Oleaginous yeasts posses the ability to accumulate increased amount of lipids under appropriate conditions. These microbial lipids vary in the composition of fatty acids which results in their wide application in the biotechnological industry. This master thesis focuses on the lipid production and fatty acids composition from waste substrates by the yeasts Metschnikowia depending on various cultivation conditions. The influence of temperature, the ratio of carbon and nitrogen in medium, and the concentration of different carbon sources was studied. The cheap and easy available waste substrates as glycerol and animal fat were used for the cultivation. The production characteristics of the yeasts were monitored by various technique including gas chromatography, Raman spectroscopy and fluorescence microscopy FLIM. Moreover, the partial optimalization of the pulse field gel electrophoresis was applied in order to characterize the karyotype of the yeasts Metschnikowia. All the studied strains were able to use the waste substrates and at the same time to produce lipids. The amount of lipids and mainly their compositions vary depending on the yeast strain and on the culture conditions. Nevertheless, the ability of the yeasts to produce significant amount of unsaturated fatty acids by manipulation of culture conditions was proved. The maximum lipid yield was achieved by M. pulcherrima 149 on glycerol medium and by M. andauensis 129 on medium containing waste animal fat.

Edible insects have been part of the diet of many countries across the world for several decades. It is becoming increasingly popular in the europian developed countries, mainly because of the content of nutritionally important substances. The theoretical part of the thesis deals with the characterization of edible insects in terms of nutritional benefits, explains the reasons and method of breeding edible insects. For the analysis of nutritionally active substances, flour of house cricket (Acheta domesticus) was used. A. domesticus belongs to the representatives of insects legalized from 1.1. 2018 on the EU market. Of the significantly nutritional substances, proteins, lipids, fatty acids, fiber and selected minerals were analyzed. In this study, optimalization of vertical electrophoresis SDS-PAGE was performed to determine protein fragment sizes. In the second part the influence of acricket flour addition protein bars was investigated by sensory analysis. In the experimental part that was found that cricket flour contains protein, lipids and fatty acids in significant amounts. It also contains important minerals for human health such as magnesium, potassium, iron or zinc. However, the sensory properties of protein bars for consumers were not very attractive.

This diploma thesis deals with the changes in content of fatty acids in long-lasting stored sterilized processed cheeses. Cheeses were stored under different temperatures 6 ± 2 °C, 23 ± 2 °C and 40 ± 2 °C during 23 months. Measurements was made in 16th and 23rd month of storage. The samples of sterilized processed cheeses were extracted by using mixture of solvents (ethanol, diethylether and petrolether). The obtained fat was then saponified by methanolic solution of potassium hydroxide. The methylesthers were analyzed by gas chromatography with flame ionization detection. Lauric, myristic, palmitic, oleic and stearic acids were the most abundant in cheeses. The yield was measured in these main acids. To summarize the content of fatty acids decreased during time of storage and also by influence of storage temperature higher than in refrigerator.

This thesis deals with the identification and quantification of free and bound fatty acids in model cheese samples of the Moravský bochník type, which were produced at Tomas Bata University in Zlín by a standard technological procedure. In a theoretical part of the thesis, the issues of lipids, fatty acids, as well as the characteristics of high cooked cheeses including Moravský bochník, microbial cultures used in their production, and the possibilities of fatty acids determination are summarized. The ČSN EN ISO 1735:2005 method was chosen for the lipid extraction from cheese samples. Present free and bound fatty acids were identified and quantified by gas chromatography with flame-ionization detection after conversion to methylesters by acid esterification with bortrifluoride as a catalyst. In total 32 fatty acids were identified in cheese samples, the highest amount of caproic, caprylic, capric, lauric, myristic, palmitic, stearic and oleic acids.

This master thesis deals with the monitoring of fatty acids during production of natural Edam cheeses, i.e. natural hard cheeses with low heat curd, and of processed cheeses, which were produced from them. The theoretical part briefly characterizes the natural Edam cheese and processed cheese, the main attention is paid to lipids, fatty acids and the possibility of their determination. In the experimental part the content of fatty acids in the model samples of natural and processed cheese produced at Tomas Bata University in Zlín was determined. The method according to ISO 1735 was chosen for extraction of lipids from the samples, the method according to ISO 5509 using methanol solution of potassium hydroxide was chosen for the esterification of fatty acids. The resulting methyl esters were determined by gas chromatography with FID detection.

The determination of non-esterified fatty acids (NEFA) in plasma is possible by many routine biochemical methods. For more detailed metabolic studies it is required to analyse the fatty acid (FA) profile. The NEFA profile determination is usually performed by the capillary gas chromatography (GC), but the sample preparation is not uniform. NEFA may be isolated by the extraction process or by the preparative thin-layer chromatography (TLC). The aim of this study was to compare these two separation procedures. The samples of pooled plasma from volunteer donors were analyzed by capillary GC after previous separation procedure by the TLC and the liquid-liquid extraction. The results were compared by the t-test for both the absolute concentration of individual FA provided by the internal standard (margaric acid) method and the relative abundance (Rel%). The reproducibility of the results was significantly better for the liquid-liquid extraction method than the TLC. The relative standard deviations (RSD) of the FA groups (unsaturated, monosaturated and polysaturated) were from 4.3 to 11.3 % vs. 8.6 to 33.8 % for the relative abundance and from 16.5 to 25.5% vs. 15.4 to 47.4% for the absolute concentration. For each FA, which were represented by more than 0.1 Rel %, RSD ranged from 4.5 to 47.8% vs. 9.5 to...

Magister Scientiae (Medical Bioscience) - MSc(MBS) / The incidence of non-communicable diseases is increasing worldwide, with South Africa being no exception. Non-communicable diseases are classified as non-infectious and are often referred to as lifestyle diseases as they are caused by common, modifiable risk factors such as unhealthy diet, obesity, tobacco use and lack of physical activity. Due to the increasing prevalence of childhood obesity diseases such as fatty pancreas and fatty liver are becoming more common. Cardiovascular disease, and more specifically atherosclerosis is the underlying cause in most adult deaths. Disease pathogenesis starts in childhood and can be detected via Aortic intima-media thickness (IMT). The developmental origins of health and disease hypothesis (DOHaD) proposes that exposures in-utero may result in persistent adaptations including alterations in metabolism.

Omega-3 Fortification of Marinara Sauce Hannah Collie, Mary Andreae, MS, RD, LDN, W. Andrew Clark, PhD, RD, LDN, Department of Rehabilitative Science, College of Clinical and Rehabilitative Health East Tennessee State University, Johnson City, Tennessee In westernized culture, there is a deficit of healthy fats in the average person’s diet. This is evidenced by many different conditions such as cardiovascular disease, cancer, and chronic inflammatory issues. The “Mediterranean Diet” has been shown as an ideal way to combat these health issues.The diet promotes fish as a protein source and a way to consume essential polyunsaturated fatty acids. Due to location and trends, fish is less often a main component of the western diet.This study investigated ways to fortify a more commonly consumed food in western culture, marinara sauce, with flaxseed oil, walnut oil, and anchovies. These fortified marinara sauces were compared to a commercial sauce, Paul Newman’s Marinara for nutritional content. We hypothesize that adding omega-3 rich ingredients to a base marinara sauce recipe will significantly vary the fatty acid profile and increase the amount of omega-3 fatty acids as well as decrease the omega-6 to omega-3 fatty acid ratio. Sauces were prepared and analyzed for nutrient content using proximate analysis methods. Preparation included cooking and freeze drying the variants. Nutrient content tests performed included: bomb calorimetry, Kjeldahl protein analysis, Soxhlet fat analysis, ash inorganic analysis, FRAP Assay for antioxidant content, and gas chromatography to characterize fatty acid composition. Each variant sauce had a fatty acid profile that was unique. Two of the three variants showed a better omega-6 to omega-3 fatty acid ratio than the (control) Paul Newman’s sauce. Samples with the highest to lowest omega-6 to omega-3 ratio were respectively, flaxseed oil, Paul Newman’s commercial sauce, walnut oil, and anchovy. From gas chromatography, omega three fatty acid composition as a percentage of total fatty acids were approximately in the flaxseed oil variant, 21% in the Paul Newman’s sauce, 30% in the walnut oil variant, and 81% in the anchovy variant. Two of three variant sauces, walnut and anchovy, when compared to the commercial Paul Newman's sauce, showed more favorable omega-3 fatty acid content and lower omega-6 to omega-3 fatty acid ratios. Unexpectedly, the flaxseed oil variant had less omega three fatty acids and a higher omega-6 to omega-3 fatty acid ratio than the commercial sauce. In conclusion, simple additions of omega-3 ingredients to marinara sauce can decrease the omega-6 to omega-3 fatty acid ratio in the diet.

Adipose tissue is a key regulator of energy metabolism and glucose homeostasis by promoting triglyceride storage and breakdown in various physiological states. Obesity, however, alters adipose tissue metabolism, inducing chronic inflammation, followed by excessive lipolysis. This results in higher systemic free fatty acid (FFA) levels, leading to desensitization of insulin signaling and ultimately to insulin resistance. Although the link between obesity and progression of insulin resistance and type 2 diabetes mellitus (T2DM) remains unclear, tumor necrosis factor-alpha (TNF-alpha) has been proposed to be a key player in promoting obesity-related development of T2DM through chronic inflammation of adipose tissue. TNF-alpha has direct and indirect mechanisms by which it elicits insulin resistance in adipocytes. TNF-alpha attenuates insulin signaling by directly inhibiting insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1). Indirectly, TNF-alpha activates signaling pathways to increase lipolysis and FFA release into circulation, leading to insulin resistance. Lipid droplet-associated fat specific protein 27 (FSP27) protects adipocytes from lipolysis by regulating the lipolytic capacity as well as transcription of adipose triglyceride lipase (ATGL). It has been observed that TNF-alpha promotes lipolysis by reducing the expression of FSP27 in murine adipocytes. The effect of TNF-alpha on lipolysis human adipocytes has also been studied; yet its effect on promoting insulin resistance in human adipocytes still remains elusive. In the present study, we examined the effect of FSP27 on TNF-alpha induced lipolysis and insulin resistance in human adipocytes. TNF-alpha enhanced lipolysis in cultured human adipocytes. In addition, TNF-alpha reduced the expression of endogenous FSP7 and the phosphorylation of AKT, inhibiting the activation of insulin signaling pathway in cultured human adipocytes. FSP27 overexpression, however, attenuated TNF-alpha induced lipolysis and restored activation of insulin signaling through phosphorylation of AKT in cultured human adipocytes. Taken together, these data suggest that FSP27 has a protective effect against TNF-alpha induced lipolysis and insulin resistance through regulating lipolysis and insulin signaling in human adipocytes.

N-acylethanolamines (NAEs) are a family of signaling lipids derived from a minor membrane lipid constituent N-acylphosphatidylethanolamine (NAPE). In Arabidopsis, NAE mediates physiological functions such as seedling growth, flowering, and response to stress via abscisic acid (ABA) –dependent and –independent signaling pathways. The function of NAEs is terminated by a highly conserved fatty acid amide hydrolase (FAAH). Studies in model plant Arabidopsis showed the significant role of NAEs that makes it relevant to elucidate the conserved metabolic pathway of NAEs in crop species such as tomato. It is hypothesized that there is a functional FAAH in tomato that hydrolyzes NAEs. To test this hypothesis, AtFAAH was used as a template to identify putative FAAH sequences in tomato, using BLASTX. Six SlFAAH sequences with the conserved amidase signature sequence and the catalytic triad, formed by Lys205, Ser281, and Ser305 in AtFAAH, were identified. Phylogenetic analysis of putative SlFAAH homologs and other FAAH family proteins (Arabidopsis, rice and moss), using CLUSTALW, revealed the two sequences that are closely related to the functionally characterized AtFAAH1. Using molecular visualization system (PyMOL), protein structures of putative SlFAAH1and 2 were predicted and compared with AtFAAH; both sequences showed similar domain structure to AtFAAH, with minor differences in spatial arrangement. For further biochemical characterization, full-length coding sequence of SlFAAH1 and SlFAAH2 were isolated and cloned into a heterologous expression system. The expressed protein will be characterized for its hydrolytic activity against radiolabelled NAE substrates. Furthermore, transcript levels for SlFAAH1 and SlFAAH2 will be quantified and correlated with the NAE levels in various tissues to predict their role in tissue-specific NAE hydrolysis. Together, these molecular and biochemical characterization studies in tomato are expected to further validate the conserved nature of NAE metabolic pathway in plants.