In 2007, Kock and co-workers published the Acetylsalicylic acid (ASA) Antifungal Hypothesis that indicated a definite link between oxylipin production [3-hydroxy (OH) oxylipins], mitochondrial activity [mitochondrial transmembrane potential (ÎÏm)] and ASA sensitivity in respiring as well as non-respiring yeasts. Here an increase in mitochondrial activity was observed in the sexual phase where an increase in energy production is probably needed for multiple ascospore development. This hypothesis has since been expanded to also include various anti-mitochondrial drugs as well as the fungi and fungi-like organisms Aspergillus, Rhizopus and Mucor. In this study the yeast Nadsonia fulvescens as well as some species of the notorious plant pathogen Phytophthora was evaluated for their ability to fit the expanded hypothesis now called the Anti-mitochondrial Antifungal Hypothesis. The yeast N. fulvescens is characterized by a unique life cycle. After conjugation between the parent cell and the first bud, the zygote moves into a second bud formed at the opposite end of the parent cell. This second bud is then delimited by a septum and becomes the ascus according to literature. Usually one, rarely two spherical, brownish, spiny to warty ascospores are formed within the ascus giving rise to brown coloured colonies. In this study the parent cell with attached first bud showed increased mitochondrial activity when compared to the ascus. When anti-mitochondrial compounds were added, the mitochondrial activity was inhibited in the parent cell with attached first bud followed by the formation of less asci with ascospores (many not fully developed and white coloured). It is suggested that sufficient mitochondrial activity in the parent cell and first bud is necessary to produce enough energy for the formation of a proper ascus with brown coloured ascospore(s). If the parent cell and first bud is regarded as part of the yeast sexual phase, then N. fulvescens also fits the hypothesis. Further investigations were conducted to study the asci of the yeast N. fulvescens containing mature and fluconazole-treated malformed ascospores using nano scanning Auger microscopy (NanoSAM) in combination with transmission electron microscopy (TEM). This is the first application of NanoSAM to biological material. Transmission electron microscopy exposed a variety of malformed ascospores in asci treated with fluconazole. Here some ascospores produced degenerated spiky protuberances with relatively large inclusions carried inside wrinkled asci. Other ascospores contained no walls or protuberances and were enclosed within smooth spherical shaped asci. The majority of ascospores contained less dense hollow areas surrounded by cytoplasmic material. Nano scanning Auger microscopy studies on these asci corroborate the TEM results although less structural detail was obtained. Nano scanning Auger microscopy showed a decrease in elemental intensities during etching which assisted structural analysis of ascospore less dense hollow areas. In this study it is shown that the oomycete, Phytophthora nicotianae also fits the hypothesis. Fruiting structures (zoosporangia) of this oomycete showed increased beta (β)-oxidation when probing levels of 3-OH oxylipins with specific polyclonal antibodies. In addition increased mitochondrial activity was also observed in the zoosporangia when the ÎÏm probe, Rhodamine 123 was added to the culture. This indicates increased mitochondrial activity in the zoosporangia when compared to the hyphae. When the anti-mitochondrial ASA was added to cultures of this oomycete, the zoosporangia were, as expected most susceptible and were drastically inhibited in the presence of 1 mM of this compound. Similar ASA inhibition results were recorded for P. citrophthora. Anti-mitochondrial compounds may now find application in combating these devastating plant pathogens and urgent further research is needed in this direction.

Geobacillus thermoleovorans GE-7 was isolated from the West-Driefontein goldmine in South Africa. It is a Gram-positive rod showing optimal growth at 65°C. This isolate was found to be able to grow on olive oil as the sole carbon source and on a variety of other lipid substrates, a feature which indicated that the bacterium produces lipases. In 2004, studies aimed at elucidating factors that would improve lipase production by G. thermoleovorans revealed that when the bacterium was cultured in medium optimized for lipase production, a production profile characterized by two enzyme activity peaks was observed. In 2005, a lipase (LipA) open reading frame (1251 bp) was amplified from the bacteriumâs genome. Furthermore, lipase purification studies from the GE-7 culture grown in media containing olive oil resulted in the purification of a lipase (~45 kDa), which corresponded to the LipA ORF that had been amplified from the GE-7 genome. However, lipase activity staining with the supernatant from GE-7 cultured in media supplemented with olive oil revealed the presence of two lipolytic protein bands of different sizes. These observations and reports on the purification of a smaller lipase in addition to LipA from the culture supernatant of related Geobacilli led to the hypothesis that the two lipase production peaks observed with GE-7 represent two distinct lipases differentially expressed by the bacterium. The aim of this study was to characterize the lipolytic activity from this bacterium using molecular and proteomic techniques. The primary objective was to identify from GE-7 a lipase different from LipA and to profile its expression relative to that of LipA. Three authors had already reported on the purification of a smaller lipase (>20 kDa), however only two authors had published sequence information in this regard (Schmidt-Dannert et al., 1994; Lee et al., 2001). Since bacterial lipolytic enzyme families are characterized by very high intra-family sequence similarity, the published N-terminal amino acid sequences were used for similarity searches on the database to identify homologs of this protein from related Geobacilli. Similarity searches revealed that the published sequences shared very high similarity with a hypothetically conserved protein (HCP) from G. kaustophilus and no homology to any other known lipase. The nucleotide sequence of the HCP was used to design primers to facilitate PCR amplification of the homolog from GE-7. The âsmall lipaseâ ORF (440 bp) was amplified from the GE-7 genome, however, functional expression using tributyrate/olive oil-Rhodamine plate assays revealed that the protein was not lipolytic. Furthermore, secondary structure predictions using the âsmall lipaseâ ORF revealed that the sequence shared significant identity to the type II secretory pathway pseudopilin protein from related Bacillus species contrary to what was reported by the two authors. As a result of the above-mentioned findings a protein purification strategy aimed at isolating a lipase different from LipA was pursued. Since LipA was purified in a previous study from the second lipase peak and given that it was hypothesized that the first peak indicated the production of a lipase different from LipA, GE-7 was cultured, lipase production monitored and the cells harvested prior to the onset of the second peak. Lipase purification experiments from the culture supernatant corresponding to the second peak resulted in the isolation of a ~45 kDa protein. The protein band was analyzed by peptide mass fingerprinting and the amino acid sequence determined. Sequence analysis revealed that the protein that was purified from the first production peak is LipA, which served as an indication that LipA was produced at both peaks. GE-7 was cultured in media with (induced) and without (uninduced) the lipid substrate (olive oil) and the lipase production profiles compared. It was observed that under induced conditions the two production peaks persisted while only one peak was observed in the uninduced. However, reverse transcription-mediated PCR on RNA isolated from GE-7 cultured as described above with primers specific for lipA revealed that LipA is produced under both culture conditions. Moreover, LipA is present at both production peaks under induced conditions which supports the purification results. Detection of LipA from the uninduced culture indicates the GE-7 is constitutively expressed, contrary to the previous reports. Bioreactor studies aimed at relating the effect of media components on lipase production by GE- 7 revealed that, under induced conditions, significant lipase production is only observed after the depletion of glucose and that the second activity peak is only observed upon the uptake of the free fatty acids by the cells. This observation suggested the fatty acids could act as a signal for further lipase production which was supported by the one lipase production peak observed under uninduced conditions (no fatty acids in the culture medium). The observation that significant lipase production was observed at the stationary phase when the glucose concentration became limiting served as a possible indication of a mechanism of gene expression regulation known as catabolite repression. Which suggests that lipA could be down-regulated in the presence of simple sugars (glucose) and up-regulated upon depletion of glucose to afford the bacterium the ability to utilize olive oil as a carbon source. A conserved sequence (CRE-box) has been identified on the promoter regions of a number of genes that are subject to this mode of regulation. As a result, the lipA promoter region was amplified and sequenced. Analysis of the sequence revealed the presence of the CRE-box. Experiments were conducted to investigate whether catabolite repression was a possibility for the GE-7 lipA. GE-7 was cultured under induced conditions, lipase activity monitored and the cells spiked with additional glucose at the onset of the stationary phase. A significant drop in the lipase production was observed subsequent to the spike. The above-mentioned observations and experiments where olive oil (used as a sole carbon source) or stearic acid was used as the inducer revealed that changing certain components of the media changed the lipase production profile. All the findings in this study suggest that LipA is the only lipase produced under the culture conditions investigated and that the changes in the production profile could be due to the regulation of the gene in response to changes in the culture medium.

Cytochrome P450 monooxygenases (P450) are a diverse, ubiquitous family of important enzymes which catalyze a variety of activities. Their outstanding abilities to hydroxylate non-activated hydrocarbons make them attractive enzymes for applications in the fields of chemical synthesis and bioremediation. Large scale applications of P450s are however largely limited by: (i) their requirement for expensive cofactors; (ii) dependence on co-proteins; and (iii) limited enzyme stability. These problems are largely circumvented by using whole-cell biocatalysis. Therefore the identification of appropriate hosts for heterologous expression is important. E. coli has several limitations in its ability to express eukaryotic P450s, therefore yeasts are attractive alternatives, but have scarcely been used for whole-cell applications (Zollner et al, 2010). The aim of this study was therefore to investigate several ascomycetous yeasts for their potential as P450-expressing whole-cell biocatalysts. To perform parallel, unbiased comparisons, the vector, cultivation conditions and assay conditions needed to be consistent between the different yeasts. This was facilitated by a broad-range vector system designed in our group, which allowed genomic chromosomal integration of foreign DNA at the 18S rDNA regions, and expression using a constitutive TEF promoter. Cultures of different yeasts were grown for the same duration at the same temperature in a medium common among strains, prior to activity assays. Using CYP53B1 as a reporter enzyme we demonstrated whole-cell activity for all of the yeasts tested, except for H. polymorpha. The native reductase systems allowed detectable activities of CYP53B1 in all of the other yeasts, with the highest activity (2.3 μmol.h-1 .gDCW -1) found in A. adeninivorans. Coexpression of cytochrome P450 reductases (CPR) from Yarrowia lipolytica (YlCPR), Rhodotorula minuta (RmCPR) and Ustilago maydis (UmCPR) all led to improvements of the CYP53B1 activities, with the highest activity (11.5 μmol.h-1.gDCW -1) obtained with CYP53B1 and UmCPR coexpressed in A. adeninivorans. The effects of the cloned UmCPR also differed between the hosts, with the biggest improvements observed in A. adeninivorans and Y. lipolytica. RmCPR and UmCPR improved the CYP53B1 activity more dramatically than YlCPR, possibly because CYP53B1, RmCPR and UmCPR are all of basidiomycetous origin. The CYP53B1 activity achieved in this study was considerably better than results obtained previously in our group. In the previous study, a Y. lipolytica strain with multiple copies of CYP53B1 and an additional copy of YlCPR both under the regulation of strong inducible promoters was used, under optimised and constant induction conditions. (Shiningavamwe et al, 2006). The activity obtained in this study on the other hand was achieved using an A. adeninivorans strain carrying presumably only one or two copies of CYP53B1 and UmCPR, expressed under the control of a constitutive promoter, and under non-optimised conditions. Activity of the self-sufficient P450s CYP102A1 and CYP505A1 was assayed using hexylbenzoic acid (HBA) as a non-natural substrate, since wild-type β-oxidation pathways would not permit the use of fatty acids as substrates. HBA is hydroxylated at the Ï-1 and Ï-2 positions of the alkyl chain by both CYP102A1 and CYP505A1 during this study. Better expression of the bacterial CYP102A1 was obtained using K. marxianus and S. cerevisiae, than in A. adeninivorans; whereas expression of the eukaryotic CYP505A1 was better in Y. lipolytica and especially A. adeninivorans. The best activity observed with a self-sufficient P450 was obtained once more with A. adeninivorans expressing CYP505A1 (33 μmol.h-1 gDCW -1). Overall, the vector system allowed successful expression of P450s and CPRs from bacterial, ascomycetous and basidiomycetous fungal origin, in multiple ascomycetous yeast hosts. The differential effects of different CPRs on a class II P450 were demonstrated, and the UmCPR was in this case found to be an excellent P450 reductase. We report heterologous P450 expression in A. adeninivorans for the first time, and it proved to be, of the yeasts tested, the host with the highest potential for efficient P450 expression and whole cell biocatalysis. The findings of this study provide insight for the improvement of the field of eukaryotic P450 research and particularly whole-cell biocatalysis, and could potentially assist in enhancing the applications of these promising enzymes.

Species of the genus Chryseobacterium (family Flavobacteriaceae) occur widely in clinical, environmental and industrial ecosystems. In the clinical environment, they are uncommon etiologic agents, but their infections may be serious in immunocompromised patients. They are often resistant to multiple antimicrobial agents making infections due to these organisms potentially difficult to treat. In the food environment, they are known to cause spoilage of foods such as canned products, milk and dairy products, fish, meat and poultry. It is therefore necessary to be able to solve or anticipate and avert possible problems caused by Chryseobacterium species. This genus also has positive characteristics which include synthesis of a number of enzymes potentially useful in industry (e.g. keratinolytic enzymes), medicine (e.g. prion degradation) and turnover of organic matter in soil, water and sewage plants. Taxonomic studies are key to solving such problems by characterization and identification of such organisms. This also sets the foundation for investigation of the organismâs beneficial roles and applications. In this study, some Chryseobacterium strains isolated from poultry feather waste and raw chicken, were phenotypically characterized and identified using conventional tests and the BIOLOG Omnilog Gen II system. Phylogenies of seven selected isolates were determined using the 16S rRNA gene sequence analysis and they were further characterized using the BIOLOG Omnilog Gen III identification system. They fell into four taxonomic groups (Group 1: 1_F178; Group 2: 5_R23647; Group 3: 6_F141B and 7_F195; and Group 4: 8_R23573, 9_R23581 and 10_R23577) which did not show affiliation to any currently recognised type species of the genus Chryseobacterium suggesting that these groups were possible representatives of novel species. Three selected strains (8_R23573, 9_R23581 and 10_R23577) were subjected to a polyphasic taxonomic study to determine their exact taxonomic identities. Results of the predominant respiratory menaquinone, fatty acid methyl esters and DNA base composition supported the affiliation of the strains to the genus Chryseobacterium. When subjected to DNA-DNA hybridization, the strains gave relatedness values of more than 81% among the three strains and less than 57% similarity between the strains and their two nearest phylogenetic neighbours C. shigense (DSM 17126T) and C. luteum (LMG23785T). A novel species emerged after a comparison of the phenotypic, chemotypic and genotypic results. The new species was described and the name Chryseobacterium carnipullorum sp. nov. was proposed. Analysis using the BIOLOG Phenotype MicroArray (PM) system, revealed that C. carnipullorum has the potential to cause food spoilage mainly by utilizing carbohydrates, carboxylic acids and amino acids by producing metabolites which lead to souring, butyric spoilage defects, alkalinisation, bitter tastes and sulphide spoilage. The organism was also shown to have potential for biotechnological applications in food and stockfeed technology; coffee extraction, oil drilling and detergent industries; manufacture of artificial antigens and chemical diagnostic agents and release of oligosaccharides and oligopeptides in (ultra) oligotrophic freshwater environments. It was found that the new species was able to produce extracellular keratinases that were able to extensively degrade chicken feather waste in 48 h. This has the potential of contributing toward solving the disposal problem which is experienced by the poultry industry that produces huge amounts of the recalcitrant feather waste as a by-product. Currently, a very small percentage of feather waste is steamed, treated chemically and ground to form dietary protein supplement for stockfeeds. Degradation of feathers using keratinolytic organisms is a more economical and environmentally friendly alternative. Chryseobacterium carnipullorum also has the potential for application in other biotechnological processes involving keratin hydrolysis. Hydrolysed feathers can be converted to fertilizers, glues, films, and they can be used as the source of rare amino acids, such as serine, cysteine and proline.

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 βâ-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.

The histone tails have for decades been regarded as unstructured polypeptide chains which simply served as molecular beacons to protein effectors which modify chromatin. However some experimental evidence shows that the tails may contain structure. Thus we conducted a Molecular Dynamics study of the Histone H3 tail and itâs most important post translationally modified isoforms. The 500 ns experiments showed the evolution of different secondary structure conformations for the different modified isoforms. More interestingly the active isoform showed a statistically significant longer reach compared to the inactive isoform. We next conducted a molecular docking study of the 15 â residue tip of the H3 tail to the nucleosome surface. The starting structures were sampled from the Molecular Dynamics trajectories. The tips showed binding to nucleosome where the H3 tail exits the nucleosome, between the DNA and the octamer. This binding position did not cha nge between the different isoforms. We thus propose a molecular mechanism whereby chromatin compaction is carried out at a nucleosome level, and is regulated by transitions in the N-terminal H3 tail structures, which, in turn, are modulated by specific epigenetic PTM patterns.

The mining industry has provided researchers access to the deep subsurface. The deep subsurface is known to harbor a treasure of novel genes and proteins that can be exploited in the biotechnology industry. It has been established that microorganisms in the deep subsurface are potentially novel and are able to endure high temperatures and extreme pH with limited nutrients for survival. Unfortunately most of these organisms are unculturable. Due to the lack of nutrients these microorganisms utilize reduced metals and minerals from the environment as a source of survival in a process known as biogeochemical cycling. Two fissure water samples were collected from two borehole sites at the Northam platinum mine and analyzed through molecular approaches. Microbial biodiversity was determined for borehole NO24FW030908 fissure water sample. The microbial biodiversity was based on the 16S rRNA and 18S rRNA gene clone libraries determined through phylogenetic clustering analyses using ARB software and a comparative analysis was done using DGGE profiling. The prokaryote and eukaryote diversity revealed low diversity at the species level but a high intraspecies diversity probably associated with the novelty of the biome. Unique isolates were cultured from borehole NO212FW050508 fissure water sample. Five isolates showed novelty at the species level and one isolate showed novelty at the genus level. Two isolates, Geobacillus sp. A8 and Geobacillus sp. A12 were sent for characterization at the DSMZ, Germany. Both isolates exhibited similarities at the genus level but significant differences at the species level based on the type strain to warrant different taxonomical positions. These isolates along with Thermus scotoductus SA-01 were analyzed for platinum reduction and the possible formation of metallic platinum. All isolates showed the ability to reduce platinum (IV) to platinum (0). Geobacillus sp. A8 was selected for further characterizations of platinum nanoparticle formation. Platinum nanoparticles were characterized with various tools to show the size and shape using TEM and SEM, to show the composition using XRD and EDS, and to show the particle size distribution using the NanoTrac and NiComp 380 ZLS systems. It has been proposed that a classical hydrogenase activated by a cytochrome c3 is responsible for the two-step reduction of platinum. Therefore, hydrogenase inhibition tests and the TTC test for the presence of an active hydrogenase were done to confirm the presence of a hydrogenase in Geobacillus sp. A8. It was then selected for the construction of the metabolic pathway genome database to study the metabolism of the microorganism in order to identify alternate or novel pathway(s) associated with the genome. Platinum reduction activity tests based on subcellular fractionation revealed platinum reduction and deposition that occurred in the periplasm; therefore, the putative protein involved in platinum reduction was probably periplasmic. The periplasmic fraction was separated into three fraction sizes, greater than 30 kDa, between 10 and 30 kDa and less that 10 kDa. The 10 â 30 kDa fraction revealed positive platinum reduction. The active fraction was analyzed on a SDS-PAGE and revealed three bands that were digested by trypsin and the peptides were analyzed by protein mass spectrometry. Two proteins were identified, an oxidoreductase commonly known as the old yellow enzyme previously shown to reduce chromate (VI), and a hypothetical YajQ protein. Both proteins were expressed and purified and both proteins showed the ability to reduce platinum. Further work would be to elucidate the in situ mechanism involved in the reduction of platinum with hydrogen as the electron donor.

ABSTRACT White-rot fungi and their enzymes are receiving increasing attention for biotechnological applications in the pulp and paper industry as alternatives to conventional bleaching. Laccase has been identified as one of the enzymes that plays a major role in lignin degradation. Laccase only attacks phenolic subunits of lignin, but its substrate range can be extended to non-phenolic subunits by the inclusion of a mediator. The use of this enzyme was, therefore, not successful in pulp bleaching trials until the discovery of mediators. Although the existence of natural mediators has not been confirmed, various components have been identified that are able to act as mediators. Improved methods of laccase production could benefit the industrial utilisation of the enzyme. White-rot fungi constitutively produce low concentrations of laccase, but higher concentrations can be obtained with the inclusion of inducers in the cultivation media. The enzyme is mainly produced during the stationary growth phase of the fungi, but various factors such as glucose, nitrogen and pH can influence levels of laccase production. The enzyme does not only hold potential for biological pulp bleaching operations, but also has application in bioremediation, the textile dye industry as well as the food and beverage industries.

YEAST DIVERSITY IN BLUE MOULD RIPENED CHEESES. ABSTRACT During the ripening process of blue veined cheese, different microbial groups interact and contribute to the final product. One of the most important of these groups are yeast. Further studies are needed to clarify their specific contribution to the ripening process. In order to accomplish this, a suitable and satisfactory enumeration medium is needed. Consequently, ten selective media were evaluated for their potential to inhibit and suppress the growth of moulds and bacteria without affecting the yeasts. Based on statistically compared data, no significant difference could be found amongst the ten media, except for one. Further studies were performed on the three media considered to be the most effective, MEA + Ox, MEA + NaCl and MEA + BP based on qualitative results. Accordingly, the three selected media were evaluated based on their ability to support the growth of the five most frequently occurring yeast species in blue veined cheese. No significant difference was obtained between two of the three media. MEA + NaCl however, was unable to support the growth of two of the five most dominant yeast species. MEA + Ox and MEA + BP proved to be superior for the enumeration and isolation of yeasts from blue veined cheese. DRBC, RBC and DG18 proved satisfactory regarding the enumeration of yeasts, whereas OGGY, MEA + SP and molybdate containing media are not recommended. Key words: Selective media, Yeasts, Moulds, Enumeration

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.