutrients were found to be as follows: 0.10 g L −1 of MgSO 4 , 1.67 g L −1 of NaNO 3 and 10 mL L −1 of minor nutrients which resulted in a 280% increase in C-PC yield with predicted and actual values of 43.97 and 45 mg g −1 , respectively. Euhalothece sp. KZN is a strong potential candidate for C-PC production and can be further exploited to produce this industrially valuable compound.

Cyanobacteria are photosynthetic microorganisms that inhabit diverse ecological habitats and are capable of producing wide range of natural products and bioactive metabolities including peptides, vitamins, enzymes and pigments such as phycobiliproteins. Amongst the group of phycobiliproteins, C-Phycocyanin (C-PC) is a light-harvesting accessory pigment known to possess excellent biotechnological applications due to their intense colour, fluorescent properties and health benefits. This study has focused on the characterisation and full genome analysis of a unique indigenous halophilic cyanobacterium capable of overproducing the pigment phycocyanin (C-PC). Further, development of a cost-effective extraction method for high purity C-PC and characterisation of the purified C-PC was accomplished. The strain was isolated from a hypersaline environment in KwaZulu-Natal, South Africa and was found to possess several unique traits such as its ability to accumulate high amount of phycocyanin, tolerance to high salinity (up to 180 g/L), ability to grow under varying growth conditions and high growth rate. The taxonomic identity of the isolate was revealed using a polyphasic approach including cell morphology, growth conditions, pigment composition, 16S rRNA analysis. The cells were oval to rod-shaped, 14-18 μm in size, and contained majority of C-PC, as well as some allophycocyanin and chlorophyll. The strain was moderately thermotolerant (35°C), alkalitolerant (pH 8.5) and was halophilic with an optimum NaCl of 120 g/L. Based on the 16S rRNA gene sequence phylogeny, the strain was found to be related to members of the 'Euhalothece' subcluster (99%). Further, the whole genome sequence was also determined, and the annotated genes have shown sequence similarity (90%) to the gasvacuolate, spindle-shaped Dactylococcopsis salina PCC 8305. Based on the above results, the strain is considered to represent a novel species of Euhalothece. The size of the genome was determined to be 5,113,178 bp and contained 4332 protein-coding genes and 69 RNA genes with a GC content of 46.7%. The full genome sequence analysis also provided important information about the strain which facilitated the identification of key genes and proteins necessary for C-PC synthesis and salt acclimation. Genes encoding osmoregulation, oxidative stress, heat shock, persister cells, and UV-absorbing secondary metabolites, among others, were also identified. Further, single factor experiments were performed to optimise the factors (extraction buffers, freezing time, biomass:buffer ratio and lysozyme concentration) essential for C-PC extraction from cyanobacteria. A range of buffers viz., acetate, potassium phosphate (PPB), sodium vi phosphate (SPB), phosphate buffered saline (PPBS), Tris-chloride and double distilled water (control) with different pH and concentrations were investigated. Cell lysis was carried out by freezing the cells at different temperatures viz., at-196, and-80, and-20°C, and by thawing at 4 and 25°C. The freezing and thawing time varied from 0.5-24 h. Based on the results obtained, thawing temperature, enzyme concentration and biomass-buffer ratio were further selected for optimisation for maximum C-PC yield and purity using response surface methodology (RSM). Under optimised conditions, the yield of crude C-PC was increased to 78 mg/g (>90% percentage increase) with a purity index of 2.5 compared to extraction prior to optimisation. The crude C-PC was further purified using 6% w/v of activated charcoal combined with a two-step ammonium sulphate (NH 4 SO 4) precipitation and ultrafiltration which resulted in high yield analytical grade C-PC with a purity index of 5. The purified C-PC showed a single absorption peak at 620 nm and emission at 640 nm. Based on the amino acid analysis the calculated molecular weight of α-and β-subunits were found to be 17.7 and 18.4 kDa respectively, which corresponded to the two bands seen on the SDS-PAGE. Additionally, the primary, secondary and tertiary structures of the C-PC was also evaluated based on the amino acid sequence obtained from the genome sequence. The C-PC physiochemical properties such as the molecular weight, isoelectric point, extinction coefficient, half-life, aliphatic index, amino acid property, instability index and Grand Average of Hydropathicity was predicted based on the in-silico analysis of the amino acid sequences. The physicochemical properties revealed that these proteins are non-polar and stable. Multiple sequence alignment analyses of the α-and β-subunits displayed significant differences amongst the amino acid residues of hypersaline/marine and freshwater cyanobacteria. These amino acids play a vital role in the stability of the C-PC. The secondary structure prediction of the α-and β-subunits consisted of > 50% of amino acid residues in α-helices, with 9-13% of amino acid residues in the extended strand. The stability of the purified C-PC under different conditions were investigated. The optimum pH range for purified C-PC was found to be 5.0-7.0 and was found to be stable up to 45ºC. However, the relative concentration C-PC (C R %) and thermostability of the purified C-PC was observed to be pH dependent, a lower pH improved the stability at higher temperatures and vice-versa. An IC 50 value of 0.540 ± 0.02 mg/mL was also observed using the DPPH assay indicating a higher antioxidant potential of the C-PC. C-phycocyanin exhibited a maximum absorbance of 1.37 ± 0.05 by ferric ion reducing assay. The presence of a high level of nonvii polar and aromatic residues such as Ala, Gly Glu, Leu, Arg, Ser, and Val could be regarded as an indication of higher antioxidant activity levels of the C-PC. Addition of preservatives sodium azide and sodium citrate (at 4°C) proved to be suitable for preservation of C-PC for up to 42 weeks. This research contributed to our understanding of molecular, cellular and biochemical mechanisms of the C-PC biosynthesis as well as newly identified metabolites in cyanobacteria. The study has also demonstrated an efficient extraction method for analytical grade C-PC from cyanobacterial strains for potential applications in biotechnological biomedical industries. viii

Cyanobacteria contain phycocyanin a light harvesting pigment found to have numerous biotechnological applications, such as: a natural colorant in food and cosmetics, fluorescent tags employed in clinical and immunological research and also in therapeutic processes. Successful phycocyanin production depends on growth characteristics, ability to accumulate high quantities of the pigment, and an effective downstream process. Therefore, the aim of this research was to optimize the extraction method and production by determining the optimal cultivation conditions for phycocyanin producing cyanobacterium. This cyanobacterium was isolated from a hypersaline water body in Kwa-Zulu Natal, and subsequently purified using traditional streak and spread plate techniques. Different cell disruption techniques and a range of buffers were evaluated for the extraction of phycocyanin. The buffer concentrations and pH was subsequently optimized. Results showed that maximum phycocyanin was extracted when cells were suspended in 50mM sodium phosphate buffer (pH-7.5) supplemented with 10 % lysozyme and then disrupted using the freeze-thaw method at-20 & 4°C. The UV-Vis absorption spectral scan of the crude extracted pigments showed a peak at 620 nm. This corresponds to phycocyanin production. Unwanted proteins were removed using a 25and 50% saturated ammonium sulphate precipitation, followed by dialysis. SDS-PAGE showed two subunits with molecular masses of 19 and 20 kDa. These masses corresponded to phycocyanin α and β subunits. Furthermore, a food grade purity ratio (A615/A280) of 1.20 was achieved. The effects of various abiotic factors (temperature, light and pH) on growth and phycocyanin production of the Cyanothece sp. was investigated. Temperature ranging from 20-45°C and pH (5-10) was evaluated for 2 weeks. Cultures were then subjected to four photoperiods (24:0, 18:06 12:12 and 8:16 h light: dark) three light intensities (25, 75 and 125 µmol photons per m 2 per-s) at varying wavelengths i.e. blue, red and green and Grolux light. Ideal conditions were observed at 35°C, 125 µmol photons.m 2 .s-1 of Grolux light for a 16:8 light and dark photoperiod. It was observed that the highest biomass and phycocyanin production was found to be at 35°C, temperatures below or above resulted in a decrease in both growth and pigment synthesis. Phycocyanin concentration changed in response to light quality and intensity. A significantly higher (p<0.05) phycocyanin yield was found when the culture was exposed to 125 µmol photons.m 2 .s-1 of Grolux light compared with the other three light conditions. Using Design vii of experiments, a series of fractional factorial experiments were carried out to optimize media components for pigment production. The final optimized growth medium was determined from a central composite design using response surface plots together with a mathematical point-prediction tool and consisted of 2g/L NaNO 3, 0.06g/L K 2 HPO 4, 0.12 g/L MgSO 4 .7H 2 O, 0.033 g/L CaCl 2 .2H 2 O, 100g/L NaCl, 12mL minor nutrients and 0.5 trace metal. A 72 % increase in phycocyanin was observed. This research revealed that this particular Cyanothece sp. shows great potential as a reliable source of phycocyanin. viii DEDICATION This dissertation is dedicated to: My husband, Kirshlin Mogany for his endless love and encouragement. The road we travel together was not easy but it was worth every step. Through good times and the worst times, you have been a there with me. & My son, Drelin Cahil Mogany, my true inspiration and hope. He has taught every challenge can be overcome with hardwork, patience and determination, ix ACKNOWLEGEMENTS First and foremost, I would like to thank God, the provider of the blessings, for giving me the ability, faith, health and patience to accomplish this goal. To my parents, who has being with me from the very start till this day, thank you for giving me strength, wisdom, and for making this dream come true for me. I am grateful to have continued support in my pursuit of happiness in my career. My sister and little brother deserve my whole hearted thanks as well. Special thanks to my husband, you are one in a million. I am forever grateful for your neverending support, love, patience, understanding and inspiration to achieve my goals in life. I wouldn't have made it this far without you. Thanks to Prof Bux for giving me this opportunity to research under his guidance and support. Your mentorship has helped me through my master's program and will continue to be useful throughout my career. Many thanks to Dr. Swalaha for the guidance of the understanding statistic methods, critically reading of my research and all the input you have given me throughout. Your open door has helped me through many difficult times that research can entail. Also thanks to Dr. Kumari for assisting with molecular techniques, and your valuable input. My gratitude goes to all colleagues and friends at the Institute for Water and Wateswater Technology, both past and present for their invaluable help and companionship while performing this research. Knowing your support is behind me has made this possible. To the National Research Foundation and Durban University of Technology for funding this research project and providing the facilities to make this study possible. Money is not everything but your financial support saw me through this all, my sincere appreciation goes to you.

Concerns 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.

Concerns 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.

Optimization of process parameters for phytase production by Enterobacter sp. ACSS led to a 4.6-fold improvement in submerged fermentation, which was enhanced further in fed-batch fermentation. The purified 62 kDa monomeric phytase was optimally active at pH 2.5 and 60°C and retained activity over a wide range of temperature (40-80°C) and pH (2.0-6.0) with a half-life of 11.3 min at 80°C. The kinetic parameters K m , V max , K cat , and K cat /K m of the pure phytase were 0.21 mM, 131.58 nmol mg-1 s-1 , 1.64 9 10 3 s-1 , and 7.81 9 10 6 M-1 s-1 , respectively. The enzyme was fairly stable in the presence of pepsin under physiological conditions. It was stimulated by Ca ?2 , Mg ?2 and Mn ?2 , but inhibited by Zn ?2 , Cu ?2 , Fe ?2 , Pb ?2 , Ba ?2 and surfactants. The enzyme can be applied in dephytinizing animal feeds, and the baking industry.

Ultraviolet (UV) light, specifically UV-C, has been successfully used as a process for the disinfection of air, water and surfaces (Guerrero-Beltran and Barbosa-Canovas, 2004; Keyser et al., 2008). In recent years, the use of UV-C to sterilize liquids has been studied (Choi and Nielsen, 2005). The UV disinfection system (SurePure Photopurification System) used in this study emits ultraviolet light (UV-C) in the germicidal wavelengths between 240 and 280nm that is of sufficient energy and time to inactivate contaminating microorganisms, such as bacteria, yeasts, moulds and viruses in treated liquids. In this range, UV-C is able to cause DNA lesions by the formation of dimers between pyrimidine units. These dimers inhibit replication and transcription processes, which lead to no cell growth and ultimately cell death of the organism (Koutchma et al., 2009). UV-C has proven to be effective in inactivating contaminating organisms without any secondary effects on the nutritional, sensory ...

Amadumbe (Colocasia esculenta), commonly known as Taro is an indigenous underutilised crop of Southern Africa. Limited utilisation of amadumbe grown in the region may be attributed to the lack of enough information on its corms and storage proteins. In this study, albumin, and globulin fractions from three landraces were investigated for their physicochemical and functional properties. Crude protein contents of amadumbe flours ranged between 2-4%. Amadumbe flours were rich in lysine (5.3 g/100 g) and leucine (9.1 g/100 g). Three major protein families were identified corresponding to albumin and globulin with approximately 55-60 kDa, 20-22 kDa and 14 kDa, respectively. FTIR revealed a protein secondary structure dominated by β-sheets and β-turns. Intrinsic fluorescence results suggested that all albumin and globulin fractions had well-defined tertiary structures. The protein solubility of albumin and globulin fractions were minimal at pH 5. Hydrophobicity data seemed to correlate with emulsification behaviour of the protein fractions. Amadumbe proteins and its albumin and globulin fractions can be a promising source for food protein applications.

Methyl-2-(3-bromophenylamino)-4-(4-chlorophenyl)-6-methyl-1,4dihydropyrimidine-5-carboxylate DHPM 2 Methyl-4-(4-chlorophenyl)-2-(2-hydroxy-4-nitrophenylamino)-6methyl-1,4-dihydropyrimidine-5-carboxylate DHPM 3 Methyl-2-(3-bromo-4-fluorophenylamino)-4-(4-chlorophenyl)-6methyl-1,4-dihydropyrimidine-5-carboxylate DHPM 4 Methyl-4-(4-chlorophenyl)-2-(4-cyanophenylamino)-6-methyl-1,4dihydropyrimidine-5-carboxylate DHPM 5 Methyl-4-(4-chlorophenyl)-6-methyl-2-(3-(trifluoromethylthio) phenylamino)-1, 4-dihydropyrimidine-5-carboxylate DHPM 6 Methyl-2-(4-bromophenylamino)-4-(4-chlorophenyl)-6-methyl-1,4dihydropyrimidine-5-carboxylate DHPM 7 Methyl-2-(3-chloro-5-hydroxyphenylamino)-4-(4-chlorophenyl)-6methyl-1,4-dihydropyrimidine-5-carboxylate DHPM 8 Methyl-4-(4-chlorophenyl)-2-(2,4-dimethoxybenzylamino)-6-methyl-1,4-dihydropyrimidine-5-carboxylate DHPMs Dihydropyrimidines DISC Death-inducing signaling complex DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DPPH 2, 2-diphenyl-1-picrylhydrazyl EtOH Ethyl alcohol FADD Fas-Associated protein with death domain FCS Foetal calf serum Gln Glutamine Glu Glutamate

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