The <i>de novo</i> formation of long-chain fatty acids and the reduction of long-chain fatty acids to the corresponding fatty alcohol were studied in the calanoid copepod <i>Calanus finmarchicus (Calanus</i>) and the fresh-water fish, <i>Trichogaster trichopterus</i> (the gourami). <i>De novo</i> biosynthesis of fatty acids by the enzyme fatty acid synthase (FAS) was localised in the 6.3 x 10⁶g-min supernatant ('cytosolic' fraction) of homogenates of whole <i>Calanus</i> and was characteristic of a Type 1 multienzyme complex. The formation of fatty acids required the presence of acetyl-CoA, malonyl-CoA and was specific for NADPH as the reductant. The main products of the reaction were the saturated long-chain fatty acids stearic acid and palmitic acid, accounting for 54% and 25% of the total fatty acid product, respectively. The alcohol-forming enzyme, NADPH-fatty acyl-CoA oxidoreductase, from both <i>Calanus</i> the gourami roe was membrane-associated. Differential ultracentrifugation showed that a 6.3 x 10⁶g-min pellet (the 'particulate' fraction) was most active in the formation of long-chain fatty acohols. The specific activity of the enzyme in the gourami roe particulate fraction was over 100-fold higher than that in the <i>Calanus</i> particulate fraction. Both systems required the presence of an acyl-CoA generating system in the form of exogenous CoASH, ATP and Mg⁺⁺. The reaction was specific for NADPH as the reductant in the case of the gourami roe system. However, NADH substituted for NADPH in the <i>Calanus</i> system. Palmitic acid, in the presence of an acyl-CoA generating system, was the most effective substrate with the 18:0, 18:1 and 22:1 substrates giving progressively lower activities.

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Orientador: Gabriela Alves Macedo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-12T23:26:09Z (GMT). No. of bitstreams: 1 Lopes_DanielleBranta_M.pdf: 1927712 bytes, checksum: 4c84529dc29400d6dfb671ad68c0abc8 (MD5) Previous issue date: 2009 / Resumo: O aumento da demanda por esteres obtidos naturalmente ou por processos biotecnologicos, para aplicacoes como aditivos em alimentos, cosmeticos, industrias farmaceuticas e de lubrificantes, faz necessario o desenvolvimento de catalisadores altamente especificos. Por esta razao, o uso de enzimas como catalisadores para a sintese de esteres de alto valor agregado vem sofrendo um rapido desenvolvimento, por proporcionar a possibilidade de obtencao de um produto atraves de um processo biologico. Embora destinada pela natureza para realizar a hidrolise de lipideos, lipases (E.C. 3.1.1.3) podem, sob condicoes apropriadas de reacao, promover a formacao de esteres atraves de reacoes de acidos e álcoois (esterificacao). Comparadas com processos quimicos ja realizados em uma escala industrial, reacoes enzimaticas ocorrem sob condicoes brandas (e ecologicamente mais favoraveis) apesar de poderem ser mais lentas. A grande vantagem e a especificidade apresentada pelas enzimas, que permite a formacao de derivados lipidicos, os quais nao sao facilmente preparados por processos laboratoriais convencionais. Neste trabalho, foi avaliado o desempenho de lipases microbianas e vegetais, nao-comerciais, e comparadas a uma lipase comercial, na síntese de esteres com possiveis propriedades emulsificantes, bem como a influencia das condicoes de reacao (concentracao de enzima, quantidade de agua, relacao molar dos substratos, tipos de meios reacionais, e cinetica) no processo de esterificacao. A etapa seguinte do estudo foi constituida de um planejamento experimental fatorial completo com o proposito de otimizar a producao do ester oleato de oleila, em meio livre de solvente organico, com a lipase de Rhizopus sp. selecionada por apresentar melhor desempenho catalitico. O mesmo estudo foi realizado tambem com a lipase comercial Lipozyme TL IMR, com finalidade comparativa. Os parametros reacionais estudados foram: razao molar entre os substratos (acido:alcool) e concentracao de enzima (% m/m em relacao a massa de reagentes). Destes ensaios, verificou-se melhor habilidade catalitica da lipase de Rhizopus sp. utilizando maior proporcao de alcool (2:1) e maior quantidade de enzima (9,8%). O mesmo foi observado com a enzima comercial Lipozyme TL IMR, a qual tambem apresentou maior producao de ester quando se fez uso de uma maior proporcao de alcool (3:1) e maior quantidade de enzima (7,0%). A porcentagem de esterificacao obtida pela lipase de Rhizopus sp. (93,1%) foi muito proxima a porcentagem obtida pela lipase comercial Lipozyme TL IMR (94,2%), evidenciando o grande potencial desta enzima nao-comercial na sintese do oleato de oleila. Por fim, foi realizada a caracterizacao parcial do ester quanto as atividades emulsificante e antimicrobiana. Resultados indicaram que o ester produzido neste trabalho demonstrou atividade emulsificante, porem esta nao foi tao alta quando comparada aos emulsificantes comerciais: Dodecil Sulfato de Sodio (SDS) e Tween 80. Observou-se tambem que o oleato de oleila nao foi capaz de inibir o crescimento microbiano de Bacillus subtilis, sendo assim, ate o presente momento, nao foi detectada atividade antimicrobiana para este ester / Abstract: The increasing demand for esters obtained either through natural or biotechnological processes for use as food additives, cosmetics, pharmaceuticals and lubricants requires the development of highly specific catalysts for ester production. For this reason, the use of enzymes as catalysts for the synthesis of esters with high added value is undergoing rapid development. Although the main function of lipases in nature is to catalyze the hydrolysis of lipids, it can also be made to promote ester formation through the reaction of acids and alcohols (esterification) under specific reaction conditions. Although on an industrial scale enzymatic reactions may be longer when under milder and ¿greener¿ conditions compared to chemical processes. The great advantage of working with enzymes is their great specificity, which often allows for the preparation of lipid derivatives not easily obtained using conventional laboratory procedures. This work first evaluated a few variables affecting the synthesis of esters and the esterification process, namely the influence of: the use of commercial and non-commercial lipases of vegetable and fungi origin, reaction conditions, enzyme concentration, amount of water, molar ratio of substrates, types of organic solvents and reaction kinetics. In a second step, a complete factorial design was considered in order to optimize ester production in solvent-free systems using Rhizopus sp. lipase and Lipozyme TL IMR which were chosen because of their good performance. The reaction parameters chosen were molar ratio of substrates (acid:alcohol) and enzyme concentration. Lipase from Rhizopus sp. displayed the best catalytic activity at a molar ratio of 1:2 (acid:alcohol) and an enzyme concentration of 9.8%, whereas Lipozyme TL IMR had the best catalytic activity at a molar ratio of 1:3 and enzyme concentration of 7.0%. The rate of the sterification reaction yielded 93.1% using lipase from Rhizopus sp., very close to what it is obtained using commercial Lipozyme TL IMR that is 94.2%. This result shows a high potential for this non-commercial enzyme to be used as a catalyst for the oleyl oleate synthesis. The characterization of the ester included evaluation of emulsifier and antimicrobial activities. Oleyl oleate synthesized in this study proved to have lower emulsifier activity compared to commercial emulsifiers like sodium dodecyl sulfate (SDS) and Tween 80. Also, oleyl oleate did not inhibit the growth of Bacillus subtilis, thus no antimicrobial activity was observed for this substance / Mestrado / Mestre em Ciência de Alimentos

Two differently structured food webs can be distinguished in the pelagic habitat of aquatic systems; the classical one (autotrophic) with phytoplankton as a base and the microbial food web (heterotrophic) with bacteria as a base. Energy (produced at the basal trophic level) reaches higher trophic levels, i.e. zooplankton, directly in the classical food web in contrast to the microbial food web where it passes through additional trophic levels before reaching zooplankton. Energy is lost between each trophic level and therefore less energy should reach higher trophic levels in the microbial food web than in the classical food web. However, factors such as edibility of prey, temperature and properties of the predator, might also influence the food web structures and functions. In this thesis I studied which factors are important for an efficient carbon transfer and how a potential climate change might alter the food web efficiency in pelagic and pelagic-benthic food webs in the Baltic Sea. Furthermore, one of the most dominant zooplankton in the northern Baltic Sea, Limnocalanus macrurus, was studied in order to establish the seasonal pattern of lipid reserves in relation to food consumption. My studies showed that the carbon transfer efficiency during summer was not directly connected to the basal production, but factors such as the ratio between heterotrophs and autotrophs, the relationship between cladocerans and calanoid copepods and the size and community structure of both phytoplankton and zooplankton were important for the carbon transfer efficiency. In a climate change perspective, the temperature as well as the relative importance of the microbial food web is likely to increase. A temperature increase may have a positive effect on the pelagic food web efficiency, whereas increasing heterotrophy will have a negative effect on the pelagic and pelagic-benthic food web efficiency, reduce the fatty acid content of zooplankton and reduce the individual weight of both zooplankton and the benthic amphipod Monoporeia affinis. During the seasonal study on the calanoid copepod L. macrurus, I found that this species is mainly a carnivore, feeding on mesozooplankton during most of the year but switches to feeding on phytoplankton when these are abundant. Furthermore, when food is scarce, it utilizes lipids that are built up during the course of the year. From these studies I can draw some major conclusions; there are many factors that influence how efficient carbon is transferred in the food web and different factors are probably of various importance in different areas. In order to determine the carbon transfer efficiency, the various strategies exerted by different organism groups have to be considered, as for example that some zooplankton utilize lipid reserves instead of feeding all year around. Also, in a climate change perspective, the pelagic-benthic food web efficiency will decrease, as will the quality of zooplankton and M. affinis, possibly having implications for higher trophic levels such as fish.

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Wax esters are substantial constituents of natural waxes, which can be found in many living organisms. Properties of lipids, including wax esters, may be significantly influenced by the position of double bond. In this diploma thesis the location of double bonds was determined by dimethyl disulfide (DMDS) derivatization followed by detection using tandem mass spectrometry with atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). We managed to measure the APCI and ESI MS/MS spectra of 8 different wax esters with different position of double bond. Diagnostic ions determining double bond position were identified. This method could be used in HPLC/MS analysis of wax esters, which cannot be analyzed by GC/MS. It was shown that the DMDS derivatization reaction and mass detection with APCI ionization is also suitable for locating double bonds in alkenes.

Obesity is tightly connected with metabolic diseases including insulin resistance, type 2 diabetes or dyslipidemia. Peroxisome proliferator-activated receptor (PPAR)-α is a key transcription factor involved in the regulation of lipid metabolism, while its activity is stimulated by a variety of hypolipidemic drugs. n-3 polyunsaturated fatty acids (PUFA), including eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid, are endogenous ligands of PPARα, and they are used in the form of fish oil as dietary supplements in order to lower blood lipid levels and to prevent cardiovascular disease. Wax esters represent a novel lipid form of EPA and DHA, and according to recent studies they could exert more potent effects than the classical fish oil (i.e. triacylglycerols). Mice of the 129S1/SvImJ inbred strain were used in the present experiment, and included wild-type (WT) mice, as well as transgenic mice either with the exclusive expression of the human form of PPARα (hPPARα) or mice completely lacking PPARα (PPARα-KO). Mice were fed for 8 weeks the following diets: (i) a control low-fat diet, (ii) obesogenic high-fat diet (cHF), and (iii) the cHF diet supplemented with the n-3 PUFA concentrate in the form of wax esters isolated from marine zooplankton Calanus finmarchicus (ω3Cal). Mice were subjected to...