Thesis (MSc)--University of Stellenbosch, 2007. / Bibliography.

Thesis (Ph. D.)--University of Delaware, 2007. / Principal faculty advisor: Colin Thorpe, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

Six auxotrophic mutants of A. nidulans were crossed in a dialell cross system to obtain heterokaryous and heterozygous diploids. In order to ascertain their lipid accumulation ability, some of these mutants and diploids were tested in a minimal medium (with 3% glucose + 0.6% NaNO3), either in a shaker or in an incubator without agitation. Both, the mutants and diploids, exhibited only 4.6% lipid, on a dry weight basis. With the aim of optimising culture conditions for lipid accumulation, a wild type was cultivated in a range of different media and cultural conditions. The best yield (about 24%), was achieved in a modified minimal medium (MM + 12% glucose + 0.1% NaNO3), with a vortex stirrer device. The lipid composition of wild type 16 grown in a fermenter was determined. The results obtained from cells grown in two different media and using two extraction methods were compared. Fractionation of the total lipid on a Florisil column showed that this strain is composed of 86% neutral lipid, 7% glycolipid and 7% phospholipid, after isopropanol (IP) extraction, whilst chloroform-methanol (CM) extraction gave 75% neutral lipid, 8% glycolipid and 17% phospholipid. A further fractionation on hydrated Florisil showed that CM extracted sterols (both free and esterified) more efficiently than IP. Therefore, CM was considered a better extraction method, particularly for protein-bound lipids. The separation of the neutral lipid fraction into sub-classes also showed that the enhanced lipid content achieved in modified minimal medium, compared with a previously reported medium, was accounted for mainly by an increase, not in the triglycerides as was expected, but in the amount of sterols. TLC analysis of glycolipid and phospholipid from IP and CM extraction demonstrated two major glycolipid components (monoglycosyl and diglycosyl diglycerides) and that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the principal phospholipids with lesser amounts of phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, cardiolipin and phospbatidic acid (PA) after CM extraction, whilst after IP extraction only PC, PE and PA were found. Another significant difference between the two extraction methods is the large amount of PA found after CM extraction, but not after IP, showing that, almost certainly, phospholipase D activity had occurred during the process of extraction and/or storage of the lipid. It was also found that the principal phospholipid attacked by the enzyme was PC. The fatty acid composition was determined by GLC. The major fatty acids found in the total lipid were: 16:0 =21%; 17:0 =5%; 18:0 =18%; 18:1 = 20%; 18:2 = 35%. Each lipid class showed a different and distinctive fatty acid composition, exhibiting variation with the growth medium and extraction method used. Of particular interest was the sterol ester fraction which contained margarinic acid (17:0) as its only fatty acid.

The objective of this study was to determine the effect of different fat inclusion levels and fat types on the physical and chemical attributes, lipid oxidation, fatty acid composition and sensory characteristics of warthog cabanossi. To achieve this, three types of cabanossi with different pork backfat levels (10 percent, 20 percent and 30 percent) were produced for the first experiment. The results from the study showed that different inclusion levels of pork backfat had an influence (P ≤ 0.05) on the physicochemical and fatty acid composition of warthog cabanossi but did not influence lipid oxidation (P > 0.05). The highest (P ≤0.05) pH, weight and moisture decline was observed in the 10 percent pork backfat cabanossi compared to the 20 percent and 30 percent treatments. However, no differences (P > 0.05) in the water activity of the product were observed. As expected total fat was lower in the 10 percent fat treatment and increased concomitantly. Similarly, protein, ash and salt were higher in the 10 percent fat cabanossi and decreased concomitantly. Differences in the fatty acid composition were observed between treatments. Furthermore, backfat level affected the sensory attributes and consumer acceptance of the cabanossi. Ten percent backfat cabanossi was scored higher (P ≤0.05) for most sensory attributes. Consequently, it was observed that the consumer panel preferred and scored the 10 percent fat cabanossi higher with regards to appearance and taste. In the second experiment, two cabanossi treatments of different fat types (pork backfat and fat-tailed sheep backfat) were produced. The weight loss, moisture content, pH, water activity and salt content did not differ (P > 0.05) between the two cabanossi products. However, there were differences (P ≤0.05) in the protein, fat and ash contents; where protein and ash were higher in the pork backfat cabanossi whilst fat was higher in the sheep backfat cabanossi. Thiobarbituric reactive substances (TBARS) were similar (P > 0.05) between the two fat types cabanossi which could be explained by similar fatty acid profiles being reported for the two cabanossi although the n-6:n-3 ratio was higher (P ≤0.05) in sheep backfat cabanossi. Results from the descriptive sensory analysis showed two distinct products (P ≤0.01) where pork backfat cabanossi scored higher for most attributes. However, the lower scores for sheep backfat cabanossi were within an acceptable range. Sheep backfat cabanossi were also scored for unique attributes that were not detected in the pork backfat cabanossi. This study concluded that fat-tailed sheep backfat can be used to produce an unique cabanossi product of acceptable quality.

This thesis investigates the fatty acid production from carbohydrates using C. butyricum. In nature a common route for the anaerobic degradation of carbohydrate in the environment is via methanogenesis. At the heart of these processes however, is the metabolism of a diversity of carbohydrate materials that produce a few fatty acids (acetate and butyrate) which are then slowly converted to methane. In this context, fatty acids can be considered as a common end- product/intermediate from carbohydrate degradation that could be used to produce chemicals. Already, acetic and butyric acid are important feedstock chemicals in the pharmaceutical, food and industrial sectors and there is potential to expand this further. As a first step to investigate the conversion of waste carbohydrate to fatty acids for chemical production, C. butyricum, a strictly anaerobic bacterium, was investigated as a model system for the potential production of acetic and butyric acid. The production efficiency of C. butyricum relies on the type of substrate, production methodology, the strain and environmental conditions. Pure cultures of C. butyricum were investigated for fatty acid production from carbohydrates. Initial studies involved medium optimization in test tube culture for high growth rate and maximum biomass production (ODmax)- In this medium, glucose was selected as the main substrate together with yeast extract, KH2PO4 and NH4(SO)4. The studies were carried out in three types of pH controlled reactors; batch stirred tank (SRT), continuously stirred tank (CSTR) and membrane bioreactor (MBR) A comparison the fatty acid production kinetics and productivity in each reactor was undertaken and the effect of glucose concentration and where appropriate, glucose feed rates, were also investigated. The results show that fatty acid production could be carried out in all three fermentation systems. A common observation in these systems was that fatty acid production was influenced by the glucose concentration in that at low glucose concentration the ratio of acetate to butyrate was about 30:1 while at higher concentrations the ratio was reduced to about 3:1 on a molar basis. The detailed kinetic studies generated unique data for this organism and shows that the maintenance coefficient (ms) increase with increasing glucose concentration (0.02 to 1.1 g substrate/g cell/h), due to mainly to end product inhibition and the true yield (Yx/s was around 0.2 for all glucose concentrations tested. Meanwhile substrate saturation (KJ decreased with increasing glucose concentrations (2.06-6.41 g/L). This observation was atypical to that observed in other anaerobic fermentations by previous workers. A comparison of fatty acid productivities using a l0g/1 glucose feed in the 3 reactors for acetic acid were 0.95 g/l/h for STR. 4.41 g/l/h for CSTR and 37.88 g/l/h for MBR and for butyric acid 0.15 g/l/h for STR, 1.27 g/l/h for CSTR and 14.34 g/l/h MBR. Although, previous work in this area is limited the data obtained in this study was also compared with other published work and this suggests that the production of fatty acid, especially acetic and butyric acid in the MBR system is by far the most productive yet reported. The results are discussed in the context of the waste treatment process for fatty acid production and its application to waste conversion and its further development.

Insulin resistance is associated with a number of metabolic abnormalities that increase the risk of developing coronary heart disease. Dietary fat has been linked with insulin resistance, with alterations in the quality as opposed to the quantity of dietary fat now thought be more important in instigating improvements in insulin resistance. The aim of this work was to investigate the effect of alterations in the dietary fat intakes of middle-aged men (n = 32) on the insulin sensitivity of glucose disposal and postprandial lipid metabolism and to explore the mechanistic links between these insulin responsive pathways. Three separate dietary interventions were conducted; the first involved an increase in the intake of n-3 polyunsaturated fat, the second a decrease in saturated fat and an increase in carbohydrate and the third a decrease in saturated fat and an increase in monounsaturated fat intake. Compliance was monitored by the measurement of red blood cell phospholipid fatty acid composition, postprandial lipid metabolism was measured over 9 hours following a high-fat breakfast (80 g fat), and insulin resistance was measured using the short insulin tolerance test. The results of the study showed that while insulin sensitivity was inversely correlated with red blood cell saturated fatty acid concentration at baseline, the insulin sensitivity of glucose disposal was unaffected by any of the dietary interventions conducted. In measurements of postprandial lipaemia, improvements were observed following the low-saturated fat / high-monounsaturated fat diet and the n-3 polyunsaturated enriched diet, however the low-saturated fat/ high-carbohydrate diet was associated with a worsening of postprandial lipaemia through an increase in the concentrations of triglyceride-rich-lipoproteins. Changes in fasting biochemical measurements were most evident in the low-saturated / high-monounsaturated diet, with an 11 % reduction in total cholesterol and a 15.4 % reduction in fasting triglycerides. There were no observed changes in the activity levels or the gene expression of lipoprotein lipase. There was an unexpected positive association between the degree of insulin sensitivity and the extent of postprandial lipaemia, indicating that the link between these pathways is complex and warrants further investigation. Overall this work supports the view that dietary guidelines should be directed towards a change in the composition of fat, to a lower saturated fat intake, a higher monounsaturated fat intake and a lower n-6 : n-3 ratio through an increase in the intake of long chain n-3 polyunsaturated fatty acids.

M.Sc. / The senescence of climacteric flowers such as carnations is accompanied by an increase in ethylene synthesis during the later stages. This increase in ethylene synthesis is preceded by an increase in the sensitivity of the flowers to ethylene. The increase in ethylene sensitivity is accompanied by a concomitant increase in the levels of short-chain saturated fatty acids (SCSFAs). Treatment of carnation flowers with SCSFA results in an increase in ethylene sensitivity. It appears that these acids act by increasing membrane fluidity, causing slight conformational changes in membrane associated proteins and thereby increasing the ability of the tissue to bind ethylene to its membrane associated receptor molecules. The levels of SCSFAs in senescing carnation petals is controlled by the activity of the enzyme acetyl-coenzyme A carboxylase (ACCase). A decrease in the activity of this enzyme results in an increase in the levels of the SCSFAs and vice versa. During the senescence of carnation flowers, ACCase activity fluctuated from day to day. This fluctuation can be correlated to the fluctuations in the ethylene sensitivity of the flowers on a daily basis. In carnation petals, ACCase is located mainly in the plastids. ACCase activity could be controlled via feedback inhibition by long-chain fatty acids such as oleic acid. Treatment of carnation flowers with oleic acid resulted in a concomitant inhibition of ACCase activity, an increase in SCSFA-levels and an increase in ethylene sensitivity. Oleic acid is a competitive inhibitor of ACCase activity, and changes in the levels of oleic acid will affect the activity of the enzyme. An increase in oleic acid concentration resulted in a decrease in enzyme activity. However, in carnations it appears that ACCase activity is not controlled via feedback inhibition by long chain saturated fatty acids. The results of this study clearly show that ACCase activity is controlled directly by the expression of at least the biotinylated (BCCP) subunit of the enzyme. A decrease in the expression of the gene during the early stages of senescence coincided with a decrease in ACCase activity and was accompanied by a concomitant increase in ethylene sensitivity. These results indicate that the increase in ethylene sensitivity caused by an increase in SCSFA levels is directly controlled by the expression of the ACCase genes.

Detailed analyses of the lipid class and fatty acid composition were carried out for the marine microalgal species Nannoch/oropsis oculata (Droop) (CCAP strain no. 849/1) of the division Eustigmatophyceae (Hibberd). The alga was grown in batch and continuous culture using a novel culturing apparatus, the cage culture turbidostat, the construction of which is detailed in full. The total lipid extract yielded by the alga varied in a growth-phase dependent manner within the range 25 % to 80 % of the lyophilised cell mass. Of this between 40 % and 70 % was recovered as fatty acid methyl esters (FAME) upon transesterification. The total fatty acid composition of N. oculata consisted mainly of 16:0, 16:1 and 20:5(n-3), these three fatty acids often accounting for greater than 80 % of the total fatty acid mass. Between 9 % and 50 % of the mass of total FAME was accounted for by 20:5(n-3), the balance being accounted for by variations in the relative proportions of 16:0, 16:1, 18:1, 18:2 and 20:4. During periods of low cellular division rate, such as the lag- and stationary-phases, the proportion of polyunsaturated fatty acids (PUFA) (mainly 20:5(n-3» decreased. The total fatty acids became increasingly saturated as higher proportions of shorter chain length fatty acids accumulated, mainly in triacylglycerols (TAO). Increased cellular proportions of total lipid resulted from TAO accumulation which occurred on account of preferential partitioning of carbon into TAO biosynthesis whilst cellular division was suspended. The fatty acid composition of the TAO was more saturated at high synthesis rate and vice-versa at lower rates. The galactolipids, monogalactosyldiacylglycerol (MODO) and digalactosyldiacyl glycerol (DODO) were rich in 20:5(n-3) during exponential cell division containing up to 77 % and 53 % 20:5(n-3) respectively. Phosphatidylcholine (PC) was the only cl~s to contain significant proportions of CIS fatty acids during exponential growth, thus implicating its involvement in the acyl chain elongation reactions between the Cl6 and C20 fatty acids. Culture incubation temperature in the range 5 °C to 25°C did not influence the fatty acid composition of N. oculata. The effect of temperature upon culture dynamics at the lower culture incubation temperatures gave an apparent decrease in the PUFA content of the total fatty acid at a given point on the cultures growth curves. By expressing the data in tenns of culture doubling periods during the exponential-phases of growth it was found that temperature had no real effect upon fatty acid unsaturation or chain length. at either the total or the individual lipid class FAME level after the cells had passed through five doubling periods. Increasing the culture medium salinity from one quarter to one and a half times that of normal seawater decreased the un saturation and chain length of the fatty acids at both total and individual lipid class levels. The change resulted from the progressive accumulation of 18:1 and 18:2"at the expense of 20:5. Variation of salinity did not affect the dynamics of the cultures in the same respect as temperature in that a lag-phase was not observed on the cultures growth curves. However. such a phase was evident in the fatty acid profile of the cells in the period following inoculation. The 'effects of culture illumination intensity in the range 45 Jill m-2 sec-I to 170 Jill m 2 sec-! were examined under continuous culture conditions using the cage culture turbidostat Accumulation of saturated TAG by the cells at the higher illumination intensities gave an apparent decrease in the rate of PUFA biosynthesis. The polar lipid classes were found to be more highly unsaturated at higher illumination intensities. At lower illumination intensity TAG accumulation was reduced and the total fatty acid composition was accordingly more unsaturated. The fatty acid composition of the TAG component was more unsaturated but those of the polar lipid classes were less unsaturated than at higher illumination intensity. Increased illumination increased the degree of un saturation of the polar lipid cl~sses. Excess fixed carbon was partitioned into TAG biosynthesis. primarily as 16:0 and 16:1. The net accumulation of this lipid class even at high cell division rates resulted in a low overall unsaturation level. The effects of decreasing nitrate concentration in the range 1.0 mM N03 - to 0.001 mM N03 - had a similar basis to those of illumination in that the changes in the total fatty acid composition were largely governed by the rate of TAO accumulation. At high nitrate concentrations the cellular division rate was relatively high and the proportion of TAO in the total lipid extract was low. Consequently, both total and individual lipid classes contained high proportions of unsaturates, particularly 20:5(n-3). However, when the nitrate concentration was decreased, such that it began to limit the rate of cellular division, TAG accumulated Cursory analyses of the molecular species of the galactolipid classes, MODO and DODO, and phospholipid class PC are presented. The effects of environmental variables are discussed in tenns of the changes which may occur in the growth phase distribution of the cells in asynchronous culture, along with the concommitant changes in the lipid composition of the cells. The potential linkage of the elongation and desaturation reactions with both MODO and PC is also discussed briefly with reference to future research.

The present study was carried out to determine the effects and possible mechanisms of action of the essential fatty acids (EFAs) (linoleic acid (LA), gamma-linolenic acid (GLA) and arachidonic acid (AA)) and ascorbic acid (Asc) on BL6 murine melanoma growth in cell culture and in mice. Interactions between the nutrients in influencing melanoma growth as well as possible mechanisms of the interactions were also examined in the above systems. Cell culture studies revealed that all three EFAs (0-SOμg/ml) and Asc (0-200μg/ml) significantly inhibited melanoma growth at the concentrations used. The EF As were also found to significantly inhibit growth, although to a lesser extent than BL6 cells, of monkey kidney (LLCMK) cells which were used as a non-malignant control cell line. Asc in contrast was found not to inhibit growth of these cells. Supplementation of Asc (lOO)μg/ml) to EFA containing (0-50μg/ml) medium was found to significantly increase inhibition of cell growth in both cell lines, and in the BL6 cells in particular, after taking into account the growth inhibitory effects of Asc in the absence of EFAs. The mechanism of cell growth inhibition by the EF As appeared to involve lipid peroxidation but not enhanced prostaglandin (PG) or leukotriene (LT) synthesis. While Asc was found to increase both lipid peroxidation and PG synthesis in the cells, these mechanisms and enhanced LT synthesis did not appear to have played a role in the inhibition of cell growth by Asc or in the growth inhibitory interaction between Asc and the EF As. In vivo studies revealed that diets containing essential or polyunsaturated fatty acids (EFAs/PUFAs) in the form of vegetable oils, and in particular GLA in the form of evening primrose oil, significantly promoted melanoma growth in mice when compared with an EFA/PUFA free diet containing predominantly saturated fats (SF). Supplementary dietary Asc in contrast was found to significantly inhibit melanoma growth in mice fed EFA/PUFA, and in particular GLA, containing diets but not in mice fed SF cont~g diets. This result appears to indicate the occurrence of an interaction between the two nutrients. Ul The mechanism of tumour promotion by the EP As/PUP As did not appear to have involved enhanced PG or LT synthesis or lipid peroxidation. Since dietary EPA/PUPA manipulation was found to significantly alter the EPA content of tissues, including the melanomas, the mechanism of tumour promotion may have involved changes in the EPA composition of the tumour cells. While supplementary Asc was found to significantly increase the Asc content of certain tissues, including the melanomas, which may have played a role in tumour growth inhibition by Asc, it was found not to affect the EPA content of tissues. Enhanced PG or LT synthesis and lipid perox:idation did not appear to have been involved in the tumour growth inhibitory interaction between Asc and the EP As/PUP As. THe activity of the enzyme delta-6-desaturase, a key enzyme in EF A metabolism which catalyses the desaturation of LA to GLA, and the influence of Asc on activity of the enzyme were also examined. The cultured cells, and BL6 cells in particular, were found to contain significant activity of the enzyme. Whereas murine liver microsomal fractions were found to contain delta-6-desaturase activity, microsomes from melanomas grown in mice were found to lack activity of the enzyme. The significant tumour promoting effects of the GLA containing EPO diet may have been the result of the lack of delta-6-desaturase activity in tumour cells grown in mice. Asc was found to stimulate activity of the enzyme in cultured BL6 cells but not in LLCM.K cells, while dietary Asc and EF A/PUP A manipulation did not influence activity of the enzyme in microsomal fractions. This study has confirmed previous reports of the in vivo tumour promoting effects of dietary EP As/PUP As and the tumour growth inhibitory effects of Asc. The in vitro cell growth inhibitory effects of Asc and the EP As also confirm the results of previous reports. Previous studies investigating possible interactions between Asc and EP As/PUP As in influencing tumour cell growth could not be located in the relevant literature. This study may therefore be one of the first investigations of any such interaction between these nutrients in tumour cells. While this study was not able to identify the mechanisms involved in the different tumour promoting or tumour growth inhibitory effects of the two nutrients in the two systems, it did eliminate a number of potential mechanisms. The results of this study also emphasise the difficulty of attempting to compare the results of in vitro and in vivo studies.

No description available.