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Konjac glucomannan (KGM), the main biologically active constituent of konjac flour extracted from corms of Amorphophallus konjac (konjac), can be used to prepare functional foods and may also have potential as a pharmaceutical product to combat obesity. The current study employed three experimental approaches to study the biology and chemistry of konjac, namely (1) glasshouse experiments to study the morphogenesis, growth and productivity of konjac plants, (2) a histological and immunocytochemical investigation of the localisation and developmental regulation of the deposition and metabolism of KGM in developing corm tissues, and (3) a comparative study of methodologies for the extraction and analysis of KGM. The current data demonstrated a morphological and functional separation between the ventral and dorsal regions of corms. The ventral region appeared to function as a source during the initial period of shoot development, while the dorsal region appeared to operate as a sink after the development of mature canopy. Once the corm reached maturity, both an inflorescence and a leaf were produced within a single season. It has also been demonstrated that the age of the ‘mother’ corm is an important factor affecting the quality of offsets produced. An anti-mannan antiserum detected a temporally regulated pattern of mannan epitope production within glucomannan idioblasts in developing corm tissues, with increased expression as the corm approached maturity/dormancy. The current observations also suggest that the mobilization of KGM initiates at the periphery of the corm and proceeds inwards towards the centre of the corm. Compositional analysis showed that the purified konjac flour (PKF) produced using a modified extraction procedure contained 92% glucomannan, with a weight average molecular weight (Mw), polydispersity index (PDI) and degree of acetylation (DA) of 9.5 ± 0.6 x 105 gmol-1, 1.2 and 2.8 wt. %. These data, plus Fourier-transform infrared spectral (FTIR) and zero shear viscosity analyses of the extract (PKF) were all consistent with the literature. Comparison of three existing methodologies for the quantitative analysis of the KGM content, namely 3,5-dinitrosalicylic acid (3,5-DNS), phenol-sulphuric acid and enzymatic colorimetric assays; indicated that the 3,5-DNS colorimetric assay was the most reproducible and accurate method, with a linear correlation coefficient of 0.997 and recoveries between 97% and 103% across three spiking levels of starch. In summary, this study has provided a better understanding of aspects of the biology and cultivation of A. konjac and has also produced methodologies which can be used as the basis for an improved good laboratory practice (GLP) for the commercial extraction and analysis of this multifunctional natural polymer.

Soybean oil is commercially obtained through an extraction process using hot commercial hexane. This toxic solvent, derived from petroleum, must be subsequently evaporated before the oil can be submitted to refining steps prior to storage and transportation or directly moving to biodiesel synthesis. Extraction of soybean oil with ethanol, a renewable, low-toxicity solvent, results in the production of an oil-rich miscella that can be directly esterified to produce ethyl esters (biodiesel) without the need for oil-refining steps and evaporation of the solvent. This extraction also generates an oil-poor miscella that has been successfully reinserted in further extractions as the solvent. The theoretical bases of the ethanolic extraction of soybean oil have been established by the works of many authors, focusing on the insurance of high oil yields. However, the effects of the ethanolic extraction of vegetable oils on the antioxidant profile and oxidative stability of the extraction products have not yet been addressed. This study aimed to increase the knowledge on the antioxidant composition of the rich and poor miscella derived from the extraction of soybean oil using ethanol as solvent. Results showed that although the rich miscella presents lower amounts of tocopherols in comparison to oils extracted with hexane, it contains antioxidant compounds of polar character with high affinity to ethanol, which would not be found in soybean oils extracted with hexane. Among these compounds isoflavones have been identified and quantified. The reuse of the poor miscella in subsequent extractions promotes an increase in the oxidative stability of the rich miscella, possibly, by adding antioxidant compounds with high affinity to ethanol. This effect was also observed in the use of the oil-poor miscella in liquid-liquid washings with waste cooking oil, resulting increase of the induction period. However, consecutive extractions of soybean oil have shown that the increase in the induction period of the oil-rich miscella reaches a limit. This phenomenon may be an indication of pro-oxidant effects, resulting from the increase in the content of the compounds present in the rich-miscella. For this reason, the study of the interactions between different classes of antioxidant compounds present in the soybean ethanolic extraction products may be an excellent way to understand the mechanisms involved in the oxidative stability of complex lipid systems. / O óleo de soja é obtido comercialmente através de um processo de extração a quente utilizando a hexana, também conhecida como hexano comercial. Esse solvente tóxico, derivado de petróleo, deve ser posteriormente evaporado para que o óleo passe por etapas de refino antes de seguir para o armazenamento e transporte ou diretamente para a síntese de biodiesel. A extração de óleo de soja com o etanol, um solvente renovável e de baixa toxicidade, resulta na produção de uma miscela rica em óleo que pode ser diretamente esterificada para produzir ésteres etílicos (biodiesel), sem a necessidade de etapas de refino de óleo e evaporação do solvente. Essa extração gera também uma miscela pobre em óleo que pode ser reutilizada em extrações posteriores como solvente. As bases teóricas da extração etanólica de óleo de soja foram estabelecidas pelos trabalhos de muitos autores, visando maiores rendimentos de extração. No entanto, o perfil antioxidante e a estabilidade oxidativa dos produtos da extração etanólica de óleos vegetais não foram abordados até o momento. Este estudo visou ampliar os conhecimentos sobre a composição antioxidante das miscelas ricas e pobres, oriundas da extração de óleo de soja, utilizando o etanol como solvente. Os resultados evidenciaram que embora a miscela rica apresente quantidades inferiores de tocoferóis em relação a óleos extraídos com hexano, a mesma é acrescida de compostos antioxidantes de natureza polar, devido a afinidade com etanol. Entre esses compostos foram identificadas isoflavonas, que normalmente não são encontradas nos óleos de soja obtidos pela extração com hexano. A reutilização da miscela pobre promove o aumento da estabilidade oxidativa das miscelas ricas, através da adição de compostos antioxidantes de maior afinidade pelo etanol. Esse efeito foi também observado no uso da miscela pobre em lavagens do óleo de fritura, resultando em aumento no período de indução. No entanto, a reutilização de miscelas pobres em extrações consecutivas demonstrou atingir um limite no aumento do período de indução das miscelas ricas. Esse fenômeno pode ser um indício de efeito pró-oxidante decorrente do aumento das concentrações de compostos presentes na miscela rica. Por essa razão, o estudo das interações entre diferentes classes de compostos antioxidantes nas miscelas oriundas da extração de óleo de soja pode ser uma excelente via para a compreensão dos mecanismos envolvidos na estabilidade oxidativa de sistemas lipídicos complexos.

The following contains two projects focused on the fungal pathogen, Fusarium oxysporum. The first project was purely computational in the examination of transposable elements (TEs), which are mobile sequences with the ability to multiply and move in their host genome. In F. oxysporum, TEs such as miniature impala elements are associated with the secreted in xylem gene that are related to its virulence over its host. The F. oxysporum species complex can be utilized as a model system for the examination of TE content and TE expression during the infection cycle. To find whether TEs play a role in the infection process and if their expression changes when fungi are in planta, a comparison was made using RNA-seq data from a pathogenic (Fo5176) and a non-pathogenic strain (Fo47) of F. oxysporum interacting with the model plant Arabidopsis thaliana. Complementary to this, the copy numbers of the same TEs were calculated in the two aforementioned strains and in F. oxysporum f.sp. lycopersici 4287 (Fo4287) to find if there was a correlation between expression and copy number. Using these two different datasets together showed that TE expression and copy number are lower in the non-pathogenic strain and unlinked in the infection course. The second project examined the growth inhibition of Fusarium oxysporum isolates Fo32931 (the isolate pathogenic to immunocompromised humans) and Fo4287 with the use of extracts from chilies of Capsicum chinense. Pepper plants were grown from seed and the peppers were harvested for an ethanol (100%) extraction. After preparation, the optical density of growth of the F. oxysporum isolates was measured for a 48-hour period with 96-well plate containing varying concentrations of the extracts and controls. Growth curves were analyzed and normalized to a growth control. After doing High Performance Liquid Chromatography, an estimated concentration of capsaicin (the causal agent of the burning sensation from hot chilis) was established. A correlation between the amount of growth inhibition and the concentration of capsaicin was made. Taken together, the data suggests that an increase of capsaicin concentration in extracts is correlated with reduced growth for the two tested isolates of F. oxysporum.

O araçá é um fruto, pertencente à espécie Psidium grandifolium Mart. ex DC, nativo da mata atlântica brasileira pouco caracterizado pela literatura ciêntífica. Esta pesquisa visou estudar o rendimento de extração e avaliar as atividades antioxidante e antimicrobiana do óleo essencial de araçá obtido por extração com dióxido de carbono supercrítico (SFE) e dos diferentes extratos obtidos por extração com etanol pressurizado (PLE) seguindo um delineamento composto central rotacional com temperatura e tempo estático como variáveis independentes estudadas. Avaliou-se também o perfil de voláteis do óleo essencial, o teor de compostos fenólicos totais dos extratos etanólicos e as alterações causadas na matriz vegetal por diferentes processos de extração. Com a SFE conseguiu-se extrair toda a fração lipídica do fruto (2,33% ± 0,04.10-2%), a análise dos compostos voláteis do óleo essencial obtido permitiu inferir que este possui diversos compostos com potencial bioativo tais como E-cariofileno (17.56%), α-humuleno (16,26%) e β-sesquifelandreno (14.27%); porém este óleo essencial não apresentou atividade antioxidante relevante. O extrato etanólico com maior rendimento (17, 49%) foi obtido a 74 °C por 6 min, mesma condição em que obteve-se a maior atividade antioxidante (EC50 = 6,37 mg/mL), porém o maior teor de compostos fenólicos totais (136,95 mg EGA/100 g de massa seca) foi obtido quando aplicou-se 60°C por 9 min. Em relação à atividade antimicrobiana, tanto os extratos obtidos por PLE quanto por SFE apresentaram atividade contra cepas de P. aeruginosa, S. aureus e B. cereus, somente extratos etanólicos apresentaram atividade contra cepas de L. monocytogenes e nenhum dos extratos apresentaram atividade contra cepas de B. subtilis, E. coli e Salmonella. Em ambos os extratos foi possível identificar compostos com potencial aplicação farmacológica. O estudo de processos de extração que envolvam inovação tecnológica na obtenção extratos com atividade biológica visa contribuir com o extrativismo consciente ou estimular o plantio em maior escala de mais um fruto tropical empregado na medicina popular brasileira. / The araçá is a fruit, belonging to the species Psidium grandifolium Mart. ex DC, native of the Brazilian Atlantic Forest, is not largely characterized by the scientific literature. This research work aimed to study the extraction yield and to evaluate the antioxidant and antimicrobial activities of the araçá essential oil obtained by supercritical carbon dioxide extraction (SFE) and the different extracts obtained by pressurized ethanol extraction (PLE) following a central composite design with temperature and static time as independent variables studied. It was also evaluated the volatile profile of the essential oil, the content of total phenolic compounds of the ethanolic extracts and the changes caused in the vegetal matrix by different extraction processes. SFE was able to extract the whole lipid fraction of the fruit (2.33% ± 0.04.10-2%), the analysis of the volatile compounds of the essential oil obtained allowed to infer that it has several compounds with bioactive potential such as E-caryophyllene (17.56%), α-humulene (16.26%) and β-sesquipelandene (14.27%); but this essential oil had no relevant antioxidant activity. The highest ethanolic extract (17, 49%) was obtained at 74 °C for 6 min, the same condition in which the highest antioxidant activity was obtained (EC50 = 6.37 mg/mL), but the higher content of total phenolics contente (136.95 mg EGA / 100 g dry mass) was obtained when 60 °C was applied for 9 min. In relation to the antimicrobial activity, both extracts obtained by PLE and SFE showed activity against P. aeruginosa, S. aureus and B. cereus strains, only ethanolic extracts showed activity against L. monocytogenes strains and none of the extracts presented activity against B. subtilis, E. coli and Salmonella strains. In both extracts it was possible to identify compounds with potential pharmacological application. The study of extraction processes that involve technological innovation in obtaining extracts with biological activity aims to contribute to the conscious extraction or stimulate the larger scale cultivation of another tropical fruit used in Brazilian popular medicine.

O araçá é um fruto, pertencente à espécie Psidium grandifolium Mart. ex DC, nativo da mata atlântica brasileira pouco caracterizado pela literatura ciêntífica. Esta pesquisa visou estudar o rendimento de extração e avaliar as atividades antioxidante e antimicrobiana do óleo essencial de araçá obtido por extração com dióxido de carbono supercrítico (SFE) e dos diferentes extratos obtidos por extração com etanol pressurizado (PLE) seguindo um delineamento composto central rotacional com temperatura e tempo estático como variáveis independentes estudadas. Avaliou-se também o perfil de voláteis do óleo essencial, o teor de compostos fenólicos totais dos extratos etanólicos e as alterações causadas na matriz vegetal por diferentes processos de extração. Com a SFE conseguiu-se extrair toda a fração lipídica do fruto (2,33% ± 0,04.10-2%), a análise dos compostos voláteis do óleo essencial obtido permitiu inferir que este possui diversos compostos com potencial bioativo tais como E-cariofileno (17.56%), α-humuleno (16,26%) e β-sesquifelandreno (14.27%); porém este óleo essencial não apresentou atividade antioxidante relevante. O extrato etanólico com maior rendimento (17, 49%) foi obtido a 74 °C por 6 min, mesma condição em que obteve-se a maior atividade antioxidante (EC50 = 6,37 mg/mL), porém o maior teor de compostos fenólicos totais (136,95 mg EGA/100 g de massa seca) foi obtido quando aplicou-se 60°C por 9 min. Em relação à atividade antimicrobiana, tanto os extratos obtidos por PLE quanto por SFE apresentaram atividade contra cepas de P. aeruginosa, S. aureus e B. cereus, somente extratos etanólicos apresentaram atividade contra cepas de L. monocytogenes e nenhum dos extratos apresentaram atividade contra cepas de B. subtilis, E. coli e Salmonella. Em ambos os extratos foi possível identificar compostos com potencial aplicação farmacológica. O estudo de processos de extração que envolvam inovação tecnológica na obtenção extratos com atividade biológica visa contribuir com o extrativismo consciente ou estimular o plantio em maior escala de mais um fruto tropical empregado na medicina popular brasileira. / The araçá is a fruit, belonging to the species Psidium grandifolium Mart. ex DC, native of the Brazilian Atlantic Forest, is not largely characterized by the scientific literature. This research work aimed to study the extraction yield and to evaluate the antioxidant and antimicrobial activities of the araçá essential oil obtained by supercritical carbon dioxide extraction (SFE) and the different extracts obtained by pressurized ethanol extraction (PLE) following a central composite design with temperature and static time as independent variables studied. It was also evaluated the volatile profile of the essential oil, the content of total phenolic compounds of the ethanolic extracts and the changes caused in the vegetal matrix by different extraction processes. SFE was able to extract the whole lipid fraction of the fruit (2.33% ± 0.04.10-2%), the analysis of the volatile compounds of the essential oil obtained allowed to infer that it has several compounds with bioactive potential such as E-caryophyllene (17.56%), α-humulene (16.26%) and β-sesquipelandene (14.27%); but this essential oil had no relevant antioxidant activity. The highest ethanolic extract (17, 49%) was obtained at 74 °C for 6 min, the same condition in which the highest antioxidant activity was obtained (EC50 = 6.37 mg/mL), but the higher content of total phenolics contente (136.95 mg EGA / 100 g dry mass) was obtained when 60 °C was applied for 9 min. In relation to the antimicrobial activity, both extracts obtained by PLE and SFE showed activity against P. aeruginosa, S. aureus and B. cereus strains, only ethanolic extracts showed activity against L. monocytogenes strains and none of the extracts presented activity against B. subtilis, E. coli and Salmonella strains. In both extracts it was possible to identify compounds with potential pharmacological application. The study of extraction processes that involve technological innovation in obtaining extracts with biological activity aims to contribute to the conscious extraction or stimulate the larger scale cultivation of another tropical fruit used in Brazilian popular medicine.