Doctoral Degrees (Microbial, Biochemical and Food Biotechnology)

Permanent URI for this collection

http://hdl.handle.net/11660/446

Browse

Now showing 1 - 20 of 59

Open Access
Activation of the SARS-CoV-2 spike protein by 𝘤𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 secreted proteases
(University of the Free State, 2023) Mjokane, Nozethu; Sebolai, O. M.; Pohl, C. H.; Albertyn, J.; Gcilitshana, O. M. N.
The thesis is not structured in a classical way. As such, it is composed of a literature review section (Chapter 1) and two research chapters (Chapters 2 and 3). A general discussion section (Chapter 4) and addendums are also included. As some chapters are in a publication format, repetition of essential information could not be avoided. Chapter 1 reviews the emergence of SARS-CoV-2 and its impact. In particular, it considers the co-infection of this virus with respiratory fungal pathogens, which are major independent risk factors that complicate COVID-19 by causing a more severe infection resulting in higher mortality than that of either infection on its own. These fungal pathogens secreted furin-like proteases to further their virulence during host invasion. In this context, the thesis argues that it is foreseeable that the virus could also access these fungal furin-like proteases and pervert them in order to activate its latent spike protein. Therefore, this set up a number of questions, which are addressed in the thesis concerning the possible activation of the viral latent spike protein by fungal furin-like proteases. In Chapter 2, it was sought to characterise 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 (𝘊.) neoformans proteases and assess if they could theoretically bind to the SARS-CoV-2 spike protein. To be specific, previous papers reporting on cryptococcal serine proteases were perused, and this made it possible to select a number of proteases, namely cryptococcal serine carboxypeptidase (CNBF4600), cryptococcal cerevisin (CNBJ2870) and cryptococcal peptidase (CNBA1340), cryptococcal peptidase (CNAG_00150) and cryptococcal cerevisin (CNAG_04625). By designing specific primers, it was possible to show that these serine proteases were expressed in 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 H99, the prototypical cryptococcal strain used in this thesis. Therefore, the expressed gene products were expected to be secreted into the culture media. This was important for the work that follows in Chapter 3. Through using the computational programme, High Ambiguity Driven protein-protein DOCKing (HADDOCK), it was possible to show that some of the selected cryptococcal serine proteases could interact with the coronavirus spike protein and yield a binding affinity greater than and comparable to furin. However, as HADDOCK is a computational programme, the predicted binding affinities might not correlate with the experimental binding affinities in solution, more so since the used 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 proteases structures were predicted and not solved. To account for this, Chapter 3 sought to provide enzymatic evidence using the collected culture media – in the form of supernatant. To do this, a mimetic fluorogenic peptide of the SARS-CoV-2 spike protein was designed and modified to have intra-molecular fluorescence quenching capability using 7-methoxycoumarin-4-yl acetyl (MCA) at the N-terminus and N-2,4-dinitrophenyl (DNP) at the C-terminus. The assay was performed using the cryptococcal supernatant. For reference, recombinant furin was included as this is the serine protease present in humans that catalyses the activation of the spike protein. Here, it was determined that cryptococcal serine proteases present in the supernatant could cleave the mimetic spike protein at S1/S2 site with biochemical efficiency comparable to furin. To test the veracity of these data, SARS-CoV-2 pseudovirion containing a full-length spike protein was used. It was possible to show that the pseudovirion could be transduced into HEK-293T cells in the presence of the cryptococcal supernatant. Chapter 4 takes into account the obtained results and provides a summary of the major observations. Of note, the thesis theorises that yeast kexin proteases are responsible for the observed activity. This is because there is a functional homology between yeast kexin proteases and furin (both are convertases); thus, it is reasonable that the supernatant (which contains yeast kexin proteases) could activate the latent SARS-CoV-2 spike protein. The thesis further proves that other respiratory fungal pathogens have yeast kexin proteases that activate the spike protein. This evidence is documented in Addendum no. 1. All things considered, the findings point to the regulation of protease activity as a viable approach to control the activation of the spike protein by either mammalian protease or fungal proteases. To this end, protease inhibitors could be used to control unwanted proteolysis. Addendum no. 2 attempted to show this. Here, it was possible to show that the South African-based medicinal plant Artemisia tea infusion extract and its active compound artemisinin could control the activation of the mimetic SARS-CoV-2 spike protein by furin but not the supernatant. The latter highlights the need to purify the supernatant and isolate yeast kexin proteases. The idea of exploring the control of unwanted proteolysis is also an interventional measure considered by Pfizer, the pharmaceutical company. This American multinational pharmaceutical and biotechnology corporation successfully piloted Paxlovid to control SARS-CoV-2. This drug contains an anti-protease (PF-07321332) that inhibits the protease (SARS-CoV-2 3CLp) responsible for viral replication.
Open Access
Biocatalytic resolution of epoxides: epoxide hydrolases as chiral catalysts for the synthesis of enantiomerically pure epoxides and vic diols from α-olefins
(University of the Free State, 1999-06) Botes, Adriana Leonora; Smit, M. S.; Litthauer, D.
English: The synthesis of chiral pharmaceuticals in an enantiopure form had become increasingly important in the last few years. This same trend is now found in the synthesis of agrochemicals. Epoxides, due to their high reactivity with a large number of reagents, and vie diols, employed as their corresponding cyclic sulfates or sulfites as reactive intermediates, are versatile chiral synthons in the synthesis of many bioactive compounds. Extensive research efforts have thus been directed towards the synthesis of optically active epoxides and viel diols. Kinetic resolution of racemic epoxides by epoxide hydrolases has recently emerged as a very attractive strategy for the synthesis of enantiopure epoxides. Both chemical and biological catalysts that may be employed to obtain enantiopure epoxides from relatively inexpensive racemic substrates had been reviewed (Chapter 1). The potential use of microbial epoxide hydrolases, including those from yeasts as elucidated during this study, was emphasised in this review. At the onset of this study, epoxide hydrolase activity had been identified in only one yeast, Rhodotorula glutinis. The broad range of substrates that were hydrolyzed with excellent enantioselectivity by this yeast, indicated that yeast epoxide hydrolases might be very interesting catalysts. This had indeed been found to be true during the course of this study. Enantioselective hydrolysis of a homologous range of aliphatic 1,2- epoxyalkanes was accomplished in collaboration with the group of Jan de Bont (Division Industrial Microbiology, Wageningen AU, The Netherlands). No other microbial epoxide hydrolases have been found that display this unique enantioselectivity for epoxides lacking other substituents (Chapter 2). Extensive screening of yeasts from the renowned UOFS Yeast Culture Collection revealed that epoxide hydrolase activity was constitutively present in about 20% of the yeasts screened, and that other basidiomycetous yeasts from the genera Rhodotorula, Rhodosporidium and Trichosporon shared this unique enantioselectivity for 1,2- epoxyoctane with Rhodotorula glutinis (Chapter 3). he apparent association between carotinoid production and epoxide hydrolase activity in bacteria as well as the red yeasts Rhodotoru/a and Rhodosporidium, prompted us to investigate the epoxide hydrolase activity of the yellow pigmented bacterium Chryseomonas /uteo/a in our collection. Indeed, this bacterium displayed epoxide hydrolase activity, and moderate enantioselectivity for 1,2-epoxyalkanes (E =20) by a bacterial epoxide hydrolase was found for the first time (Chapter 4). A survey of the enantioselectivities of yeasts for a homologous range of 1,2- epoxyalkanes, 1,2-epoxyalkenes as well as the 2,2-disubstituted 2-methyl-1,2- epoxyheptane and benzyl glycidyl ether was conducted. Excellent biocatalysts for C-5 to C-8 epoxyalkanes and the C-8 epoxyalkene were found. The epoxide hydrolases from all the enantioselective yeasts were found to be membrane-associated (Chapter 5). The epoxide hydrolase from the yeast Rhodosporidium toru/oides was purified in an elegant one-step protocol from the microsomal fraction, using affinity chromatography (Chapter 6). However, initial attempts to obtain amino-acid sequences failed. In lieu of information about the primary structure of yeast epoxide hydrolases, inactivation of the enzyme by modification of specific amino acids was studied. Asp/Glu and His residues were found to be essential for catalytic activity. In addition, it was found that one or more Ser residues in the catalytic site are indispensible for catalytic activity. These results indicate that yeast epoxide hydrolases probably belong to the same subfamily of a,l3- hydrolase fold enzymes as the microsomal epoxide hydrolases from other eukaryotes. Unusual kinetic behaviour was observed during the hydrolysis of 1,2-epoxyalkanes by purified epoxide hydrolase. Hydrolysis was characterised by a strong dependence of enantioselectivity on the presence of the substrate as a second (Iypophilic) phase. The purified epoxide hydrolase was not very stable, with a half-life time at 35°C of 18 hours (Chapter 7).
Open Access
Biological synthesis of gold nanoparticles by Thermus scotoductus SA-01
(University of the Free State, 2010-01) Van Marwijk, Jacqueline; Van Heerden, E.
English: The usual strategy to prepare gold nanoparticles involves the reduction of a gold salt in solution by various reducing agents in the presence of a stabilizer. These particles are mostly spherical with poor monodispersity. An alternative means is to use biological material to mediate particle synthesis. Microorganisms such as fungi have demonstrated the ability to produce nanoparticles of different shapes and sizes extending beyond the scope of chemical means, and the microbial interaction with metals also supply eco-friendly methods for nanoparticle production. It has been hypothesized that the proteins involved in nanoparticle synthesis require a co-factor such as NADH / NADPH, as previous studies have indicated that NADH- and NADPH-dependent enzymes are important factors in the biosynthesis of metal nanoparticles. Thermus scotoductus SA-01, a thermophilic bacterium, isolated from an AngloGold Ashanti mine near Carletonville, Republic of South Africa, was used for purification of a gold(III) reducing and nanoparticle synthesizing protein. This bacterium has the ability to produce gold nanoparticles, and more than one pathway can be followed to produce these particles. A protein was purified to homogeneity by using a combination of several liquid chromatography resins. The N-terminal sequence was obtained by using automated Edman degradation. The protein purified is not a classical oxido-reductase and was identified as an ABC transporter peptide-binding protein (~70kDa). This discovery shows that gold nanoparticles can be produced by proteins other than oxidoreductases. The interaction of the protein extracted and purified from Thermus scotoductus SA-01, as well as the recombinant proteins, with liquid gold under varying physico-chemical conditions have been studied using TEM, EDS, and by measuring the plasmon resonance band, to illustrate the effect on particle morphology and to elucidate the protein mechanism. The size and the shape of particles could, to an extent, be manipulated by controlling the environmental parameters. The purified protein as well as the recombinant proteins was only able to produce nanoparticles in the presence of sodium dithionite and it is thus hypothesized that the donation of electrons via the disulphide bridge in the protein is involved in the reduction of the gold ions. Even though the recombinant proteins had the ability toproduce nanoparticles they were not as efficient as the native protein, but when the optimum parameters for the recombinant proteins are established they could be used in the upscale production of gold nanoparticles or gold nanosheets.
Open Access
Biology of botryosphaeria dothidea and sphaeropsis sapinea as endophytes of eucalypts and pines in South Africa
(University of the Free State, 2001-12) Smith, Hendrik; Wingfield, M. J.; Coutinho, T. A.; Crous, P.W.
English: Botryosphaeria dothidea and Sphaeropsis sapinea both very important pathogens in the South African forestry context. These fungi are well established in the country and contribute substantially to annual losses incurred. Currently very little can be done to control the fungi and the damage they cause. The understanding of their respective disease etiologies is thus of great importance to develop relevant counter measures. The overall aim of this dissertation was to investigate various poorly understood aspects of these two fungi and to try and relate the results to practical contributions towards controlling the impact the two pathogens have. Studies have been conducted during the course of five years and each study represents an independent research investigation. The introductory chapter presents a review of the literature pertaining to all aspects of biology, history and taxonomy of B. dothidea and S. sapinea. The two fungi are clearly very similar in all these aspects and perhaps the only clear difference is that S. sapinea is restricted to pines in South Africa. Many other similarities and some differences between these two important pathogens are highlighted and many of these have provided the background for further investigations. In chapter two the presence of B. dothidea and S. sapinea lS demonstrated as symptomless endophytes in healthy, pine and eucalypt tissue. Botryosphaeria dothidea was found to be common in all the Eucalyptus spp. tested, occurring at high percentages in symptomless leaves of Eucalptus smithii, E. camaldulensis, E. grandis and E. nitens. Sphaeropsis sapinea was, in contrast, only present in young, green Pinus patuIa and P. radiata cones, but virtually absent from the cones of P. elliottii and P. taeda. Botryosphaeria dothidea is associated with die-back and canker diseases of eucalypts in South Africa. Despite this fact, little is known about the infection process. The fungus is known to occur endophytically in leaves of various Eucalyptus species in South Africa. In chapter three I consider the ability of B. dothidea to infect apparently healthy Eucalyptus leaves and the subsequent location and structure of these infections once inside leaf tissue. Scanning electron microscopy revealed that conidia of B. dothidea can infect healthy leaves through stomata. These infections ultimately reside amongst mesophyll cells and constitute a number of individual infections per leaf. Two morphologically similar fungi are associated with die-back and canker of eucalypts in South Africa. The one was identified as part of the Botryosphaeria dothidea-complex. In chapter four, the identity of the second fungus was determined by comparing morphology, pathogenicity and DNA sequence analysis of isolates of both taxa. Based on results obtained, Botryosphaeria eucalyptorum, and its anamorph Fusicoccum eucalyptorum, are described as a new species. I found that the teleomorph is morphologically similar to other taxa in the B. dothidea-complex, but conidial characteristics of the anamorph are distinct, as well as the sequences of the nrDNA internal transcribed spaeers ITS 1 and ITS2. As is the case with B. doth idea , the fungus is pathogenic to Eucalyptus, there do not, however, appear to be differences in pathogenicity between the two. Sphaeropsis sapinea is the most important pathogen of pines in South Africa. The fungus, which reproduces only asexually, occurs only on exotic pines. In chapter five, I investigated the diversity of the S. sapinea population in South Africa and compared it with a population from Northern Sumatra. Both populations were obtained from exotic P. patuIa plantations. The phenotypic diversity of these populations was assessed using vegetative compatibility tests. The percentage maximum genotypic diversity, based on Stoddard and Taylor's index, for the South African population was much higher than the Northern Sumatran population, thus indicating that the South African S. sapinea population was more diverse than the Northern Sumatran population. These results support the hypothesis that the population of S. sapinea in South Africa has been introduced from various parts of the world, during the last century. In chapter six, I investigated the role that latent S. sapinea infections in seed cones of P. patuIa, play in post-hail associated die-back. Pinus patuIa seed cones were found to be infected during the second year of development, with extensive colonization only occurring m the third year when cones mature, prior to seed discharge. Vegetative compatibility tests revealed that the presence of S. sapinea in individual third year seed cones is confined to a single genetic entity. Sphaeropsis sapinea colonisation of third year seed cones thus, apparently results from a single successful infection per cone. The probable role of latent infections by S. sapinea indicated that tree age and by implication, increased numbers of attached seed cones, contributes to more severe die-back after hail damage. The control of damage caused by S. sapinea is highly dependant on a dynamic hybridisation programme. Alternative species of pines is thus constantly evaluated for potential. In chapter seven, 65 families representing both the northern and southern populations of P. greggii were evaluated for their tolerance to infection and subsequent die-back caused by S. sapinea. Families were evaluated following natural infection after hail damage, as well as by artificial inoculation. Variation in tolerance occurred and was highly significant between the two provenances, with the northern provenance proving to be very tolerant. Pinus greggii trees of the southern provenances were comparable with P. patula. The potential of the families from northern origins has to be investigated further. Cultures of Cytospora isolated from Eucalyptus trees in South Africa, Congo, Thailand, Venezuela, Mexico, Uganda and Australia, as well as Cytospora-like isolates from Indonesia were compared in chapter eight. Comparisons were based on the homology of the internal transcribed spaeer regions and the 5.8S ribosomal DNA of the nuclear ribosomal DNA repeat unit. Isolates clustered into at least three unrelated groupings, with a fourth grouping that included isolates that morphologically resembled Cytospora. Results from this chapter indicated that the current description of Valsa ceratosperma encompasses several distinctly different species and needs to be further refined. Botryosphaeria dothidea and S. sapinea are two of the most important pathogens of eucalypts and pines in South Africa. The fact that they exist as symptom less endophytes in trees has added a fascinating aspect to our understanding of their role in tree diseases. In the past, they have generally been considered to be wound infecting opportunistic fungi. Results of these studies have shown that this is not so and that they are clearly able to infect healthy trees. They are unlikely to be able to infect dead or moribund tissue. The investigations presented in this dissertation have added considerable knowledge to our understanding of B. dothidea and S. sapinea and will also promote efforts to reduce disease caused by them. However, there are many questions that remain to be answered pertaining to them and it is my hope that this study will provide a foundation and stimulus for further work.
Open Access
Bioremediation of a bleach plant effluent from the pulp and paper industry
(University of the Free State, 2003-11) Van Driessel, Brian; Christopher, L.
English: Bleach plant effluent was characterised by physico-chemical methods. The chemistry of the bleach plant effluent was examined to devise effective treatment methods. Effluent contained trace amounts of nitrogen as well as carbohydrates and no ortho phosphate could be detected in the wastewater. The best decolourisation activities were obtained using adsorption as treatment method, with activated carbon removing > 99% colour from effluent. Chitosan (81%) and chitin (77%) could remove appreciable levels of colour from bleach plant effluent, followed by biomass from Rhizomucor pusillus, a mucoraelean fungus (71%). Chitosan and chitin from the cell wall of R. pusillus might be involved in the fungus decolourisation ability. Effluent pH was inversely related to effluent decolourisation when R. pusillus, chitosan or chitin was used as adsorbents. This might in part be due to acid catalysis during nucleophilic addition reactions, where amino groups of chitin/chitosan react with carbonyl groups in Eo-effluent. Also, chitin and chitosan amino groups can be protonated under acidic conditions and acquire positive charges that can interact with the chromophores found in Eo-effluent. However, pH exerted no significant effect on decolourisation when activated carbon was employed as adsorbent of effluent colour. Decolourisation employing commercial adsorbents seemed to be mainly due to chemisorption. Adsorption experiments conducted at various ionic strengths indicated that coulombic interactions are responsible for a fraction of the decolourisation activity of chitosan and chitin. Nevertheless, decolourisation obtained with RM7 and activated carbon was unaffected by the ionic strength. Flocculation of coloured compounds from Eo-effluent by chitosan containing solutions resulted in a maximum decolourisation of 75%. Anion-exchange treatment removed 96% colour from Eo-effluent. Ultraviolet irradiation could decolourise the Eo-effluent by about 42 to 43%. Decolourisation using organic solvent extraction proved ineffective with a highest colour removal efficiency of only 21% being achieved. Biological methods used for effluent remediation were: 1) Trickling filters, 2) Activated sludge reactors and 3) Rotating biological contactor reactors (RBC). Treatment using one biological system was followed by treatment in another system With trickling filters containing immobilised white-rot fungi, the highest decolourisation (61%) was obtained with Coriolus versicolor. This fungus required a co-substrate to efficiently decolourise the effluent. Effluent treatment in an activated sludge reactor reduced toxicity, COD and chlorophenol levels. However, colour and high molecular mass compounds were not affected significantly by this method of treatment. Decolourisation was studied in a RBC using immobilised C. versicolor and R. pusillus, respectively. The decolourisation rate by both fungi was proportional to initial colour intensities. Decolourisation was not adversely affected by colour intensity, except at the lowest level tested. Decolourisation of 53 to 74% could be attained using a hydraulic retention time of 23 h. Rhizomucor pusillus, removed 55% of AOX compared to a 40% AOX reduction by C. versicolor. Treatment employing R. pusillus and C. versicolor, respectively, rendered the effluent essentially non-toxic. Addition of nutrients to the decolourisation media stimulated colour removal by C. versicolor, but not significantly in the case of R. pusillus. Ligninolytic enzymes (manganese peroxidase and laccase) were only detected in effluent treated by C. versicolor. Decolourisation mechanisms were investigated using gel permeation chromatography. Rhizomucor pusillus decolourised the effluent by adsorption and C. versicolor removed effluent colour by adsorption as well as by biodegradation. Coriolus versicolor could decolourise the effluent for a period of 34 d whereas R. pusillus decolourised the effluent up to 54 d. Further improvements in effluent quality could be attained when treatment using one system was followed by treatment in another system, possibly because of toxicity reduction in the pre-treatment steps.
Open Access
Characterisation of arsenic hyper-resistance in bacteria isolated from a South African antimony mine
(University of the Free State, 2009-11-20) Botes, Elsabé; Van Heerden, E.; Litthauer, D.
English: Soil and water sites were sampled at a South African antimony mine with elevated levels of arsenic due to the refining process. Enriched media yielded six pure bacterial cultures able to grow in both arsenite and arsenate. These bacteria were identified as two strains of Bacillussp. (SA Ant 10(1) and SA Ant 14) with close relatedness to B. maltophilia and B. thuringiensis, another as Stenotrophomonas maltophilia SA Ant 15 and two isolates as Serratia marcescens (SA Ant 10(2) and SA Ant 16). Bacillus sp. SA Ant 14, S. maltophilia SA Ant 15 and S. marcescens SA Ant 16 were used for further investigation. All three isolates were able to grow in arsenite and arsenate respectively and S. marcescens SA Ant 16 grew in up to 500mM arsenate, making it the most arsenic resistant organism described to date. During growth, addition of arsenate or arsenite anions adversely affected biomass production and maximum specific growth rate and, in some instances, longer lag phases were induced. Reduction of arsenate to arsenite partly accounted for the high tolerance of the bacteria to arsenate. It was attempted to isolate the arsenate reductase from S. marcescens SA Ant 16 by making use of a PCR based approach using a both documented as well as degenerate primers based on sequence similarities of related Gram negative bacteria as well as Gram positive bacteria. After many unsuccessful attempts, this line of investigation was abandoned in favour of constructing genomic libraries. An Escherichia coliarsenate reductase knockout strain as well as a variety of laboratory strains was used for screening purposes. After screening of more than 5 x 104 colonies, no positive transformants were obtained. It may be possible that since S. marcescens SA Ant 16 exhibited hyper-tolerance to arsenate, the screening hosts used may not have been able to recognise and express the arsenate reductase from this organism successfully. The growth optima with regards to pH and temperature were established for S. marcescens SA Ant 16 grown under aerobic conditions as well as a suitable electron donor and electron acceptor concentration when grown under anaerobic conditions. The surface characteristics of S. marcescens SA Ant 16 cells, grown both in the presence and absence of oxygen, was investigated to infer adhesion capacity. It was found that both types of cells exhibited a negatively charged, highly hydrophilic and acidic character which would imply successful and similar adhesion of both aerobically and anaerobically grown cells to sand grains. Arsenate reduction was optimised in a factorial design layout with regards to electron donor (glucose) and substrate (arsenate) concentration under both aerobic and anaerobic conditions. Optimum contact time between cells and sand and loading capacity of the sand were determined. Cells were tracked through the sand columns and parameters for in situarsenate reduction established. Successful conversion of up to 50% arsenate to arsenite was demonstrated from an initial 5mM starting concentration. This hyper-resistant bacterium could be the solution to water contaminated with extremely high arsenate concentrations.
Open Access
The chemical, microbial and sensory evaluation of lucerne (Medicago sativa L.) for human consumption and acceptability
(University of the Free State, 2014-06) Mielmann, Annchen; Hugo, C. J.; Bothma, C.
English: Lucerne (Medicago sativa L.) has not recently been investigated as an alternative protein source for human consumption in SA. The chemical (degrees Brix, macro- and micro-minerals, protein, AA, dry matter, moisture, ash, fat, fibre, carbohydrates and energy content) composition, microbial content, and shelf-life of three lucerne cultivars, ‘SA Standard’ (‘SAS’), ‘WL525’ and ‘WL711’, were investigated. It was compared to SB (Beta vulgaris var. cicla L.), to identify the lucerne cultivar to use for future research purposes. The Brix content of the lucerne cultivars was significantly (p<0.01) higher than SB. Raw lucerne cultivars had significantly (p<0.01; p<0.001) higher Ca and S contents than raw SB. The K, S and Mn content for the raw lucerne cultivars were significantly (p<0.001) higher than the cooked cultivars. The protein content of the raw lucerne cultivars were significantly (p<0.001) higher than the raw SB. Cooked ‘SAS’ and ‘WL525’ had significantly (p<0.001) higher protein contents than cooked SB. Both raw and cooked ‘SAS’ had a significantly (p<0.001) higher carbohydrate and energy contents than both raw and cooked ‘WL525’, ‘WL711’ and SB. The lucerne cultivars contained higher values of essential and non-essential AA than SB. The total bacterial count, coliform count, lactic acid bacteria count and mould count conformed to standards, while cooking significantly (p<0.001) reduced the bacterial load. The shelf-life of cooked lucerne and SB samples were of satisfactory microbiological quality until day seven. The sensory descriptive attributes of the three lucerne cultivars were determined, by using GDA, and consumers’ acceptance (degree of liking for aroma, taste, mouthfeel and overall acceptability). ‘SA Standard stew’ showed the lowest value, of the lucerne samples, for fibrous appearance, chewy and fibrous mouthfeel, bitter taste, and bitter and metallic aftertaste. Principal component analysis showed that, three stew samples (SB, ‘SAS’ and ‘WL525’) and plain SB displayed ‘positive’ attributes, such as soft and wet appearance, spinach beet aroma and salty taste. ‘WL711 stew’ and three plain samples (‘SAS’, ‘WL525’ and ‘WL711’) were associated with ‘negative’ descriptors, namely grassy aroma, fibrous and chewy texture, fibrous appearance, bitter taste, and bitter and metallic aftertaste. ‘SA Standard stew’ was the most preferred lucerne cultivar, as it’s values for degree of liking for aroma, mouthfeel, taste and overall acceptability were numerically higher than ‘WL525’ and ‘WL711’. External preference mapping indicated that three consumer clusters, with different lucerne preferences, were obtained by AHC. ‘SA Standard stew’ was the lucerne sample preferred by all the consumers, except those with some primary education and a yearly income of R501 000 – R750 000 per year, who indicated only a high acceptance for this sample. While 63.1% of the respondents knew what lucerne was, 77% was unfamiliar with the term ‘alfalfa’ and 90.3% never consumed lucerne before. Younger respondents of the white population group, with a grade 12 or higher education, were significantly (p<0.001) more knowledgeable about lucerne. The predictors, sensory qualities and synonyms, contributed significantly (p<0.001) to consumers’ attitudes towards lucerne. While respondents believed that ‘health general’ (40.1%) was the most important advantage of eating lucerne, ‘sensory properties’ (35.4%) and ‘vegetables spinach’ (24.5%) were regarded as the most important disadvantage and association, respectively. The differences in lucerne knowledge of respondents from different demographic backgrounds indicated a need to inform SA consumers, on the benefits of consuming lucerne. Based on these findings, lucerne could be implemented as an alternative vegetable protein and therefore, cultivar ‘SAS’ was proposed for future studies and product development.
Open Access
A combined systems biology and genomics approach to the study of metabolism in Kluyveromyces marxianus
(University of the Free State, 2016-10) Schabort, Du Toit Willem Petrus; Du Preez, J. C.; Kilian, S. G.
English: The yeast Kluyveromyces marxianus has become an important micro-organism for industrial applications, as have other non-conventional yeasts. It has the advantages over Saccharomyces cerevisiae (baker’s yeast) in that it is more thermotolerant, has a much higher growth rate and can utilise a wider range of sugars, including the pentose D-xylose, which is found abundantly in lignocellulosic biomass. Although considerable advances have been made in engineering S. cerevisiae strains to ferment pentose sugars, their performance in this respect still does not approach that of glucose fermentation. S. cerevisiae is the model Crabtree positive yeast, meaning that it naturally ferments glucose even if oxygen is present at a high level. Crabtree negative yeasts, such as K. marxianus, have to be forced into a fermentative metabolism by imposing oxygen-limited conditions, which is impractical on industrial scale. Thus, a tremendous amount of knowledge needs to be gained regarding the regulation of metabolism in this non-conventional yeast before success could be expected in the re-programming of K. marxianus strains into xylose fermenting, Crabtree positive strains. The challenge of bringing a non-model species such as K. marxianus to the point of identifying key regulators affecting central metabolic pathways seems formidable. The aims of this work was to firstly harness the new technology of next-generation sequencing (NGS) to create a first draft genome for K. marxianus strain UFS-Y2791 and to generate high-quality RNA-seq differential transcriptome datasets, simultaneously capturing a tremendous amount of information. Efficient analytical methods and software implementations were also developed to explore these large datasets in an efficient manner, revealing new insights into the response of this species to glucose and xylose as carbon sources. RNA-seq data revealed a striking resemblance with the pattern of glucose derepression in the xylose medium, with up-regulation of genes for alternative carbon source utilisation, especially in the peroxisomes. Subsequently, two independent approaches were taken to identify differentially active transcription factors regulating the response. The first was the enumerative method of heptamer frequency comparisons, revealing the most likely regulators of differentially expressed genes. Secondly, a likelihood statistical approach was designed that employs multiple sources of evidence, which resulted in the construction of the first genome-wide gene regulatory network for K. marxianus. The method bridges the gap between the new NGS-based methods, which can rapidly generate data on any non-model species, and the wealth of experimental data that exist for a model species such as S. cerevisiae. Gene set enrichment statistics of the transcription factor target sets showed a general pattern that the activities of differentially active transcription factors were regulated primarily by post translational modifications instead of gene regulation. The use of RNA-seq was further expanded to the elucidation of the kinases that regulate transcription factors. The chromosomal context of differential gene expression was also investigated. Clusters of genes were identified, similar to the sub-telomeric regions previously identified in S. cerevisiae, but not close to telomeres. These regions contain industrially important enzymes and the potential binding sites for differentially active transcription factors. Finally, the possible roles of cofactor balances were investigated. Flux balance analysis was demonstrated here in rationalising the genetic response observed in RNA-seq transcriptomics and to understand the complex interplay between ATP, NADPH and NADH, the cofactor specificity of the oxidative pentose phosphate pathway, as well as the role of fructose-1,6-bisphosphatase. New roles are proposed for the latter enzyme, which differs from the currently accepted norm. A strategy for the metabolic engineering of a future xylose fermenting K. marxianus strain is also presented. The integrated analysis presented here expands our knowledge base of this yeast species, which is set to become increasingly important in a future bio-economy.
Open Access
Control of Listeria monocytogenes in an avocado processing facility
(University of the Free State, 2015-11) Strydom, Amy; Witthuhn, R. C.; Gouws, P. A.; Albertyn, J.
Listeria monocytogenes contamination of food is a growing concern for the food industry since it is the causative agent of human listeriosis. Despite increased awareness and strict microbiological standards for this pathogen, countries such as France, Austria and Germany have reported increases in listeriosis outbreaks. The research in this thesis shows how Listeria contamination in a South African avocado processing was almost eradicated. The first aim of this project was to isolate and genetically type the L. monocytogenes strains isolated from the facility. Pulsed field gel electrophoresis (PFGE) was used to group a subset of strains (n=80) according to the digestion of their genomes with the restriction enzyme AluI. These results were compared to polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) of the inlA gene (n=140). The results of the two methods compared well with each other but both indicated a lower genetic diversity among the isolates than expected. These strains were isolated over a period of four years and only two major groups were identified with the PFGE and the PCR-RFLP resulted in only three banding patterns. Also, the origin of the bacterial contamination could not be identified, but results indicated that cross contamination played a role in the persistence of these bacteria in this food processing facility. Secondly, the commercial product ListexTM P100 was assessed for possible use as a biocontrol agent in the facility. The host range of the phage product was determined based on 239 L. monocytogenes strains isolated from this facility, but only 26.7% of these strains were susceptible to the bacteriophage. The strains were also analysed for serotype and no correlation was observed between typing methods or isolation source and date of isolation of the strains, as well as the susceptibility to the phage product. This could indicate that cross contamination played a role in the transfer of bacterial cells since these strains were distributed randomly in the facility. Inoculation of the phage product with L. monocytogenes T162 in brain heart infusion (BHI) broth resulted in significant reductions in the bacterial concentration. However, activity of the phage in avocado pulp and guacamole was very low and no significant reduction in the L. monocytogenes concentration was measured. Lastly, the population of the Listeria strains in the facility were continuously monitored over five years. The final products and processing environment, including floors, equipment, work areas and personnel were tested on-site for Listeria with the ISO 11290-1 method. Based on the prevalence of Listeria, the facility introduced new strategies in processing to counter cross contamination. Results from the 2014 guacamole production season showed almost complete eradication of Listeria spp. in final products (0.17%, n=1170) and the processing facility (0.79%, n=1520). These results indicate that successful management of Listeria spp. in an avocado processing facility can be accomplished with in-house monitoring of the bacterial population and subsequent adjustments to the processing system. The results from this project indicated that the cause of contamination by L. monocytogenes in the facility was due to cross contamination, although a strict quality control system was followed. Despite low genetic variability between the L. monocytogenes strains, the commercial phage product was only effective against 26.7% of strains tested. This is surprising since literature reported very high percentages of susceptible strains to this specific product. Although bacteriophage biocontrol with ListexTM P100 was not effective in this facility, it cannot be concluded that this will be the case for other facilities. Also, bacteriophage product with a broader host range such as a cocktail of different phages, may work well in the processing environment to minimise transfer of bacterial cells to the final product. Control of L. monocytogenes will, however, only be effective if the processing conditions counter cross contamination.
Open Access
Cytochrome P450 monooxygenases from extremophiles
(University of the Free State, 2012-01) Müller, Walter Joseph; Smit, M. S.; Van Heerden, E.; Albertyn, J.; Piater, L. A.
English: Information indicate that CYP450s are prevalent in members of the bacterial phylum Deinococcus-Thermus as well as the archaeal family Halobacteriaceae that belong to the phyulm Euryarchaeota. A property shared by these phylogenetically distant extremophiles is the production of carotenoid pigments. It became the purpose of this study to use genome sequence information to clone and study new CYP450s from the genera Thermus and Halobacterium and to explore the role of these CYP450s in pigment production. The non-pigmented thermophilic bacterium Thermus scotoductus SA-01 was screened by PCR for the presence of a cytochrome P450 monooxygenase (CYP450). No CYP450 could be found and subsequent genome sequencing confirmed this finding. However, a CYP450 gene (CYP175A) was isolated from the closely related yellow pigmented strain Thermus sp. NMX2.A1 using oligonucleotides based on the DNA sequence of the β-carotene gene cluster from three Thermus strains. The genome sequence of T. scotoductus SA-01, revealed a ferredoxin (Fdx) and ferredoxin reductase (FNR) that were almost identical to those of Thermus thermophilus HB27. In T. thermophilus HB27 the Fdx and FNR are the native redox partners for CYP175A1, a β- carotene hydroxylase. After heterologous expression in Escherichia coli, we attempted to hydroxylate β-carotene with the CYP450 from Thermus sp. NMX2.A1 and the redox partners of T. scotoductus SA-01 using cell free extracts, but no products were detected. Thirty two CYP450s have been identified in the sequenced genomes of thirteen extremely halophilic archaea. Initial attempts to clone and heterologously express a CYP174A2- homologue from a Haloarcula LK-1 strain in E. coli and Pseudomonas fluorescens were unsuccessful. In order to study the physiological role of CYP450s in halophilic archaea and to create a strain that can be used for heterologous expression of CYP450s from halophiles CYP174A1 was deleted from H. salinarum R1. CYP174A1 is the only CYP450 in H. salinarum R1 and H. salinarum R1 is a genetically tractable strain. Upon culturing the wildtype and deletion strains, a difference in red pigmentation of stationary phase cultures was observed; implying that CYP174A1 might play a role in carotenoid synthesis. Microarray analyses revealed that the bop gene, which codes for bacterioopsin (BO) was severely repressed in stationary phase cultures of the deletion strain and sucrose gradient experiments showed a consequent loss of purple membrane (PM) in the deletion strain. The classical causes of bop repression e.g. insertion elements in the bop open reading frame as well as in the brz gene was ruled out by PCR screening. In addition to bop repression, the neighboring vng1459 and vng1468 genes (both part of the bopregulon) were also down regulated, but the genes normally involved in regulation of the bop gene were not affected. Currently the functions of vng1459 and vng1468 are unknown. Retinal, together with BO, is a key component of bacteriorhodopsin (BR) and essential for PM synthesis. Retinal is formed by the central cleavage of β-carotene which can be achieved by monooxygenases or dioxygenases.The Blh and Brp proteins in H. bacterium salinarum are very closely related to a confirmed bacterial 15,15′-β-carotene dioxygenase and studies have shown that deletion of both brp and blh results in complete abolishment of retinal and BR. It is therefore unlikely that CYP174A1 plays a role in retinal biosynthesis. Another possible function for CYP174A1 might be the hydroxylation of β-carotene, since it is known that H. salinarum strains produce hydroxylated carotenoids such as transastaxanthin, but no genes encoding typical β-carotene hydroxylases or ketolases have been identified in the genomes of H. salinarum strains. This will imply that hydroxylated carotenoids play a role in the regulation of bop.
Open Access
Determining the canning quality of small seeded white beans (Phaseolus vulgaris L.)
(University of the Free State, 1999-05) De Lange, Anna Francina; Osthoff, G; Labuschagne, M. T.
English: It is important to the producers, processors and consumers in south Africa to have dry bean cultivars with acceptable canning quality. Therefore, dry bean breeders needs sui table screening methods to evaluate the various lines at an early stage (F4) when only small amounts of seed are available. A micro-canning method to evaluate canning beans .in tomato sauce has been developed and compared to the commercial processing procedures with comparable results. External factors that influenced the canning quality like the water quality were investigated. This micro-canning method could therefore be used to investigate the effect of genotype and environment interactions that significantly influenced the canning quality. The objectives of this study were to: o Evaluate different qeriet i.c material for use as parents in the breeding program. $ To obtain a better understanding of canning quality characteristics of beans and to ensure that the most important characteristics are evaluated and the component interrelationships. • Determine the genotypic, environmental and genotype x environment interactions that influenced the canning quality. • To ascertain the patterns of interrelationships of the canning quality parameters and chemical analysis. • To investigate the patterns and relationships between standard and choice grade cultivars. e To investigate stability of locali ties between seasons as well as the clustering of different environments and seasons. Small seeded white beans, carioca and yellow haricot beans were used to determine variability in canning quality but as a result of a lower canning quality of coloured beans, only small seeded white beans were used for further investigations. As result of the investigations of different a characteristics, only the seed size, water absorption during soaking and canning, the texture and subjective visual appearance evaluations were used to determine canning quality. These characteristics were interrelated but single no parameter could explain variation' in canning quality. Canonical correlation analysis was used to determine to what extent variation of chemical components was responsible for differences in canning quality and these results indicated that mainly potassium and calcium would influence the water absorption and texture, respectively. Canonical variate analysis was used to determine the difference between unacceptable, standard and choice grade cultivars. A model was described from these analysis that could be applied to independent data sets that results in coordinates that differentiates the lines or cultivars as unacceptable, standard and choice grade. Significant interactions between genotype, environmental and seasonal effects for canning quality traits indicated that cultivar responses to variation in localities and seasons differ. Environmental effects resulted in inconsistent quality measurements since trait expression is strongly influenced by genotype x environment interactions. Results from this study suggested that the difference between cultivars could therefore be due to a complex interaction of the chemical and structural composition, which is genetically determined and influenced by the anv i ronmen t . as well as the changes that occur during processing. The Additive Main Effects and Multiplicative Interaction (AMMI) model mainly grouped KwaZulu-Natal separately as a region with poor canning quality. The rest of South Africa's localities grouped different for each season. Resulting from this investigation, several recommendations can be made: c Breeding material should be tested fo~ more than one season in order to select superior and more stable lines. o Elimination of canning quality evaluations of KwaZulu-Natal could improve the genetic progress. • Exploitation of the interactions by breeding for specific adaptation in a region of homogeneous area. Demarcating one or two areas in South Africa for the exclusive production of small white canning beans could improve the overall canning quality of the small white bean production in South Africa.
Open Access
Development and application of a small-scale canning procedure for the evaluation of small white beans (phaseolus vulgaris)
(University of the Free State, 2004) Van Loggerenberg, Magdalena; Osthoff, G.; Pretorius, A. J.
English: Laboratory canning and evaluation of dry beans are common practices for testing canning quality of cultivars before commercial release to canning industries. Suitable laboratory canning and evaluation procedures for small white beans in tomato sauce were identified. Standard values for choice and standard grade beans for laboratory evaluation of canning quality were defined, using four small white bean cultivars from nine localities during the 2000/01 season. The cultivar Teebus was used as reference standard for choice grade beans and its canning quality complied with international guidelines when the modified canning technique (MCT) was used. From the laboratory and modified canning evaluation procedures hydration coefficient, percentage washed drained weight, visual appearance (scale 1 to 10), splits (scale 1 to 10), texture (kg.100 g -1 .12 s -1 ), size, clumping, L-values, aL-values and bL-values were identified as suitable canning parameters for small scale evaluation of beans. Beans canned with the MCT were also canned and evaluated industrially and results compared. The interpretation of the different canning parameters with laboratory and industrial canning were simplified by the use of canonical variate analysis (CVA). Canonical variate analysis indicated the same groupings for cultivars according to choice and standard grade canning quality for laboratory and industrial canned beans. Laboratory canning and evaluation could be used in the evaluation of the canning quality of beans intended for industrial canning. Canning quality of seven small white bean cultivars from 33 localities and two seasons was determined with the MCT and CVA. Cultivars with acceptable and unacceptable canning quality were identified using laboratory evaluation and CVA. The CVA resulted in a prediction model for canonical variates 1 and 2 (CV 1 and CV 2) by identifying two discriminative equations for CV 1 and CV 2 scores. The CVA for environments identified differences in the canning quality of beans from different regions, while also indicating seasonal differences. The canning quality of dry bean cultivars from different environments can be determined using CVA. The model equations for the prediction of the canning quality of small white beans were validated on four cultivar samples from four regions (2000/01 season) and 24 breeding samples from three localities (2002/03 season) that were not included in the development of the model. The CVA and the model identified the same entries from breeding trials over localities not to be significantly different from Teebus (P > 0.05) in canning quality, but were unable to group cultivars statistically correct according to choice grade. The model was however capable of grouping standard and choice grade cultivars separately. The model could be applied to identify breeding trial entries as choice grade and to identify entry x locality interactions. The use of small-scale canning and evaluation procedures in combination with CVA could be employed to classify cultivar canning quality as either choice- or standard grade and to determine environmental canning quality. These techniques could be used, with the assistance of the prediction model to compare samples from a breeding program with a reference standard.
Open Access
Development of a DNA vaccine for the prevention of Psittacine beak and feather disease
(University of the Free State, 2008-05) Kondiah, Kulsum; Bragg, R. R.; Albertyn, J.
Psittacine beak and feather disease (PBFD) is a readily recognisable dermatologic condition in wild and captive psittacines worldwide. It is caused by Beak and feather disease virus (BFDV) which is classified in the family Circoviridae and the genus Circovirus. BFDV has a circular ss-DNA genome consisting of seven open reading frames (ORFs), three being conserved in all BFDV isolates, ORF 1 which encodes the Rep protein, ORF 2 which encodes the coat or capsid protein (CP) and ORF 5 which encodes a protein whose function is as yet unknown. General symptoms of the disease include the symmetrical loss of feathers, feather abnormalities, beak and claw deformities, weight loss, anorexia and immunosuppression. The inability to grow BFDV in tissue culture or in embryonated eggs has hindered the routine diagnosis of PBFD affected birds and the development of reliable diagnostic tests and an effective vaccination program. PBFD is widespread in South Africa, leading to a loss of at least 10% of psittacine breeding stocks annually. The disease is also a major threat to the already endangered Cape Parrot (Poicephalus robustus) and the black-cheeked lovebird (Agapornis nigrigenis) and it is only a matter of time before we may see the extinction of these and other parrot species due to the lack of a preventative vaccine. The economical and natural implications of the attack by PBFD led to the aims of the present study which were to develop a potential DNA vaccine candidate, develop an expression system for production of recombinant CP as antigenic protein and establish an enzyme linked immunosorbent assay for the detection of BFDV-specific antibodies in parrots. The entire CP gene which has been suggested to encode for the epitopic protein of the virus was amplified by polymerase chain reaction (PCR) and ligated into a bacterial vector, pBAD/His B or a yeast vector, pKOV136 for expression of recombinant CP in Escherichia coli or Yarrowia lipolytica, respectively. Alternatively, CP gene PCR products were ligated into the mammalian expression vector pcDNA™3.1D/V5-His-TOPO® which was the vector of choice for DNA vaccine design and used to transiently transfect Chinese hamster ovary cells. Subsequently, the candidate DNA vaccine was used in a basic vaccine trial where budgerigars (Melopsittacus undulatus) were vaccinated either with the DNA vaccine candidate or a sub-unit vaccine consisting of purified recombinant CP. Expression of recombinant CP was monitored using polyacrylamide gel electrophoresis (PAGE), chemiluminescent and colorimetric detection on Western blots and ELISAs. While expression of the recombinant CP was unsuccessful in the yeast system using pKOV136, expression of recombinant CP was achieved in E. coli cells using the pBAD vector. Recombinant CP was partially purified and applied in both indirect and indirect competitive ELISAs as coating antigen for the detection of BFDV specific antibodies. Using the established ELISAs, BFDV specific antibodies could be detected in naturally infected parrots as well as in budgerigars vaccinated with the DNA vaccine and sub-unit vaccine. Comparable results were obtained when nonpurified recombinant CP was applied in the ELISAs in lieu of partially purified recombinant CP. Vaccinated budgerigars formed BFDV specific antibodies in response to the DNA vaccine and sub-unit vaccine that were detected using the indirect competitive ELISA established in the study. The antibody responses to the sub-unit vaccine were higher than those in response to vaccination with the DNA vaccine candidate. Although the indirect competitive ELISA could not provide an indication of whether these antibody responses are protective, the results obtained during the trial are a preliminary indication that both the DNA vaccine and sub-unit vaccine may be functional in parrots and safe to use as no adverse reactions were observed.
Open Access
Development of Yarrowia lipolytica for enhanced production of heterologous proteins
(University of the Free State, 2011-03) Ramagoma, Faranani; Smit, M. S.; Nthangeni, M. B.
English: Yarrowia lipolytica is a non-conventional yeast which is considered to be suitable for production of industrially important proteins at commercial scale. The yeast is non pathogenic and is generally regarded as safe and as such can be used to produce biotechnological products for human consumption and applications. The genetic tools for the manipulation of the yeast have been developed over the years. This includes chemical and genetic based mutagenesis techniques to develop and isolate Y. lipolytica strains with enhanced properties in the production of endogenous and heterologous proteins. Protein expression systems comprised of a variety of inducible and constitutive promoters are available; secretion signals for extracellular production of proteins and a choice of replicative or integrative expression systems have also been developed. Auxotrophic and antibiotic based selectable markers are available some of which result in single or multiple copies of the integrative expression cassettes containing the target gene. The Y. lipolytica yeast can utilise a wide variety of carbon sources and its fermentation processes in bioreactors are well established. The property of Y. lipolytica as a prolific producer of the endogenous extracellular Lip2p was exploited for the development of an expression system for production of therapeutic peptides. To this end, the 38 amino acid long RANTES I peptide with therapeutic applications in the treatment of HIV/AIDS was selected as a model peptide for co-expression with Lip2p. The sequence encoding the peptide was cloned downstream of the complete LIP2 gene. The therapeutic peptide was separated from the LIP2 gene by the sequences encoding for 6X His and the DDDDK sequence recognized by the enterokinase proteolytic enzyme. The expression cassette was under the hp4d quasi constitutive promoter with the rDNA as a target for multi copy integration using the defective Ura3d4 integration cassette. The amount of RANTES I produced by the expression system was estimated to be 0.274 μg.L-1 as determined by the CCL5/RANTES immunoassay method. The RANTES I peptide produced in this study was reactive to polyclonal antibodies raised against human RANTES and functionality assays showed that the peptide inhibited the binding of the pseudoviral particles to the TZM-bl cells through the CD4/CCR5 receptors. Another section of the study sought to identify genetic elements responsible for enhanced secretion of the extracellular Lip2p in Y. lipoyltica. Lipase hyperproducing mutants were generated using the zeta based mutagenesis system which integrates randomly within the Y. lipolytica genome. One mutant denoted Y. lipolytica Y12 which showed lipase hyperproducing phenotype was found to be disrupted in the function of the GPI7 gene, a protein encoding a polypeptide of 830 amino acids. The GPI7 protein is 33% identical to the S. cerevisiae GPI7 known to play a role in the maintenance of the yeast cell wall integrity. Consistent with the disruption of cell wall integrity through deletion of GPI7, the Y. lipolytica strain was more sensitive to cell wall hydrolysing enzymes, had defects in cell separation and a daughter cell-specific growth defect at the non-permissive temperature and exhibited hypersensitivity to Calcofluor white and Congo red which are known to interfere with yeast cell wall composition. While the Y. lipolytica Yl12 and the ”wild-type” Po1d strains grew similarly in shake flask cultures, the Y. lipolytica Yl12 strain produced 7 times more extracellular lipase activity in liquid shake flask cultures than the control Y. lipolytica Po1d strain. To investigate the mode of GPI7 action in the enhancement of protein production, a Y. lipolytica YlHmA25 strain expressing intracellular epoxide hydrolase under control of the hp4d promoter was deleted for the GPI7 gene to create a Y. lipolytica YlHmA25ΔGPI7 strain. The YlHmA25ΔGPI7 strain contained epoxide hydrolase activity in the extracellular in contrast to the Y. lipolytica YlHmA25. In addition, the Y. lipolytica YlHmA25ΔGPI7 accumulated more total extracellular protein than Y. lipolytica YlHmA25 an indication that the strain accumulates more proteins extracellulary in comparison with the Y. lipolytica YlHmA25 strain. Taken together, the results suggested that the disruption of GPI7 affects the integrity of the yeast cell wall which in turn results in leakage or “enhanced” secretion of proteins to the extracellular. The challenge in developing a host system with broad applications in heterologous production of proteins including therapeutics lies in the elimination of the endogenous hyper-mannosylated yeast glycans. The OCH1 gene encoding a protein with α-1,6- mannosyltransferase activity was deleted from the GPI7 deleted mutant of Y. lipolytica. The OCH1 and GPI7 double deletion mutant of Y. lipolytica showed similar growth patterns to the GPI7 null mutant. Glycan analyses revealed that the most abundant glycans are the Man8GlcNAc2 and Man9GlcNAc2 which are acceptable substrates of the Trichoderma reesei α-1,2-mannosidase I the enzyme widely used in the engineering of yeast glycosylation pathway to produce Man5GlcNAc2, a prerequisite intermediate for the synthesis of complex glycans in human cells. The OCH1 and GPI7 double deletion mutant of Y. lipolytica is a good candidate for further glycoengineering to enable production of extracellular therapeutic proteins with human-like glycans.
Open Access
Diseases of Acacia mearnsii in South Africa, with particular reference to ceratocystis wilt
(University of the Free State, 1998-12) Roux, Jolanda; Wingfield, M. J.
English: The Acacia mearnsii industry is a relatively small, though very profitable industry in South Africa. Wood derived from A. mearnsii is currently in greater demand than that of either pine or eucalyptus in South Africa. Despite the importance of this industry, very little attention has been given to the genetic improvement, disease tolerance or general improvement of A. meamsii as a forestry species. The result has been that, during the last few decades, pathogens have become adapted to, and spread through plantations of this tree. Although relatively little research has been conducted on the impact of pathogens on A. mearnsii, this situation has changed during the past nine years, and particularly since the identification of Ceratocystis wilt. The planting of exotics has many advantages over native plants. In South Africa, exotic forestry species, such as Eucalyptus spp., Pinus spp. and A. mearnsii were introduced to halt the uncontrolled logging of native forests. These native forests were logged mainly for furniture and building material, but also for fuel wood, resulting in the near complete destruction of South Africa's native forests. The introduced exotics prevented the further destruction of these forests and soon became a large industry. This was particularly due to the fact that it was found that they also had a superior growth rate when compared to native species. This accelerated growth rate brought rapid results from breeding trials and, thus, a relatively rapid improvement of the material planted. Because they had been separated from their natural enemies, these trees were also initially disease free. The A. mearnsii industry has, and will continue, to face many problems and challenges from pests and diseases. After the initial phase in which the tree was removed from the pathogens affecting it in it's native range, it faced attacks by native South African pests and diseases. These can spread from native Acacia species, or from any other native plants in the same, or even different families. Exotic, mono culture industries are also constantly under threat from the introduction of pathogens from other countries, including the country of origin. This can be done by the introduction of new germ plasm or on any other plant species or plant material brought into a country. Because A. mearnsii is now planted as a monoculture, in contrast to it's native situation, diseases and pests can potentially be much more severe and will spread more rapidly and widely throughout even aged and genetically uniform stands. Propagation of A. mearnsii has, recently, advanced considerably and this is concurrent with increased demand for this wood on world markets. Lessons learned from eucalypt and pine forestry need, however, to be heeded to save unnecessary losses and time. With the advent of vegetative propagation of A. mearnsii in South Africa, it is important to include disease screening trials at the early stages of the development of clones. In order to do this, a knowledge of all possible pathogens of A. mearnsii is needed. This includes pathogens known in South Africa and those that occur beyond the borders of the country. It is also necessary to have a detailed knowledge of the biology and population structure of these pathogens in order to gain an impression of the possible success of control measures. This thesis is a compilation of work conducted on some of the known pathogens of A. mearnsii in South Africa. It also includes a large component dealing with the identification and clarification of previously unknown pathogens of A. mearnsii. It, therefore, does not focus only on diseases of A. mearnsii, but includes a chapter on a disease of Eucalyptus. The causal agent of this disease has, however, also recently been found on A. mearnsii in South Africa and this chapter aims at elucidating the possible origin of the isolates from South Africa. It also illustrates the potential threat of this pathogen to the A. mearnsii industry. South Africa is a semi-arid country that regularly suffers from severe drought. Forestry activities in the country are also mainly restricted to areas with poorer soil and where agriculture cannot be pursued on a profitable basis. Factors such as drought, hail, frost and sub-optimal soil conditions can all contribute to increased stress on trees. Under these conditions, many fungi can act as opportunistic pathogens, causing large scale losses. They often live as endophytes within their hosts, not causing any negative affect until the onset of stress. At this stage, they spread throughout trees, preventing them from recovering from the stress condition and leading to cankers and tree death. Careful management, particularly site/species matching, is required to minimise losses caused by these pathogens. This thesis provides a basis for future research on the development of management strategies to control diseases of A. mearnsii in South Africa. Information, however, also provide valuable knowledge for forestry industries outside South Africa by highlighting the threat of exotic pathogens and the importance of strict quarantine measures to prevent the spread of pathogens. This is true for the movement of not only A. mearnsii material, but as was seen here, the movement of any forestry products, since many pathogens have a wide host range. Although the thesis is comprised of a series of individual entities, these all provide information regarding the hygiene of A. mearnsii plantations. This thesis thus aims at identifying future focus points for intensive research, while at the same time focusing on those pathogens that have been known to the South African industry for a longer period of time. Chapter one provides a review of the available literature on diseases affecting not only A. mearnsii, but also other Acacia spp. important to the forestry industry, world wide. It also highlights some of the uses of these species in the countries where they are planted. The multi-purpose use of Acacia spp. is an important aspect emerging from this review. In many countries, Acacia spp. are not only planted as forestry species but are also used for soil reclamation, nitrogen fixation and fodder. The main focus of the chapter, however, is on the A. mearnsii industry in South Africa, with a brief discussion on all the diseases currently known to occur in the country. It is concluded that much research is still needed to reduce the impact of these diseases and to ensure that the Industry functions optimally. Ceratocystis albofundus must be considered as one of the most important pathogens of Acacia spp., world-wide. Currently this pathogen occurs only in South Africa, but if it is to spread to other countries, large scale losses will be incurred. It may also affect, not only A. mearnsii, but most likely many other plant species. Breeding programmes for A. mearnsii in South Africa focus strongly on this pathogen. In Chapter two, the population diversity of C. albofundus was investigated and compared with data for other Ceratocystis spp., using nuclear and mitochondrial DNA fingerprinting. It was found that the C. albofundus population has a greater genetic diversity than any of the species with which it was compared. This will thus mean that intensive breeding programmes will be necessary to ensure durability of disease tolerance. It also supports previous hypotheses that C. albofundus is native to South Africa and may be a temperate species, not found in tropical areas where its close relative, C. fimbriata, commonly occurs. The first unequivocal report of C. fimbriata and Ch. elegans from A. mearnsii is presented in Chapter three. Both these fungi were isolated from dying trees with typical symptoms of Ceratocystis wilt caused by C. albofundus. Both were shown to be capable of causing disease to seedlings under green house conditions. It was, however, found that C. albofundus is more virulent than either Ch. elegans or C. fimbriata. Both isolates were identified using molecular and morphological approaches. Unfortunately only one isolate of each exists and surveys to obtain additional samples continue to be a priority. The first report of a wilt disease of Eucalyptus, caused by Ceratocystis fimbriata in the Republic of the Congo in West Africa is recorded in Chapter four. This is not only the first report of C. fimbriata as a pathogen of Eucalyptus in Africa but is also one of the few unequivocal reports of this fungus from the continent. Pathogenicity of C. fimbriata on Eucalyptus spp. was confirmed in glass house tests. In this Chapter, C. fimbriata and C. albofundus from A. mearnsii, and C. fimbriata from Eucalyptus in Brazil were also compared to the C. fimbriata from the Congo. Comparison of the lTS region of the rRNA operon showed that isolates from all three areas grouped together in a clade of C. fimbriata, separate from European isolates. Sequence data showed that C. fimbriata from A. mearnsii in South Africa is nearly identical to the fungi from Eucalyptus in Brazil and Congo, suggesting that they may have a common origin. These findings stress the importance of sound quarantine measures to prevent the introduction of potentially devastating pathogens to South Africa. It is not yet known why C. fimbriata has not caused more diseases on A. mearnsii or Eucalyptus spp. in the country, but the situation will need to be monitored closely. Apart from C. albofundus, there are many other fungi that cause disease of A. mearnsii in South Africa. Chapter five reports on a species of Seiridium that was isolated from stem cankers on A. mearnsii. Morphological and molecular comparisons, as well as pathogenicity studies have shown that the species from A. mearnsii is similar to those species responsible for Cypress canker in many parts of the world. It also confirms previous reports that the taxonomy of the three Seiridium spp. causing cypress canker needs re-evaluation, since molecular data support the view that the three species, represent a single taxon. Pathogenicity trials on mature Cuppressus lusitanica and on A. mearnsii trees showed that both the cypress and A. mearnsii isolates are capable of causing lesions on both hosts. Many of the fungi isolated from diseased A. mearnsii during the current and previous studies of diseases resulted in the isolation of fungi, commonly found as latent pathogens on other forest trees. Chapter six encompassed a survey of the endophytic fungi of A. mearnsii, with the specific aim of identifying possible pathogens. Thirty different fungal taxa were found as endophytes of the xylem and rachi. These included F. graminearum and Botryosphaeria dothidea, which are known pathogens. During periods of environmental stress, these fungi can apparently cause disease. This is especially true because A. mearnsii is often planted on marginal sites in South Africa. Chapter seven represents the first report of Fusarium graminearum from A. mearnsii and presents evidence for the fungus being involved in disease of A. mearnsii. This pathogen was first isolated during 1994-95 disease surveys, but was not identified due to the fact that cultures on artificial media did not sporulate. In the current study, additional isolates were obtained from stem cankers and die-back symptoms and the fungus was identified based on β-tubulin gene sequences. Field inoculations using F. graminearum showed extensive lesion formation in the xylem. Previously, this Fusarium sp. was known only as a pathogen of maize and wheat in various parts of the world. Results of this study are, therefore, enigmatic and intriguing.
Open Access
The effect of dietary conjugated linoleic acid supplementation on production efficiency and meat quality of pigs
(University of the Free State, 2014-01) Ferreira, Jacobus Philip; Hugo, A.; Strydom, P. E.; Kock, J. L. F.; Kanengoni, A.
The objectives of this study were to determine the effects of a commercial dietary CLA feed supplement on the production and meat quality parameters of pigs under commercial production conditions. It included the study of the chemical and sensory stability of processed meat products manufactured from the meat of such animals. One hundred and forty four Landrace x Large White crossbred pigs, weighing ± 30 kg, were randomly divided into two groups of seventy two pigs each, that were assigned to one of two dietary treatments. Diets consisted of a control diet supplemented with 1% SFO and the experimental diet where 0.5% SFO was replaced with 0.5% CLA. Each dietary group was further divided into three gender groups (boars, barrows and gilts) that consisted of twenty four pigs each. Each gender group was further divided into two slaughter weight groups (70 kg and 90 kg) consisting of twelve pigs each. Pigs were fed until the average live weight of the pigs was ± 70 kg for the porkers and ± 90 kg for the baconers. Growth performance (weight increase, ADG and FCR) and carcass characteristics (warm and cold carcass mass, dressing percentage, carcass length, shoulder and buttock circumference, pH, backfat thickness, eye muscle thickness and LMC) were assessed. Animals receiving the CLA diet had improved FCR and carcasses with thinner backfat and higher LMC, compared to animals on the SFO diets. This resulted in a higher frequency of P and O classification of carcasses from CLA supplemented pigs. Backfat, belly fat and M. longissimus thoracis quality of the dietary treatment and slaughter weight groups were compared. Baconers had improved technological properties compared to porkers. Dietary CLA supplementation resulted in improved technological properties of backfat and belly fat, demonstrated by decreased IV; RI; DBI; UFA; MUFA; PUFA; MUFA/SFA ratio; PUFA/SFA ratio; 9 desaturase index; C16:1 + C18:1/C16:0 + C18:0 ratio and increased C18:0; cis-9, trans-11; trans-10, cis-12; SFA; AI; C16:0 + C18 and ratios of C18:0/C18:2; C18:2/C18:1; C16:0/C18:2. M. longissimus thoracis from CLA supplemented pigs had higher a*-values, drip loss and WHC. Dietary CLA supplementation resulted in a decrease of health and nutritional properties of M. longissimus thoracis, demonstrated by increased SFA content and AI, while UFA, MUFA, PUFA, n-6, n-3 and ratios of MUFA/SFA and PUFA/SFA decreased. Technological and health properties were inversely related. The decreased health properties must be weighed against the numerous health benefits, ranging from improved immune function to prevention of cancer that can be attributed to CLA supplementation. Conjugated linoleic acid isomers were deposited into the neutral- and glycolipid fraction of subcutaneous adipose tissue and into the phospholipid fraction of IMF. Processed products (patties, bacon and salami) were manufactured from meat from the experimental treatment groups. The chemical stability and sensory properties of fresh meat and processed products manufactured from the experimental treatment groups were compared. Conjugated linoleic acid also demonstrated antioxidant properties in animal feed. Sensory analysis indicated the small effect of dietary CLA supplementation on the sensory properties of fresh and processed pork products. In the case of fresh pork chops and pork patties, dietary CLA supplementation had a stabilizing effect on the a*-value of the products. The lipid stability of pork patties was improved by dietary CLA supplementation as indicated by TBARS values. Salami from the CLA groups was firmer. That could be ascribed to the fat hardening effect of CLA. Pork and pork products enriched with CLA can be considered functional foods and even “nutraceuticals” with positive effects on human health. South African pig producers may therefore consider marketing CLA enriched pork products as a health food. The potential advantages and the premium that can be earned on such meat has to be balanced against the reality of increased feed cost.
Open Access
The effect of dietary conjugated linoleic acid supplementation on the physicochemical, nutritional and sensory qualities of pork
(University of the Free State, 2014-05-27) Bothma, Carina; Hugo, A.; De Kock, H. L.
English: Forty eight gilts were fed one of four dietary treatments containing 0, 0.25, 0.5 and 1% CLA, until their weight reached 95 kg and were then slaughtered. There were a lack of significant differences in pig performance and growth traits (weight increase, ADG, ADFI, FCR), and slaughter characteristics (SLW, hot carcass weight, cold carcass weight, dressing percentage, BFT, MT and LMC). There were no change in M.longissimus thoracis area, drip loss, WHC, pH45 and pH 24, while L*-and b* values decreased with increased dietary CLA. Colour a*-values and SI also did not differ between the four treatments. For the BF, IVs decreased with increased dietary CLA, while RI, colour a* and SI values remained unchanged, and colour b* values and hardness increased. Conjugated linoleic acid supplementation resulted in improved technological quality of subcutaneous fat, demonstrated by reduced IVs, unchanged RI and extractable fat content, and increased FFDM. With increase in CLA supplementation, C18:0, cis 9, trans 11, trans 10, cis 12, UFA, SFA, n-6/n-3, PUFA/SFA, dienoic acid, C16:0+C18:0, C16:0/C18:2, C16:1+C18:1c9/C16:0+C18:0 and C18:0/C:18:2 increased, C18:1c9/C18:0, MUFA, PUFA, MUFA/SFA, n-3, PI, trienoic, tetraenoic, penta + hexaenoic acid and DBI decreased, while n-6 remained unchanged. There was a tendency for sampling positions on the dorsal (neck, BF, chuck) and lateral (rib area) sides of the carcass to have higher CLA content. Differential scanning calorimetry of subcutaneous fat showed the presence of β’-crystals in fat from 0.25 and 0.5% CLA- fed pigs and β-crystals in fat from 1% CLA-fed pigs. For IMF samples, increased dietary CLA led to no change in IV, C18:0, C18:1t9, C18:1c7, C18:3n-3, PUFA/SFA, tetraenoic acid, C16:0/C18:2 and C18:0/C18:2 contents, while C16:1c9, cis- 9, trans-11, trans-10, cis-12, C22:5, C22:6, SFA, AI, PI, n-3, n-6/n-3, PUFA, n-6, dienoic acid, trienoic acid, penta- + hexaenoic acid, C16:0+C18:0 and C16:1+C18:1c9/C16:0+C18:0 contents increased and C18:1c9, C20:1c11, C18:1c9/C18:0, C18:2, C18:3n-6, C20:3n-3, C20:2, C20:4, C20:5, MUFA, UFA, MUFA/SFA and AI contents decreased. Most CLA was deposited on the lateral sides of the carcass, namely the M.triceps brachi. M.supra spinatus showed an atypical FA composition. Descriptive sensory analysis was performed on oven-broiled pork chops and fat samples by a trained panel. The control was rated most tender, confirming the results from the physical texture analysis. The control also had least resistance for first bite, with the 0.5% CLA treatment having most resistance. The 0.5% CLA treatment had a chemical aroma for the fat. The accelerated oxidation test indicated that BF from the control did not become rancid faster than BF from the three CLA treatments. Refrigerated display of pork chops for 8 days resulted in increased L*and b* values for the CLA treatments, unchanged TBARS values, while SI decreased. After frozen storage for 3 months, TBARS values remained unchanged for pork chops from the different dietary treatments. After 6 months of frozen storage, TBARS values decreased for pork chops from CLA supplemented pigs. After eight and 16 weeks of frozen storage, PVs for frozen patties decreased for the 0.5 and 1% CLA treatments. Differences in TBARS values became evident after eight weeks for the frozen patties, compared to sixth months for frozen pork chops. The TBARS values for the frozen chops were lower than the frozen patties. At the end of the ripening period, PVs for salamis from the 0.5 and 1% CLA treatments decreased, along with TBARS values. Belly fat from the CLA treatments was firmer than the control. No significant differences were observed between the four bacon treatments for either PV or TBARS values over the course of the six week refrigerated storage. According to the consumer panel, the control bacon was preferred to the 0.25% CLA bacon.
Open Access
The effect of histone H3 and H4 mutants on the chronological lifespan of Saccharomyces cerevisiae
(University of the Free State, 2015) Ngubo, Mzwanele; Patterton, Hugh-George
In this study we maintained histone mutant strains in a batch culture. We observed that some strains significantly decreased at a lower rate in the quiescent population.Some strains disappeared from quiescent population more rapidly, surprisingly, these strains did not increase in the non-quiescent population, suggesting that they may lyse and die.Residues that are implicated in lifespan extension are mostly histone tail residues and residues that are located in the solvent accessible region, suggesting that these residues may be bound by another protein(s) such as the silent information regulator, Sir3, via a BAH domain.All residues that are implicated in lifespan reduction are localised internally on the nucleosome core except for H3K18, suggesting their role in destabilising the nucleosome structure. This may allow elevated levels of DNA damage, and induce apoptosis. The gene expression studies of cells at late log phaseshowed that although all selected strains exhibited chronological lifespan extension, they had two distinct regulatory processes, suggesting that the H4K16Q and H4H18A strains, on the one hand, and the H3E50 strain on the other hand, may modulate different pathways to regulate chronological lifespan.The GO term enrichment for genes that are significantly up-regulated in the H4K16Q and H4H18A mutants, include rRNA processing and maturation, rRNA export, ribosome assembly, gene expression, cytoplasmic translation and ethanol metabolic processes. On the other hand, the GO term enrichment for induced genes in H3E50A mutant, include, mitochondrial electron transport, tricarboxylic acid cycle, ATP synthesis coupled proton transport, oxidative phosphorylation, glutamate metabolic process, glyoxylate cycle, and purine ribonucleoside triphosphate biosynthetic process. In addition, response to hydrogen peroxide, reactive oxygen species metabolic process, and response to oxidative stress appeared. It appears that many metabolic processes are upregulated in the H3E50A mutant strain. It is likely that the increased metabolism leads to storage of energy to be used in long starvation periods. The proteome studies of H4K16Q and H4H18A mutants at late stages of growth showed similar profiles. Snz1 is involved in vitamin B6 biosynthesis. It is regulated by, among others, Rap1. Jhd2 induction is interesting because it is a histone demethylase involved in global demethylation of H3K4, a histone mark added by Set1 and associated with active transcription. Gcy1 is a glycerol dehydrogenase, and was shown to increase in response to DNA replication stress. In H3E50A mutant, ribosomal protein, RPL16B, which is regulated by Rap1 was upregulated. Scs2 is involved in phospholipid metabolism, it was also shown that Scs2 over-expression rescued telomeric shortening and was de-repressed in a strain over-expressing Mec1. Tfs1 is an inhibitor of carboxypeptidase Y, and was also shown to regulate the PKA signalling pathway. We propose that Sir3, which is recruited to the silent chromatin by Rap1, cannot bind to the telomere nucleosomes, therefore, Rap1 redistributes to other regions of the genome of histone mutants. The regions, which Rap1 may relocate to, include stress response, DNA replication and damage stress response, and telomere maintenance genes.
Open Access
The effect of sodium reduction on the chemical, microbial and sensory quality of prominent South African processed meat products
(University of the Free State, 2016-08) Cluff, MacDonald; Hugo, A.; Hugo, C. J.
English: In light of the recent South African regulations limiting the sodium content of processed meat products, the latest draft of these regulations were used to establish to what extent commercial processed meat products deviated from these limits and required reformulation, two and a half years in advance of the first reduction limits coming into effect. Almost 60% of product labels already included information on sodium content almost three years before the applicable labelling regulations came into effect. Surveying nationally and regionally available products allowed for the identification of the five largest product classes. A comparison between labelled and determined Na content revealed that processors tended to overestimate Na content as a precautionary measure. A generous tolerance of 20% for underestimating the Na content, as stated in the labelling regulations draft, showed that only a small number of products would at the time, have exceeded the futuredated regulatory limits. Bacon, polony and pork bangers, representing the three most populous classes were used to evaluate the efficacy of the two-part regulatory limits as intermediate added NaCl levels without replacers or alterations in processing. Water activity, pH and moisture content were inconsistently affected with no definite links to deviations in dependent parameters such as microbial stability. Microbial and oxidative stability and sensory quality results were encouraging. Current processing techniques and ingredients other than NaCl maintained quality and stability. Changes in bacon and banger colour were found, although subjective evaluation is needed to grasp the implications. Polony texture was deemed acceptable, both quantitatively and qualitatively. Total Na levels better matching the regulatory limits may further limit the minor deviations in quality and stability. The relative success of using only 1% NaCl (w/w) in the pork bangers prompted the use of various compounds, either alone or in combination, to address the gaps in functionality of the 1% NaCl removed from the original formulation. Potassium chloride (1% w/w), K-gluconate (1% w/w), KCl (0.8% w/w) with YE (1% w/w) and lastly, KCl (0.8% w/w) with K-lactate (0.2% w/w) were compared to 1% NaCl and 2% NaCl controls. Treatments containing KCl had improved cooking losses over that of the controls. The use of K-containing compounds increased the K-content in addition to reducing Na-content. Basic chemical parameters were similar to that of the 2% NaCl control with only water activity being more similar to that of the 1% NaCl control. These replacers did not improve lipid oxidative stability and the use of YE actively deteriorated lipid oxidative stability. Colour was the most affected multi-component parameter and consumers had less favourable hedonic responses towards the use of K-gluconate. Partial replacement with 1% KCl was the most suitable solution when additional factors such as price-point, similarity to NaCl, and ease-of-use were taken into account. Lastly, the efficacy of the reduction and/or partial replacement of NaCl against the growth and survival of E. coli and S. aureus inoculated into banger batters were monitored. No effects on E. coli were observed beyond the bacteriostatic effect of sub-optimal temperatures (4 °C and 10°C) and the anti-microbial effects of the other additives in the formulations. At reduced NaCl levels, S. aureus was unable to grow and survival rate ultimately decreased. Partial replacement led to limited growth although survival rates eventually decreased. Survival rates were highest at 1% NaCl, 1% KCl and 0.2% K-lactate. Sub-optimal temperatures and other anti-microbial effects overrode that of partial NaCl replacement. Beyond the initial inoculation levels, reduction and/or partial replacement of NaCl did not increase the food safety risk of these bacterial species. This research showed that conformation with the regulatory limits warrants a back-to-basics strategy using multiple approaches that deliver better results when these approaches are linked to one another.
Open Access
Elucidation of African elephant beta casein phosphorylation state and casein micelle structure
(University of the Free State, 2017-01) Madende, Moses; Osthoff, G.; Kemp, G.
English: The exact structure of casein micelles still remains a debated subject. While most of the experimental work on cow caseins and casein micelles has provided a wealth of data, data of caseins and casein micelles of non-bovine origin provide a new insight into the structure of casein micelles. Microscopic examination of cow, sheep, horse, human and African elephant milk casein micelles show that the respective casein micelles are all spherical in shape but differ in size as well as surface appearances. Human casein micelles were the largest of the casein micelles whereas sheep casein micelles were the smallest. Apart from their smaller size, sheep micelles also had a smooth surface compared to a rough surface observed on the rest of the casein micelles. African elephant casein micelles were the second largest of the five casein micelles compared. It may be derived that, although casein micelle shape and size seem to be species specific, the differences observed may be a result of the differences in total casein content, the proportions of the individual casein types and the presence and or absence of some of the casein types. The elucidation of African elephant β-casein phosphorylation state by LC MS/MS, showed the presence of a single phosphorylation site at Ser9. In contrast, electrophoresis analysis showed that there are up to five phosphoforms of African elephant β-casein. The LC MS/MS also showed that the presence of a short length African elephant β-casein that is 200 amino acids long and that the gene sequences coded for by exons 4 and 5 have been truncated. Homology modeling of cow, sheep, horse, human and African elephant caseins showed that the secondary structure of α-caseins predominantly consist of α-helices, whereas the secondary structure of β- and κ-caseins is dominated by random coils. Alpha caseins give micelles a slightly compact structure whereas random coils result in a more open and larger size of micelles. These structural differences of caseins could possibly explain the varied size of casein micelles in milk. Comparative genomics of casein genes across mammalian species shows that several mammalian species are devoid of CNS1S1 and CSN1S2 genes. Considering the evolution of the casein gene locus organization, it appears that the CNS1S1 gene has been lost whereas the CSN1S2 gene has not been gained or developed in these species. In contrast, the CSN2 and CSN3 genes have been preserved and gained respectively, in most mammalian species. This suggests that these genes have a more important role in casein micelle formation and consequently the sequestration of large amounts of calcium and phosphate. Evidence from this study suggests that studying of non-cow caseins may shed more light on the casein micelle structure.