Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 141-163) Also available in print.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18343号 / 農博第2068号 / 新制||農||1024(附属図書館) / 学位論文||H26||N4850(農学部図書室) / 31201 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 植田 充美, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective of this work was the polyunsaturated fatty acids (PUFAs) production by submerged fermentation from the fungus Mortierella isabellina. A Plackett Burman experimental design was designed to evaluate the best conditions for PUFAs production in shake flasks. Three different components of the culture medium (yeast extract, peptone, and sucrose) and three fermentation parameters (agitation, temperature and pH) were evaluated. Analyzing the results of experimental design new assays were performed with increasing concentration of yeast extract. Fermentations were conducted in Erlenmeyer flasks with 10% (v/v) inoculums for 120h, micronutrients ((NH4)2SO4, FeSO4.7H2O, MnSO4.H2O, MgSO4.7H2O) were fixed. The scale up using STR bioreactor with 2.5 L of culture medium and 10% (v/v) inoculums for 120h was defined through better results of Plackett Burman.. A 2² central composite design was set to evaluate the effect of agitation and aeration in fungal biomass production, lipids content and fatty acids profile. The lipid was extracted from dried biomass and the fatty acids were analyzed by gas chromatography. The results of the fermentation in shake flasks showed that only yeast extract had a significant effect (p <0.1) in PUFAs production and this effect was positive. The highest percentage of PUFAs produced (28.17%) was obtained in higher yeast extract concentration (3.75 g / L). Agitation and aeration did not had a significance effect (p <0.1) in the evaluated range, in bioreactor on PUFAs and lipids production. However, agitation had a significant positive effect on biomass production.The presence of fatty acids of family n-3 (α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid) and n-6 families (linoleic acid and γ-linolenic acid) were observed. Nitrogen source is directly related to increased PUFAs production. The variables agitation and aeration, in the evaluated range, did not influence the PUFAs production. The Mortierella isabellina produced fatty acids n-3 and n-6 family, but additional studies are needed to optimize this production. / O objetivo deste trabalho foi produzir ácidos graxos poli-insaturados (PUFAs) por fermentação submersa a partir do fungo Mortierella isabellina. Um planejamento experimental Plackett Burman foi delineado para avaliar as melhores condições para produção de PUFAS em frascos agitados. Foram avaliados três diferentes componentes do meio de cultura (extrato de levedura, peptona e sacarose) e três parâmetros de fermentação (agitação, temperatura e pH). Através dos resultados obtidos no planejamento experimental, novos ensaios foram definidos com o aumento da concentração de extrato de levedura. As fermentações foram realizadas em frascos Erlenmeyers com 10% de inoculo (v/v) durante 120h e a composição de micronutrientes ((NH4)2SO4, FeSO4.7H2O, MnSO4.H2O, MgSO4. 7H2O) foi mantida fixa. Na melhor condição definida em frascos agitados foi realizada a ampliação da fermentação para biorreator STR, com 2,5 L de meio de cultura e 10 % de inoculo (v/v) durante 120h. Um delineamento composto central 2² foi definido para avaliar o efeito das variáveis agitação e aeração na produção de biomassa fúngica, lipídeos e no perfil dos ácidos graxos. A fração lipídica foi extraída a partir da biomassa seca da fermentação e os ácidos graxos foram analisados por cromatografia em fase gasosa. Os resultados da fermentação em frascos agitados demonstraram que somente a variável extrato de levedura teve efeito significativo (p<0,1) na produção de PUFAs sendo este efeito positivo. O maior percentual de PUFAs produzidos (28,17%) foi obtido na maior concentração de extrato de levedura (3,75 g/L). Os resultados obtidos na fermentação em biorreator demonstraram que as variáveis agitação e aeração não foram estatisticamente significativas (p<0,1) para o acúmulo de PUFAs e lipídeos dentro das faixas avaliadas. Entretanto a agitação teve efeito significativo positivo na produção de biomassa. Foi observada a presença dos ácidos graxos da família n-3 (ácido α-linolênico, ácido eicosapentaenóico e ácido docosaexaenóico) e da família n-6 (ácido linoléico e o ácido γ-linolênico). Em vista dos resultados obtidos, pode-se concluir que a fonte de nitrogênio esta diretamente relacionada com o aumento da produção de PUFAs. As variáveis agitação e aeração nas faixas avaliadas não influenciaram no acúmulo de PUFAs. A Mortierella isabellina produziu ácidos graxos da família n-3 e n-6, entretanto estudos adicionais devem ser realizados para otimizar esta produção.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-06-08T13:21:01Z No. of bitstreams: 2 Dissertação - Kelly Carolina Frauzino Araujo - 2014.pdf: 2783382 bytes, checksum: f8ddacd33ce41be9f223a9570381c87b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-06-08T13:21:15Z (GMT) No. of bitstreams: 2 Dissertação - Kelly Carolina Frauzino Araujo - 2014.pdf: 2783382 bytes, checksum: f8ddacd33ce41be9f223a9570381c87b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-06-08T13:21:15Z (GMT). No. of bitstreams: 2 Dissertação - Kelly Carolina Frauzino Araujo - 2014.pdf: 2783382 bytes, checksum: f8ddacd33ce41be9f223a9570381c87b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-02-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The "Microbial Models of Mammalian Metabolism" represents an alternative to use of animals on metabolism studies. Introduced in the 70s this model, also called biotranformation, has several advantages for their application as low cost, reduction of animals for experimentation and and higher number and range of metabolites produced. 1-(4-((1-(4-chlorophenyl)-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethanone (LQFM030) and 4-((1-(4-chlorophenyl)-1H-pyrazol-4-il)methyl)piperazin-1-ethyl carboxylate (LQFM018) compounds were synthesized by molecular simplification of a series of compounds with chronic myeloid leukemia antiproliferative activity already described, the Nutlins prototypes. This study aims to produce a probable human metabolites of LQFM 030 and LQFM 018 by microbial biotranformation with filamentous fungi. To do so, analytical methodologies were developed by thin layer chromatography and high performance liquid chromatography in order to monitor metabolites production. After perform a screening of tem microorganisms Mortierella isabellina NRRL 1757 strain was selected to obtain metabolites on a larger scale. Incubations were carried out with 100 mL of glucose culture medium in each flask . At the end of incubation (96 h) extraction and purification of possible metabolites was performed . In an independent assay with LQFM 030, ketoconazole (10, 20 and 30 mg) was added to inhibit P450 cytochrome . In another test 1 mL of ethanol was added every 24 hours to induce cytochrome totaling 96 hours of incubation. To evaluate the best time to finalize incubation , an experiment was conducted in 168 hours with different concentrations of the substrate, 0.25 mg / mL and 50 mg / mL. A possible role of CYP 3A was evidenced by the inhibition caused by ketoconazole addition, that can inhibit the formation of N-oxide metabolite in LQFM 030 biotransformation. Ethanol addition does not induce LQFM 030-N-oxide production, but was able to induce other metabolites formation. The best time to end LQFM 030 incubation was defined as 168 hours and 0.25 mg/mL concentration. / O “Modelo Microbiano do Metabolismo Animal”, pode representar uma alternativa ao uso de animais nos estudos de metabolismo de novos candidatos a fármacos. Introduzido nos anos 70, este modelo, também denominado de biotransformação, apresenta várias vantagens para a sua aplicação como o baixo custo, a redução da utilização de animais de experimentação e maior quantidade e variedade de metabólitos produzidos. Os compostos 1-(4-((1-(4-clorofenil)-1H-pirazol-4-il)metil)piperazin-1-il)etanona (LQFM 030) e 4-((1-(4-clorofenil)-1H-pirazol-4-il)metil)piperazin-1-carboxilato de etila (LQFM 018) foram sintetizados a partir da simplificação molecular de uma série de compostos com atividade antiproliferativa frente à leucemia mielóide crônica já descrita, os protótipos Nutlins. O presente estudo objetivou a produção de prováveis metabólitos humanos do LQFM 030 e LQFM 018 através de biotransformação microbiana com fungos filamentosos. Para isso, foram desenvolvidas metodologias analíticas por cromatografia em camada delgada e cromatografia líquida de alta eficiência, a fim de se monitorar a produção dos metabólitos. Após seleção dentre dez microrganismos a cepa Mortierella isabellina NRRL 1757 foi escolhida para obtenção de metabólitos em maior escala. A incubação foi realizada com 100 mL de meio de cultura de glicose, em cada Erlenmeyer. Ao término da incubação (96 horas) foi realizada a extração e purificação dos possíveis metabólitos. Em um ensaio independente foi adicionado cetoconazol (10, 20 e 30 mg) ao meio contendo LQFM 030 com o objetivo de inibir o citocromo P450. Em outra etapa foi adicionado 1 mL de etanol a cada 24 horas para induzir o citocromo, totalizando 96 horas de incubação. Para avaliar a melhor cinética reacional, um experimento foi conduzido em 168 horas, com diferentes concentrações do substrato, 0,25 mg/mL e 50 mg/mL. A biotransformação do LQFM 030 e do LQFM 018 produziu seus respectivos N-óxidos, caracterizados por RMN 1H e 13C e EM. Uma provável participação da CYP 3A foi evidenciada através da inibição causada pela adição de cetoconazol capaz de inibir a formação do metabólito N-óxido na biotransformação do LQFM 030. A adição de etanol não induziu a produção do LQFM 030-N-óxido, contudo foi capaz de induzir a formação de outros metabólitos. O melhor tempo para término da incubação do LQFM 030 foi definido como 168 hrs e 0,25 mg/mL de concentração.

Thesis (MSc (Microbiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Long chain omega-3 fatty acids such as eicosapentaenoic acid (EPA) are essential for the regulation of critical biological functions in humans and other mammals. Fish oil as the main dietary source of EPA holds several disadvantages and alternative sources and production processes such as microbial fermentation are increasingly being investigated. Therefore the aim of the first part of this study was to evaluate brewers’ spent grain (BSG) as substrate for the production of EPA by solid state fermentation with 29 fungal strains representing different Mortierella species. The effect of a 10% (w/w) linseed oil (LSO) supplement on EPA production was also studied. Consequently, fungal inoculated BSG was incubated at 22oC for three days to obtain optimal fungal growth, before the incubation temperature was lowered to 16oC for the following eight days. Cultures were then harvested and dried, followed by lipid extraction and analyses using gas chromatography. All the strains were found to produce EPA on BSG, while addition of the LSO improved the EPA yield of most strains. The strains which produced the highest levels of EPA on BSG supplemented with LSO were Mortierella antarctica Mo 67 and Mortierella epicladia Mo 101, which respectively produced 2.8 mg and 2.5 mg EPA per g of BSG. During the second part of the study eight Mortierella strains were used to study EPA production via solid state fermentation of sunflower press cake (SPC). Similar culture conditions and analytical methods were used as in the first part of the study. The effect of supplementing the SPC substrate with 10% (w/w) LSO was studied with regard to the supplement’s impact on EPA production and on the highly unsaturated fatty acid (HUFA) profile of the fermented substrate. Addition of LSO improved EPA yield of most strains on SPC, leading to a reduction in the average arachidonic acid (ARA):EPA ratio from 50.68 to 3.66. The ratio of HUFA to saturated and monoenoic fatty acids, was increased significantly (t=5.75, p=0.05) by the addition of LSO, with higher desaturation levels among the 20-carbon fatty acids. Addition of LSO also had a positive effect (r = 0.9291, p = 0.001) on the relative amount of long chain fatty acids (C≥20) produced. The strains which produced the highest levels of EPA on SPC supplemented with LSO were Mortierella alpina Mo 46 and Mortierella basiparvispora Mo 88, which produced 6.4 mg and 5.8 mg EPA per g of sunflower press cake, respectively. Fungi belonging to the genus Mortierella successfully converted LSO supplemented agro-processing wastes, such as BSG and SPC, to materials containing EPA, thereby adding value to these substrates. These EPA-enriched waste substrates could eventually find applications as animal or fish feed or as a source of EPA and other HUFA for the growing omega-3 market in the neutraceutical and therapeutics industry. / AFRIKAANSE OPSOMMING: Langketting omega-3 vetsure soos eikosapentaenoë suur (EPS) is noodsaaklik vir die regulasie van kritiese biologiese funksies in mense en ander soogdiere. Visolie, die mees belangrike EPS-bron in die dieet, hou verskeie nadele in en alternatiewe bronne sowel as produksie-prosesse, soos mikrobiologiese fermentasie, word dus toenemend ondersoek. Die doel van die eerste gedeelte van hierdie studie was dus om gebruikte brouersgraan (GBG) te ëvalueer as ‘n substraat vir die produksie van EPS deur soliede staat fermentasie met 29 fungus isolate wat verskillende Mortierella spesies verteenwoordig. Die uitwerking van byvoeging van 10% (m/m) lynsaadolie (LSO) op EPS-produksie is ook bepaal. Gevolglik is fungus-geïnokuleerde GBG vir drie dae by 22oC geïnkubeer om optimale fungusgroei te verkry, waarna die inkubasie temperatuur verlaag is na 16oC vir die volgende agt dae. Kulture is hierna ge-oes en gedroog, gevolg deur lipied ekstraksie en analise met behulp van gaschromatografie. Al die isolate het EPS geproduseer op die GBG substraat, terwyl byvoeging van LSO die EPS-opbrengs van die meeste isolate verbeter het. Die isolate wat die hoogste vlakke van EPS op GBG wat met LSO verryk is, geproduseer het, was Mortierella antarctica Mo 67 en Mortierella epicladia Mo 101, wat onderskeidelik 2.8 mg en 2.5 mg EPS per g GBG geproduseer het. Tydens die tweede gedeelte van die studie is agt Mortierella isolate gebruik om die produksie van EPS deur soliede staat fermentasie van sonneblom perskoek (SPK) te ondersoek. Kultuurtoestande en analitiese metodes soortgelyk aan die eerste gedeelte van die studie is gebruik. Die uitwerking van byvoeging van 10% LSO tot die SPK substraat is ondersoek met betrekking tot die impak van die byvoeging op EPS produksie asook op die profiel van hoogs onversadigde vetsure (HOVS) van die gefermenteerde substraat. Die byvoeging van LSO tot SPK het die EPS opbrengs van meeste isolate verbeter en het tot ‘n verlaging in die gemiddelde arachidoonsuur (ARS):EPS verhouding vanaf 50.69 tot 3.66 gelei. Die verhouding van HOVS tot versadigde en mono-onversadigde vetsure, is betekenisvol (t=5.75, p=0.05) verhoog deur die byvoeging van LSO, met hoër vlakke van onversadigheid onder die 20-koolstof vetsure. Byvoeging van LSO het ook ‘n positiewe uitwerking (r = 0.9291, p = 0.001) op die relatiewe aantal langketting vetsure (C≥20) gehad. Die isolate wat die hoogste vlakke van EPS geproduseer het op LSO-verrykte SPK, was Mortierella alpina Mo 46 en Mortierella basiparvispora Mo 88, wat onderskeidelik 6.4 mg en 5.8 mg EPS per g SPK geproduseer het. Fungi wat aan die genus Mortierella behoort, het LSO-verrykte agroprosesserings afvalprodukte, soos GBG en SPK, suksesvol omgeskakel na materiale wat EPS bevat, en sodoende waarde toegevoeg aan hierdie substrate. Die EPS-verrykte afvalsubstrate kan uiteindelik toepassings vind as diere- of visvoer of as bron van EPS of ander HOVS vir die groeiende omega-3 mark in die neutraseutiese en terapeutiese industrie.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23253号 / 農博第2460号 / 新制||農||1085(附属図書館) / 学位論文||R3||N5343(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 阪井 康能, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(農学) / 甲第19047号 / 農博第2125号 / 新制||農||1032 / 31998 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 喜多 恵子, 教授 栗原 達夫 / 学位規則第4条第1項該当

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19047号 / 農博第2125号 / 新制||農||1032(附属図書館) / 学位論文||H27||N4929(農学部図書室) / 31998 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 喜多 恵子, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM