O objetivo do presente estudo foi avaliar o efeito da Terapia Fotodinâmica Antimicrobiana (aPDT) com a utilização de azul de metileno a 0,01%, combinado ao laser de diodo, na viabilidade e na produção de polissacarídeos, de biofilmes de S. mutans. Biofilmes de cepas de S. mutans UA159 foram cultivados em discos de resina acrílica e expostos oito vezes por dia, durante 1 min, à solução de sacarose a 10%. Após 48 horas da formação do biofilme, as amostras foram distribuídas aleatoriamente em relação aos tratamentos (n=4): aplicação de solução salina a 0,9% (controle negativo), aplicação de 0,12% de digluconato de clorexidina (controle positivo), ou aplicação da terapia fotodinâmica antimicrobiana. Para aplicação da terapia fotodinâmica antimicrobiana, foi utilizado azul de metileno a 0,01%, combinado ao laser de diodo com comprimento de onda de 660 nm. Foram usados os seguintes parâmetros: área da secção transversal do feixe de laser 0,028 cm&sup2;, potência de 100 mW, energia de 9J, densidade de energia de 320J/cm&sup2;, durante 90s. Os tratamentos foram realizados duas vezes ao dia. Após 120 h, os biofilmes de S. mutans formados sobre cada disco de resina acrílica foram coletados, de modo a determinar o número de bactérias viáveis e a concentração do polissacarídeo extracelular insolúvel (PECI) e do polissacarídeo intracelular (PIC). A análise variância um critério (ANOVA) e o teste de Tukey revelaram que as contagens das bactérias nos biofilmes formados foram significativamente diferentes entre os tratamentos. A aPDT mostrou inibição do crescimento do biofilme, quando comparado ao grupo do NaCl (p<0,05). A concentração de PECI e PIC foi maior no biofilme exposto ao NaCl do que a encontrada nos outros grupos (p<0,05) e não foi observada diferença significativa entre os grupos digluconato de clorexidina e aPDT (p>0,05). De acordo com as condições experimentais do presente estudo, sugere-se que o tratamento realizado duas vezes ao dia com aPDT utilizando-se azul de metileno a 0,01%, combinado ao laser de diodo (&lambda; = 660 nm; 320 J/cm&sup2;; 100 mW; 90s; 9J), diminui a viabilidade do biofilme e afeta sua organização estrutural. / The aim of this study was to evaluate the effect of Antimicrobial Photodynamic Therapy (aPDT) using methylene blue 0.01% combined with diode laser on biofilm viability and polysaccharides produced by Streptococcus mutans. S. mutans UA 159 biofilms were grown on acrylic resin discs and exposed eight times/day for 1 min in a 10% sucrose solution. After the biofilms were allowed to grow for 48 h, they were treated two times/day according to the treatments (n=4): 0.9% saline solution (negative control), 0.12% chlorhexidine digluconato (positive control) or Antimicrobial Photodynamic Therapy (aPDT). For the application of Antimicrobial Photodynamic Therapy has been used methylene blue 0.01% in combination with the diode laser with 660 nm wavelength. The parameters adopted were: spot size of 0.028 cm&sup2;, fixed output power of 100 mW, energy density of 320 J/cm&sup2;, time exposure of 90 s and energy of 9 J. Treatments were performed twice daily. After 120 h of growth, the biofilm formed on each disc was collected to determine the number of viable bacteria, and concentration of the insoluble exopolysaccharide (IEPS) and intracellular polysaccharide (IPS). The analysis of variance one-way (ANOVA) and Tukey test revealed that the counts of bacteria in the biofilms formed differ significantly among the treatments and aPDT showed biofilm inhibition when compared to NaCl group (p<0.05). The concentration of IEPS and IPS was higher in biofilm exposed to NaCl than that found in the other groups (p<0.05) and no significant difference was observed among CHX and aPDT groups (p>0.05). According to experimental conditions of the present work, the results suggest that a twice-daily treatment with aPDT using methylene blue 0.01% in combination with the diode laser (&lambda;=660 nm, 320 J/cm&sup2;, 100 mW, 90 s; 9J) decreases biofilm viability and affects its structural organization.

S. pneumoniae é um importante patógeno humano que afeta sobretudo crianças. Vacinas são a principal estratégia de combate e o polissacarídeo (PS) da cápsula é o antígeno. Dentre os sorotipos do patógeno, o tipo 1 é prevalente em crianças e o PS1 componente obrigatório das vacinas. Neste trabalho objetivou-se: estabelecer um método de dosagem do PS1, selecionar a melhor cepa produtora de PS1, avaliar 3 peptonas, investigar 4 componentes do meio de cultura em planejamento experimental (PE) em reator e propor um novo meio e estratégia de cultivo. A cepa ST595/01 foi selecionada e o Phytone (Phy) foi a peptona com maior produção de PS1, 298mg/L, medido por m-hidroxidifenil com sulfamato. O PE 24-1 com extrato de levedura (EL), Phy, Asn e Gln mostrou os maiores efeitos positivos do Phy para produção de biomassa (Cx) e PS1. EL foi positivo para Cx, Asn não apresentou efeitos e Gln menor efeito positivo para PS1. Assim, testou-se o novo meio com Phy 15g/L e EL 2g/L sob duas estratégias: a batelada alimentada superou a batelada simples em 2x para Cx e 2,5x para PS1. / S. pneumoniae is an important human pathogen that affects mainly children. Vaccines are the main strategy to fight the disease and capsular polysaccharide (PS) is the antigen. Among pneumococcal serotypes, type 1 is prevalent in children. The aims of this work were: establish a method to measure PS1, screen strains for the best PS1 producer, evaluate 3 peptones, investigate the effects of 4 medium components by a design of experiment (DoE) and propose a new medium/strategy for cultivation. The strain ST595/01 was selected. Phytone (Phy) was the peptone with the highest PS1 production (298mg/L, measured by m-hydroxydiphenyl + sulfamate method). DoE 24-1 was carried out to assess the effects of yeast extract (YE), Phy, Asn and Gln. Phy showed the major positive effects for biomass (Cx) and PS1. YE demonstrated effects for Cx. Gln showed minor effect on PS1 production and Asn did not present effects. A new medium based on 15g/L Phy and 2g/L YE was evaluated by 2 strategies: fed-batch showed Cx production 2-fold higher and PS1 2.5-fold higher than simple batch.

A ingestão regular de fibras alimentares pode auxiliar na redução do risco de certas doenças crônicas, como doenças cardiovasculares, síndromes metabólicas, diabetes tipo 2 e alguns tipos de câncer. Dentre as principais fontes de fibras alimentares, destaca-se o grão de trigo, que é rico em fibras solúveis e insolúveis. O grão de trigo pode ser identificado por três macrorregiões distintas, sendo elas o pericarpo, o gérmen e o endosperma. O principal alimento oriundo do trigo é a farinha produzida através da moagem seriada do endosperma dos grãos, sendo o principal subproduto a casca do grão conhecido como farelo de trigo, cujo pericarpo é o principal constituinte. As fibras alimentares estão concentradas no pericarpo, ou seja, no farelo do trigo, e estão associadas através de uma estrutura tridimensional não uniforme e compactada, o que prejudica a solubilidade de polissacarídeos complexos diminuindo suas biodisponibilidades caso ingeridos na alimentação humana. Apesar do farelo de trigo ser um resíduo com razoável valor biológico e com alto teor de fibras, o principal destino do farelo é a alimentação animal. Devido ao fato do farelo de trigo possuir baixo custo, elevado teor de fibras e grande volume disponível no mercado, o objetivo desse trabalho é estudar três métodos físico-químicos que modifiquem a estrutura do farelo de trigo e permitam que uma maior quantidade de polissacarídeos se tornem mais solúveis em água. Uma vez diluídas, essas frações de polissacarídeos consideradas como fibras alimentares solúveis (FAS) poderão ser extraídas, isoladas e aplicadas como ingredientes na produção de alimentos funcionais, como margarinas e sucos. Assim, o projeto permitirá que a indústria de alimentos desenvolva uma fonte de fibras solúveis a partir de uma matéria prima de baixo custo e alta produção. As modificações químicas foram feitas em meio alcalino (solução de NaOH nas concentrações 0,025, 0,05 e 0,1M), meio ácido (solução de H2SO4 nas concentrações 0,25, 0,5e 1,0M) e com agentes oxidantes (solução de hipoclorito de sódio a 10%).Com relação ao rendimento da extração das frações solúveis, as extrações em meio ácido foram as melhores. Todos os polissacarídeos não amido extraídos do farelo de trigo apresentaram baixo peso molecular e composição de açúcares diferentes. Os polissacarídeos oriundos das extrações em meio ácido demonstraram mais glicose e um menor percentual de arabinose e xilose ao serem comparadas com a água, enquanto os oriundos das extrações alcalinas apresentaram um teor menor de glicose e maior de arabinose e xilose (em comparação com as frações da água). A partir da análise de açúcares (foi avaliado maiores quantidades dos monossacarídeos arabinose, xilose e glicose) e do rendimento das extrações, foram escolhidas 5 amostras, sendo elas a fração solúvel extraída em água, H2SO4 0,25 e 0,5M e NaOH0,25 e 0,1M para as análises do teor de &#946;-glicanos, compostos fenólicos, propriedades funcionais (propriedades espumantes, absorção de água, solubilidade e umidade) e efeito prebiótico. Como resultado, todas os polissacarídeos não amido apresentaram elevada solubilidade e não possuíram propriedades espumantes e de absorção de água (WAC) relevantes. O teor de compostos fenólicos foi reduzido em todas as frações solúveis, quando comparado com o teor de compostos fenólicos do farelo de trigo e o pequeno percentual remanescente de compostos fenólicos possui baixo poder redutor. Foi encontrado &#946;-glicano em todas as frações dos polissacarídeos não amido extraídas, sendo 0,82% nas frações extraídas em água, 0,97 e 2,79% nas frações extraídas com H2SO4 e 5,42 e 4,62% nas frações extraídas com NaOH 0,025 e 0,1M. Embora tenha sido encontrado &#946;-glicano em todas as amostras extraídas, somente as extrações em água e com NaOH 0,1 M apresentaram possível efeito prebiótico in vitro. / Regular intake of dietary fiber can help to reduce the risk of certain chronic diseases, such as cardiovascular disease, metabolic syndromes, type 2 diabetes and some types of cancer. Among the main sources of dietary fiber is the wheat grain, which is rich in soluble and insoluble fibers. The wheat grain can be identified by three distinct macroregions, they are the pericarp, the germ and the endosperm. The main food made by wheat is its flour produced by serial grinding of the grain endosperm, and the main by-product is the husk of the grain known as wheat bran, whose pericarp is the main constituent. Food fibers are concentrated in the pericarp and are associated through a non-uniform and compacted three-dimensional structure, which impairs the solubility of complex polysaccharides, decreasing their bioavailability when ingested in human food. Although wheat bran is a residue with reasonable biological value and high fiber content, the main destination of the bran is animal feed. Due to the fact that wheat bran has a low cost, high fiber content and a great volume available in the market, the objective of this work was to study three physic-chemical methods that modify the structure of wheat bran and allow a greater amount of polysaccharides to become more soluble in water. Once diluted, these fractions of polysaccharides considered as soluble dietary fiber (FAS) can be extracted, isolated and applied as ingredients in the production of functional foods such as margarines and juices. Thus, the project will enable the food industry to develop a source of soluble fiber from a low-cost, high-yield raw material. The chemical modifications were made in alkaline medium (NaOH solution at concentrations of 0.025, 0.05 and 0.1M), acid medium (H2SO4 solution at concentrations of 0.25, 0.5 and 1.0M) and with oxidizing agents (10% sodium hypochlorite solution). In relation to the extraction yield of the soluble fractions, acid extraction was the best. All non-starch polysaccharides extracted from wheat bran had low molecular weight and different sugar composition. The polysaccharides from acid extraction showed more glucose and a lower percentage of arabinose and xylose when compared to water, while those from the alkaline extractions had a lower glucose content and higher arabinose and xylose (compared to the fractions from water). From the analysis of sugars (higher amounts of monosaccharides arabinose, xylose and glucose) and extraction yields, 5 samples were chosen, being the soluble fraction extracted in water, 0.25 and 0.5M H2SO4 and NaOH 0.25 and 0.1M for analyzes of &#946;-glycan content, phenolic compounds, functional properties (foaming properties, water absorption, solubility and moisture) and prebiotic effect. As a result, all non-starch polysaccharides exhibited high solubility and did not possess relevant foaming and water absorption properties (WAC). The content of phenolic compounds was reduced in all soluble fractions when compared to the phenolic compounds content of wheat bran and the small remaining percentage of phenolic compounds had low reducing power. &#946;-glucan was found in all fractions of the extracted non-starch polysaccharides, 0.82% in fractions extracted in water, 0.97 and 2.79% in fractions extracted with H2SO4 and 5.42 and 4.62% in fractions extracted with 0.025 and 0.1M NaOH. Although &#946;-glycan was found in all the extracted samples, only extractions in water and with 0.1M NaOH showed possible prebiotic effect in vitro.

Materiais bioativos têm a capacidade de interagir com tecidos naturais, provocando reações que favorecem o desenvolvimento de processos como fixação e biodegradação do material implantado, além de regeneração de tecidos. Dessa forma, o objetivo deste trabalho está relacionado ao desenvolvimento de novos biomateriais, os quais foram obtidos a partir de combinações de fases orgânicas e inorgânicas em escala nanométrica, resultando em propriedades aprimoradas, como bioatividade, hidrofilicidade, resistência mecânica e biodegradabilidade. Foram sintetizadas e caracterizadas membranas autossuportadas constituídas por blendas biopoliméricas reforçadas com fosfato de cálcio. Essas blendas foram preparadas a partir da mistura, nas proporções 2,5:2,5 e 3,5:1,5 % (p/v), entre colágeno hidrolisado e polissacarídeos, como quitosana e -carragenana devido as suas notáveis propriedades e a sua semelhança com constituintes da matriz extracelular óssea nativa. A incorporação da fase inorgânica composta por fosfato de cálcio na matriz orgânica se deu por meio de três metodologias diferentes: (1) Precipitação in locu nos interstícios da matriz biopolimérica; (2) Adição das nanopartículas previamente sintetizadas; (3) Adição de hidroxiapatita bovina na matriz biopolimérica. A partir dos resultados obtidos por meio da microscopia eletrônica de varredura (MEV), espectroscopia vibracional na região do infravermelho (FTIR) e difração de raios X (DRX), observou-se que as membranas híbridas formados a partir da metodologia (1), resultaram em deposição homogênea e continua de HAp por toda a matriz biopolimérica. Como consequência, o módulo de Young desses materiais híbridos foi o maior em relação aos valores obtidos para as suas respectivas matrizes biopoliméricas na ausência do mineral, como indicado pelo aumento do módulo de Young até 130% para membranas compostas por quitosana e até 115% para as membranas contendo -carragenana. Verificou-se que a redução da concentração de polissacarídeo na composição das blendas, afeta significativamente o módulo de Young e a taxa de degradação das membranas híbridas em solução salina tamponada de fosfato (PBS, do inglês phosphate buffered saline) a 37°C. Além disso, foi observado que as membranas compostas por quitosana apresentam menor taxa de degradação quando comparadas as membranas híbridas compostos por -carragenana. A incorporação da fase mineral resultou em aumento da hidrofilicidade e energia livre de superfície. A exposição das membranas híbridas ao fluido corporal simulado (SBF, do inglês Simulated body fluid) resultou na deposição de uma camada de fosfato de cálcio sobre a superfície das amostras. A resposta biológica dessas membranas foi avaliada por cultura de osteoblastos, indicando que os materiais contendo quitosana não são tóxicos. No entanto, o mesmo não foi observado para amostras contendo -carragenana, pois essas membranas, devido a elevada taxa de degradação, apresentam baixa estabilidade e integridade no meio de cultura celular. Nanopartículas de prata (NpAg), com tamanho variando de 3-9 nm, foram sintetizadas e incorporadas nas membranas híbridas obtidas pela metodologia 1. Após essa adição, observou-se uma ação antibacteriana contra as bactérias Escherichia coli, Staphylococcus aureus e Pseudomonas aeruginosa. Esses resultados indicam que as membranas híbridas obtidas neste trabalho podem, potencialmente, ser usadas como membranas de regeneração tecidual guiada temporária em defeitos ósseos / Bioactive materials present the ability to interact with natural tissues, causing reactions that favor the development of processes such as implant and biodegradation of implanted material, as well as tissue regeneration Thus, the objective of this study is to the develop new biomaterials combining organic and inorganic phases at the nanoscale resulting in improved properties such as bioactivity, hydrophilicity, mechanical strength and biodegradation. Herein, hybrid self-supported membranes formed by polymer blends and reinforced with calcium phosphate were synthesized and characterized. These blends were prepared from the combination, in the proportions 2,5 : 2.5 and 3,5 : 1,5 wt%, between hydrolyzed collagen and polysaccharides, such as chitosan and -carrageenan due to its remarkable properties and its similarity to the constituents of the native extracellular matrix. The incorporation of the inorganic phase consisting of calcium phosphate in the organic matrix was performed using three different methodologies: (1) Precipitation in locus in the interstices of the polymer matrix; (2) Addition of previously synthesized nanoparticles; (3) Addition of bovine hydroxyapatite in polymeric matrix. From the results obtained by scanning electron microscopy (SEM), infrared vibration spectroscopy (FTIR) and X-ray diffraction (XRD), it was observed that the hybrid systems formed from the methodology (1) resulted in a homogeneous and continuous deposition of HAp throughout the biopolymer matrix. As a consequence, the Young\'s modulus of these hybrid materials was the higher in relation to the values obtained for the biopolymer matrices in the absence of the mineral, as indicated by the increase of the Young\'s modulus up to 130% for membranes composed by chitosan and up to 115% for the membranes containing -carrageenan. It was noted that the reduction of the polysaccharide concentration in the blends composition significantly affects the Young\'s modulus and the rate of degradation of the hybrid systems in phosphate buffered saline (PBS) at 37 °C. In addition, it was observed that membranes composed of chitosan have a lower degradation rate compared to hybrid membranes composed of -carrageenan. The incorporation of the mineral phase resulted in increased hydrophilicity and surface free energy. Exposure of hybrid membranes to simulated body fluid (SBF) resulted in the deposition of a layer of calcium phosphate under the surface of the samples. The biological response of these membranes was assessed by culturing osteoblasts, indicating that chitosan-containing systems are non-toxic. However, the same was not observed for samples containing carrageenan, because these membranes, due to the high degradation rate, present low stability and integrity in the cell culture medium. Silver nanoparticles (NpAg), ranging in size from 3-9 nm, were synthesized and incorporated into the hybrid membranes obtained by methodology 1. After this addition, an antibacterial action against the bacteria Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa was observed. These results indicate that the hybrid membranes obtained in this study can potentially be used as membranes for temporary guided tissue regeneration in bone defects.

Thesis (MTech (Food Technology))--Cape Peninsula University of Technology, 2015. / The aim of this study was to characterise the physicochemical, rheological, prebiotic and emulsion stabilising properties of four varieties (black-eye, brown-eye, brown and red) of Bambara groundnut (BGN) extracted using the modified wet milling method. A relatively high yield of BGN dietary fibres was obtained with soluble dietary fibres (SDFs) ranging from 15.4 to 17.1% and insoluble dietary fibres (IDFs) ranging from 12.0 to 15.6%. Black-eye and brown-eye dietary fibres showed superiority in terms of swelling capacities, water holding capacities, oil binding capacities, antioxidant properties as well as thermal stabilities than red and brown dietary fibres. In addition, black-eye and brown-eye dietary fibres were characterised by higher lightness (L*), redness (+a*), yellowness (+b*), chroma (C*) and hue. All four SDFs showed acceptable colour differences with ΔE < 8 ranging from 0.81 to 3.08. The hydrolysable polyphenolic (HPP) content of SDFs ranged from 6.89 to 20.86 mg/g GAE and that of IDFs ranged from 10.96 to 14.43 mg/g GAE. All four SDFs differed significantly (p < 0.05) in their HPP content. BGN IDFs were very low in tannins (< 2.2 mg/g).

Este trabalho teve por objetivo caracterizar polissacarídeos não amiláceos (PNAs) extraídos de bananas no início da senescência, por meio de calorimetria exploratória diferencial (DSC) e isotermas de sorção de vapor, para construir o diagrama de estado e consequentemente selecionar as melhores condições de uso e armazenamento do produto. Os PNAs foram obtidos de purê de bananas maduras. Os açúcares foram extraídos usando etanol, resultando em uma fração insolúvel rica em PNAs. Esse processo foi feito em seis condições diferentes: duas temperaturas (25 °C e 65 °C) e três tempos de extração (30 min, 60 min e 90 min). Para comparação, realizaram-se testes também com ácido poligalacturônico (APG) e arabinoxilano (AX), PNAs presentes nas bananas, adquiridos puros. Após a extração, a análise térmica foi feita em cadinhos de alumínio, sob atmosfera de nitrogênio. As amostras foram resfriadas até - 60 °C, então aquecidas até 90 °C, a 5 °C/min. As isotermas de sorção foram obtidas usando o método DVS (do inglês Dynamic Vapor Sorption), à temperatura ambiente, de aw = 0,10 até aw = 0,95, com passo de aw de 0,1, tolerância de 0,05 % na mudança de massa (weight trigger), e timeout de 24 h. As amostras com baixos conteúdos de umidade não apresentaram picos de cristalização, mas transições vítreas foram detectadas. Para construir os diagramas de estado e delimitar suas regiões, ajustaram-se os dados aos modelos de Gordon-Taylor, para transição vítrea, e de Chen, para o início da fusão. As isotermas de sorção apresentaram formatos correspondentes ao tipo III. As umidades de monocamada calculadas pelo modelo de GAB ficaram entre 6 g/ 100 g e 16 g/ 100 g de amostra em base seca. A vida de prateleira, a 25 °C em embalagem de polietileno contendo 1 kg de PNAs foi estimada entre 175 e 299 dias. / The aim of this work was to characterize non starch polysaccharides (NSP) obtained from overripe bananas, using differential scanning calorimetry (DSC), and vapor sorption isotherms, in order to build a state diagram, and select the best usage and storage conditions. NSP samples were obtained using overripe bananas puree. The sugars were extracted using ethanol, resulting in an insoluble, NSP-rich fraction. This process was carried out under six different combinations of temperature (25 °C or 65 °C) and extraction time (30, 60 or 90 min). For comparison, pure arabinoxylan (AX) and galacturonic acid (APG) samples, which are NSP components of banana, were also analysed. After the extraction, thermal analysis was carried out in aluminium pans, under nitrogen atmosphere. Samples were cooled up to -60 °C, then heated up to 90 °C, at a 5 °C/min rate. Sorption isotherms were obtained by Dynamic Vapor Sorption (DVS) method, at room temperature, from aw = 0,10 to aw = 0,95, with an aw step of 0,1, a weight trigger of, 0,05 %, and a 24-hour timeout. The samples with low moisture contents showed no crystallization peaks, but glass transitions occurred. To build state diagrams and mark their zones, Gordon- Taylor and Chen models were, respectively, adjusted to glass transition and fusion onset data. The sorption isotherms had type III formats. The monolayer moistures calculated using the GAB model were between 6 g/ 100 g sample and 16 g/ 100g sample (dry basis). The shelf life, at 25 °C in polyethylene bags containing 1 kg of NSP, was estimated between 175 and 299 days.

O presente trabalho visa compreender o processo de degradação da parede celular vegetal de hospedeiros de fitopatógenos da família Xanthomonadaceae. Criamos e aperfeiçoamos uma técnica de enumeração dos genes relacionados ao metabolismo de polissacarídeos, com enfoque na distinção entre aqueles que agem sobre os componentes da parede celular vegetal e sobre outros polissacarídeos. A história evolutiva desse conjunto de enzimas foi delineada através de inferências sobre as relações de homologia entre os genes enumerados, sua presença ou ausência nos diversos genomas abordados e comparação das taxas de mutação entre grupos de homólogos. Além disso, procuramos também, com essa etapa de bioinformática e a etapa seguinte, incrementar a anotação desses genes, muitos descritos como hipotéticos ou com vaga definição de sua função. Na segunda parte do desenvolvimento do projeto foram feitos experimentos de expressão heteróloga e verificação da atividade enzimática para validação da anotação de alguns dos genes identificados. / This study aims to understand the process of degradation of host plant cell walls by plant pathogens of the Xanthomonadaceae family. We created and perfected a technique for enumeration of genes related to the metabolism of polysaccharides, focusing on the distinction between those who act on components of plant cell wall and on other polysaccharides. The evolutionary history of this group of enzymes has been outlined through inferences about the relations of homology between the genes listed, their presence or absence in different genomes and comparison of mutation rates between groups of homologues. Moreover, we also attempted with this bioinformatics step and the next step, to enhance the annotation of these genes, many described as hypothetical or vague in the determination of its function. In the second part of the project development heterologous expression and enzymatic activity assays were made to validate the annotation of some of the genes identified.

The consumption of fungal- and plant-derived non-starch polysaccharides (NSP) have been associated with reduced risk of cardiovascular diseases and cancer. In addition to promote physiochemical effects on the gastrointestinal tract and serve as substrate for the intestinal microbiota to produce short-chain fatty acids, NSP can interact with immune system cells including macrophages, which are crucial for tissue repair, lipid metabolism and host defense against foreign substances and pathogens. However, the effects of NSP in macrophages depends on their structure. Recently, it was showed that the chayote (Sechium edule) and the fungus Pleurotus albidus are promising sources of NSP with potential immunomodulatory effects in macrophages. In this study, it was explored the effects of cooking on the composition of NSP from chayote and evaluated their biological effects in macrophages. Furthermore, it was optimized a method for the extraction of mushroom NSP and characterized the structure and biological effects of NSP from P. albidus in macrophages. Results showed that the NSP from chayote pulp regulate cytokine secretion and phagocytosis by macrophages, and minor changes in composition during cooking influences their effects in macrophages. Furthermore, NSP from chayote induces cholesterol efflux and inhibits the expression of genes required for NLRP3 inflammasome activation in macrophages previously exposed to cholesterol crystals. Then, it was showed that the optimized method for the extraction of NSP from mushroom reduces by up to half the extraction time commonly required. Furthermore, results showed that P. albidus is source of easily extractable glucans with biological effects in macrophages. Results also suggest that glucans from P. albidus inhibit lipid-induced inflammation and foam-cell formation at distinct levels, with significant effects on NLRP3 inflammasome activation. Taken together, the results suggest that the benefits of chayote NSP is beyond their physical properties on the gastrointestinal tract, and that the P. albidus NSP offers potential health benefits that might be of relevance as a functional food ingredient. / O consumo de polissacarídeos não-amido (PNA) de fungos e plantas tem sido associado a redução do risco de doenças cardiovasculares. Além de promoverem efeitos físicos no trato gastrointestinal e serem utilizados como substratos pela microbiota intestinal, os PNA podem interagir com células do sistema imune, como macrófagos, cruciais no reparo tecidual, metabolismo lipídico, e na defesa do organismo contra patógenos. Entretanto, os efeitos em macrófagos dependem da estrutura do PNA. Recentemente, foi observado que o chuchu (Sechium edule) e o fungo Pleurotus albidus são fontes de PNA com potencial efeito sobre macrófagos. Assim, foram avaliados os efeitos dos PNA do chuchu fresco e cozido em macrófagos. Além disso, foi otimizado um método para extração de polissacarídeos de cogumelo, e avaliada a estrutura e os efeitos biológicos dos PNA do P. albidus em macrófagos. Foi observado que os PNA do chuchu regulam a secreção de citocinas e o processo de fagocitose por macrófagos, e alterações na composição de PNA durante o cozimento tem um impacto em seus efeitos biológicos. Além disso, os PNA do chuchu induzem o efluxo de colesterol e regulam a expressão de genes necessários para a ativação do inflamassoma NLRP3 em macrófagos previamente tratados com cristais de colesterol. Também foi demonstrado que o método otimizado de extração de PNA de cogumelos reduz em até pela metade o tempo de extração normalmente empregado. Além disso, foi verificado que o P. albidus é fonte para extração de glicanos com efeitos em macrófagos. Os resultados também sugerem que os glicanos obtidos do P. albidus inibem em diferentes níveis a inflamação induzida por lipídeos e a formação de células espumosas, com efeitos significativos sobre a ativação do inflamassoma NLRP3. Tais diferenças parecem estar associadas à estrutura dos glicanos. Por fim, os resultados sugerem que os benefícios dos PNA do chuchu estão além dos seus efeitos físicos sobre o trato gastrointestinal, e que os PNA do P. albidus promovem benefícios que podem ser relevantes para explorar sua utilização como um alimento ou fonte para extração de ingredientes funcionais.

Koon Chi Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 158-174). / Abstracts in English and Chinese. / Acknowledgment --- p.i / Abbreviations --- p.ii / Abstract --- p.v / Chinese Abstract --- p.viii / Table of Content --- p.x / Chapter Chapter one: --- Introduction --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Classification of Plant Polysaccharides --- p.2 / Chapter 1.2.1 --- Definition of Dietary Fiber --- p.3 / Chapter 1.2.2 --- Types of Soluble Dietary Fiber --- p.3 / Chapter 1.3 --- Physiological Effect of Fiber --- p.6 / Chapter 1.3.1 --- Reduction in Absorption by Viscous Polysaccharides --- p.7 / Chapter 1.3.2 --- Gastric Emptying --- p.7 / Chapter 1.3.3 --- Effect of Viscous Polysaccharides on Intraluminal Mixing --- p.8 / Chapter 1.3.4 --- Effect of Luminal Secretions on Viscosity --- p.9 / Chapter 1.4 --- Physicochemical Qualities and Hypocholesterolemic Effects --- p.9 / Chapter 1.5 --- Gastrointestinal Events and Hypocholesterolemic Effects --- p.11 / Chapter 1.5.1 --- Mouth --- p.11 / Chapter 1.5.2 --- Stomach --- p.12 / Chapter 1.5.3 --- Small intestine --- p.12 / Chapter 1.5.4 --- Large intestine --- p.13 / Chapter 1.6 --- Proposed Mechanisms for Hypocholesterolemic Effects --- p.13 / Chapter 1.6.1 --- Altered Bile Acid Absorption and Metabolism --- p.14 / Chapter 1.6.2 --- Modified Lipid Absorption and Metabolism --- p.15 / Chapter 1.6.3 --- Effects of SCFA on Lipid Metabolism --- p.15 / Chapter 1.6.4 --- Changed Hormone Concentrations --- p.16 / Chapter Chapter Two: --- Materials and Methods --- p.17 / Chapter 2.1 --- Materials --- p.17 / Chapter 2.1.1 --- Fungus --- p.17 / Chapter 2.1.2 --- Animals --- p.17 / Chapter 2.1.2.1 --- Golden Syrian Hamster --- p.17 / Chapter 2.1.2.2 --- Rabbit --- p.18 / Chapter 2.1.3 --- Characterization of Auricularia Polytricha --- p.18 / Chapter 2.1.4 --- Chromatographic materials --- p.22 / Chapter 2.1.5 --- "Determination of Plasma TC，HDL-C, LDL-C，TG，AST and ALT" --- p.24 / Chapter 2.1.6 --- HMG-CoA Reductase Activity Assay --- p.26 / Chapter 2.1.7 --- "Quantitative Determination of Liver Cholesterol, Acidic and Neutral Sterol" --- p.27 / Chapter 2.1.8 --- Animal Diets --- p.29 / Chapter 2.1.8.1 --- Hamster Diets --- p.29 / Chapter 2.1.8.2 --- Rabbit Diets --- p.29 / Chapter 2.2 --- Methods --- p.33 / Chapter 2.2.1. --- Extraction of Water-Soluble AP Polysaccharide (APP) --- p.33 / Chapter 2.2.2. --- Characterization of Auricularia Polytricha --- p.34 / Chapter 2.2.2.1 --- Determination of carbohydrate content of AP Polysaccharide --- p.34 / Chapter 2.2.2.2 --- Determination of uronic acid content of AP Polysaccharide --- p.34 / Chapter 2.2.2.3 --- Determination of protein content of AP Polysaccharide by BCA protein assay --- p.35 / Chapter 2.2.2.4 --- Determination of component sugar units of AP Polysaccharide --- p.35 / Chapter 2.2.2.5 --- Fractionation of AP Polysaccharide --- p.36 / Chapter 2.2.2.6 --- Determination of monosaccharides of AP Polysaccharide by HPLC --- p.37 / Chapter 2.2.3 --- "Determination of plasma TC, HDL-C, LDL-C,TG,AST and ALT" --- p.39 / Chapter 2.2.3.1 --- Plasma Total Cholesterol --- p.39 / Chapter 2.2.3.2 --- Plasma HDL-Cholesterol --- p.40 / Chapter 2.2.3.3 --- Plasma LDL-Cholesterol --- p.40 / Chapter 2.2.3.4 --- Plasma Triglyceride --- p.41 / Chapter 2.2.3.5 --- Plasma Aspartate Aminotransferase --- p.41 / Chapter 2.2.3.6 --- Plasma Alanine Aminotransferase --- p.42 / Chapter 2.2.4 --- HMG-CoA Reductase Activity Assay --- p.42 / Chapter 2.2.4.1 --- Preparation of Hepatic Microsome --- p.42 / Chapter 2.2.4.2 --- HMG-CoA Activity Assay --- p.43 / Chapter 2.2.5 --- Quantitative Determination of Liver Cholesterol --- p.44 / Chapter 2.2.5.1 --- Cholesterol Extraction and its Silylation --- p.44 / Chapter 2.2.5.2 --- GLC Analysis of TMS-Ether Derivative of Cholesterol --- p.45 / Chapter 2.2.6 --- Quantitative Determination of Neutral and Acidic Sterols --- p.45 / Chapter 2.2.6.1 --- Separation of Neutral and Acidic Sterols --- p.45 / Chapter 2.2.6.2 --- Conversion of Neutral Sterols to its TMS-Ether Derivative --- p.46 / Chapter 2.2.6.3 --- Conversion of Acidic Sterols to its TMS-Ether Derivatives --- p.46 / Chapter 2.2.6.4 --- GLC Analysis of Neutral and Acidic Sterols --- p.47 / Chapter 2.2.7 --- Study of Atherosclerosis of Rabbit --- p.48 / Chapter 2.2.7.1 --- Sudan III staining of the thoracic aorta --- p.48 / Chapter 2.2.7.2 --- Measurement of atheroma formation in the aorta --- p.49 / Chapter 2.2.8 --- Animal Experiments --- p.51 / Chapter 2.2.8.1 --- Protective Effect of APP in Hyperlipidemic Study (Exp. 1) --- p.51 / Chapter 2.2.8.2 --- Therapeutic Effect of APP in Hyperlipidemic Study (Exp. 2) --- p.52 / Chapter 2.2.8.3 --- Dose Response of APP in Hyperlipidemic Study (Exp. 3) --- p.52 / Chapter 2.2.8.4 --- Hypolipidemic Effect of Short Chain Fatty Acid (Exp. 4) --- p.53 / Chapter 2.2.8.5 --- Effect of APP and SCFA on HMG-CoA Reductase Activity (Exp5） --- p.53 / Chapter 2.2.8.6 --- Hypolipidemic and Anti-atherosclerotic Effect of APP (Exp. 6) ´Ø… --- p.54 / Chapter 2.3 --- Statistical analysis --- p.54 / Chapter Chapter Three: --- Fractionation and Characterization of Auricularia Polytricha Polysaccharide --- p.55 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Fungal polysaccharides from Auricularia Polytricha --- p.55 / Chapter 3.3 --- Results --- p.57 / Chapter 3.3.1 --- Extraction and Fractionation of Auricularia Polytricha --- p.57 / Chapter 3.3.2 --- Determination of Carbohydrates Content --- p.58 / Chapter 3.3.3 --- Determination of Protein Content --- p.61 / Chapter 3.3.4 --- Determination of Uronic Acid Content --- p.61 / Chapter 3.3.5 --- Determination of component sugars of AP Polysaccharide --- p.65 / Chapter 3.3.6 --- Fractionation of AP Polysaccharide --- p.67 / Chapter 3.3.7 --- Determination of monosaccharide components of AP Polysaccharide by HPLC --- p.72 / Chapter 3.4 --- Discussion --- p.79 / Chapter Chapter Four: --- "Protective, Therapeutic and Dose Effect of Auricularia Polytricha Polysaccharide (APP) on Hyperlipidemia" --- p.83 / Chapter 4.1 --- Introduction --- p.83 / Chapter 4.2 --- Results (Exp. 1) --- p.86 / Chapter 4.2.1 --- Body Weight and Food Intake --- p.86 / Chapter 4.2.2 --- Effect of APP Supplementation on Hepatic Cholesterol --- p.86 / Chapter 4.2.3 --- "Effect of APP Supplementation on Plasma TC, HDL-C and TG" --- p.87 / Chapter 4.2.4 --- Effect of APP Supplementation on Fecal Output of Neutral Sterols --- p.94 / Chapter 4.2.5 --- Effect of APP Supplementation on Fecal Output of Acidic Sterols --- p.94 / Chapter 4.3 --- Discussion (Exp. 1) --- p.99 / Chapter 4.4 --- Results (Exp. 2) --- p.102 / Chapter 4.4.1 --- Body Weight and Food Intake --- p.102 / Chapter 4.4.2 --- Effect of APP Supplementation on Hepatic Cholesterol --- p.102 / Chapter 4.4.3 --- Effect of APP Supplementation on Plasma TC and TG --- p.103 / Chapter 4.4.4 --- Effect of APP Supplementation on Plasma HDL-C and LDL-C --- p.104 / Chapter 4.5 --- Discussion (Exp. 2) --- p.109 / Chapter 4.6 --- Results (Exp. 3) --- p.111 / Chapter 4.6.1 --- Body Weight and Food Intake --- p.111 / Chapter 4.6.2 --- Dose Response of APP Supplementation on Hepatic Cholesterol --- p.111 / Chapter 4.6.3 --- Dose Response of APP Supplementation on Plasma TG --- p.112 / Chapter 4.6.4 --- Dose Response of APP Supplementation on Plasma HDL-C and LDL-C --- p.112 / Chapter 4.6.5 --- Dose Response of APP Supplementation on ALT and AST Activity --- p.113 / Chapter 4.6.6 --- Dose Response of APP Supplementation on Fecal Output of Neutral and Acidic Sterols --- p.113 / Chapter 4.7 --- Discussion --- p.121 / Chapter Chapter Five: --- Hypolipidemic Effect of Short Chain Fatty Acids --- p.123 / Chapter 5.1 --- "Introduction (Exp. 4,5)" --- p.123 / Chapter 5.2 --- "Results (Exp. 4,5)" --- p.125 / Chapter 5.2.1 --- Body Weight and Food Intake --- p.125 / Chapter 5.2.2 --- Effect of SCFA Supplementation on Hepatic Cholesterol --- p.125 / Chapter 5.2.3 --- "Effect of SCFA Supplementation on Plasma TG, HDL-C and LDL-C" --- p.128 / Chapter 5.2.4 --- Effect of SCFA Supplementation on AST and ALT Activity --- p.128 / Chapter 5.2.5 --- Effect of SCFA supplementation on HMG-CoA Reductase Activity --- p.133 / Chapter 5.3 --- "Discussion (Exp. 4,5)" --- p.135 / Chapter Chapter Six: --- Hypolipidemic and Antiatherosclerotic Effect of APP --- p.137 / Chapter 6.1 --- Introduction (Exp. 6) --- p.137 / Chapter 6.2 --- Results (Exp. 6) --- p.139 / Chapter 6.2.1 --- Body Weight and Food Intake --- p.139 / Chapter 6.2.2 --- Effect of APP Supplementation on Hepatic Cholesterol --- p.139 / Chapter 6.2.3 --- "Effect of APP Supplementation on Plasma TG, HDL- and LDL-C" --- p.141 / Chapter 6.2.3 --- Effect of APP Supplementation on AST and ALT Activity --- p.142 / Chapter 6.2.5 --- Effect of APP supplementation on HMG-CoA Reductase Activity --- p.146 / Chapter 6.2.6 --- Effect of APP supplementation on the Formation of Atheroma --- p.146 / Chapter 6.3 --- Discussion (Exp. 6) --- p.151 / Chapter Chapter Seven: --- General Discussion and Future Perspectives --- p.153 / References --- p.158

Sit Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 135-150). / Abstracts in English and Chinese. / Acknowledgment --- p.i / Abbreviations --- p.ii / Abstract --- p.v / Chinese Abstract --- p.vii / Table of Content --- p.ix / Chapter Chapter one: --- General Introduction --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Definition of Dietary Fiber --- p.1 / Chapter 1.3 --- Classification of Dietary Fiber --- p.2 / Chapter 1.4 --- Hypocholesterolemic Effects of Soluble Dietary Fibers --- p.3 / Chapter 1.5 --- Proposed Mechanisms for Hypocholesterolemic Effects --- p.4 / Chapter 1.5.1 --- Alter Eating Pattern --- p.4 / Chapter 1.5.2 --- Delay Gastric Emptying --- p.4 / Chapter 1.5.3 --- Modify Lipid Digestion and Absorption --- p.5 / Chapter 1.5.4 --- Effects of SCFA on Lipid Metabolism --- p.6 / Chapter 1.5.5 --- Enhance Bile Acid Excretion --- p.7 / Chapter 1.6 --- Auricularia polytricha --- p.8 / Chapter Chapter Two: --- Chemical Analysis of Auricularia polytrica --- p.11 / Chapter 2.1 --- Introduction --- p.11 / Chapter 2.2 --- Materials and Methods --- p.12 / Chapter 2.2.1 --- Extraction and Fractionation of Auricularia polytricha --- p.12 / Chapter 2.2.2 --- Determination of Carbohydrate Content --- p.12 / Chapter 2.2.3 --- Determination of Protein Content --- p.13 / Chapter 2.2.4 --- Determination of Uronic Acid Content --- p.13 / Chapter 2.2.5 --- Determination of Molecular Weight by Gel Filtration Chromatography --- p.14 / Chapter 2.2.6 --- Determination of Monosaccharide Components by HPLC --- p.15 / Chapter 2.3 --- Results --- p.18 / Chapter 2.3.1 --- Yield of Auricularia polytricha polysaccharides --- p.18 / Chapter 2.3.2 --- Carbohydrate Content of APPs --- p.18 / Chapter 2.3.3 --- Protein Content of APPs --- p.18 / Chapter 2.3.4 --- Uronic Acid Content of APPs --- p.19 / Chapter 2.3.5 --- Molecular Weight of APPs --- p.22 / Chapter 2.3.6 --- Monosaccharide Components of APPs --- p.27 / Chapter 2.4 --- Discussion --- p.33 / Chapter Chapter Three: --- Hypolipidemic Effects of APPs --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- Materials and Methods --- p.38 / Chapter 3.2.1 --- Golden Syrian Hamster --- p.38 / Chapter 3.2.2 --- Animal Experiments --- p.40 / Chapter 3.2.2.1 --- Protective Effect and Dose Response of APPs (Exp. 1) --- p.40 / Chapter 3.2.2.2 --- Therapeutic Effect of APPs (High-cholesterol Diet) (Exp. 2) --- p.40 / Chapter 3.2.2.3 --- Therapeutic Effect of APPII (Normal Diet) (Exp. 3) --- p.41 / Chapter 3.2.2.4 --- Effect of APPs on HMG-CoA Reductase and AC AT Activity (Exp. 4) --- p.42 / Chapter 3.2.3 --- Determination of Plasma AST and ALT --- p.42 / Chapter 3.2.4 --- "Determination of Plasma TC, LDL-C, HDL-C and TG" --- p.43 / Chapter 3.2.5 --- Quantitative Determination of Hepatic and Heart Cholesterol --- p.43 / Chapter 3.2.6 --- Quantitative Determination of Perirenal Adipose Tissue Triglyceride --- p.44 / Chapter 3.2.7 --- Statistical analysis --- p.45 / Chapter 3.3 --- Results (Exp. 1) --- p.47 / Chapter 3.3.1 --- Food Intake and Growth --- p.47 / Chapter 3.3.2 --- Effect of APPs on Plasma AST and ALT --- p.47 / Chapter 3.3.3 --- "Effect of APPs on Plasma TC, LDL-C, HDL-C and TG" --- p.53 / Chapter 3.3.4 --- Effect of APPs on Hepatic and Heart Cholesterol --- p.59 / Chapter 3.4 --- Discussion (Exp. 1) --- p.64 / Chapter 3.5 --- Results (Exp. 2) --- p.67 / Chapter 3.5.1 --- Food Intake and Growth --- p.67 / Chapter 3.5.2 --- Effect of APPs on Plasma AST and ALT --- p.67 / Chapter 3.5.3 --- "Effect of APPs on Plasma TC, LDL-C, HDL-C and TG" --- p.67 / Chapter 3.5.4 --- Effect of APPs on Hepatic and Heart Cholesterol --- p.71 / Chapter 3.6 --- Discussion (Exp. 2) --- p.74 / Chapter 3.7 --- Results (Exp. 3) --- p.76 / Chapter 3.7.1 --- Food Intake and Growth --- p.76 / Chapter 3.3.2 --- Effect of APPII on Plasma AST and ALT --- p.76 / Chapter 3.7.3 --- "Effect of APPII on Plasma TC, LDL-C, HDL-C and TG" --- p.76 / Chapter 3.7.4 --- Effect of APPII on Hepatic and Heart Cholesterol --- p.80 / Chapter 3.8 --- Discussion (Exp. 3) --- p.83 / Chapter Chapter Four: --- Influences of APPs on Cholesterol Homeostasis --- p.84 / Chapter 4.1 --- Introduction --- p.84 / Chapter 4.2. --- Materials and Methods --- p.87 / Chapter 4.2.1 --- HMG-CoA Reductase Activity Assay --- p.87 / Chapter 4.2.1.1 --- Preparation of Hepatic Microsome --- p.87 / Chapter 4.2.1.2 --- HMG-CoA Reductase Activity Assay --- p.87 / Chapter 4.2.2 --- ACAT Activity Assay --- p.88 / Chapter 4.2.2.1 --- Preparation of Hepatic and Intestinal Microsome --- p.89 / Chapter 4.2.2.2 --- ACAT Activity Assay --- p.89 / Chapter 4.2.3 --- Quantitative Determination of Neutral and Acidic Sterols --- p.90 / Chapter 4.2.3.1 --- Extraction of Neutral and Acidic Sterols --- p.90 / Chapter 4.2.3.2 --- Conversion of Neutral Sterols to its TMS-Ether Derivative --- p.91 / Chapter 4.2.3.3 --- Conversion of Acidic Sterols to its TMS-Ether Derivatives --- p.91 / Chapter 4.2.3.4 --- GLC Analysis of Neutral and Acidic Sterols --- p.92 / Chapter 4.3 --- Statistic Analysis --- p.93 / Chapter 4.4 --- Results (Exp. 4) --- p.94 / Chapter 4.4.1 --- Effect of APPs on Hepatic HMG-CoA Reductase Activity --- p.94 / Chapter 4.4.2 --- Effect of APPs on Hepatic and Intestinal AC AT Activity --- p.94 / Chapter 4.4.3 --- Effect of APPs on Fecal Excretion (Exp. 1 & 4) --- p.98 / Chapter 4.5 --- Discussion (Exp. 4) --- p.105 / Chapter Chapter Five: --- Hypolipidemic and Antiatherosclerotic Effect of APPII in Rabbit --- p.110 / Chapter 5.1 --- Introduction --- p.110 / Chapter 5.2 --- Materials and Methods --- p.113 / Chapter 5.2.1 --- New Zealant White Rabbit --- p.113 / Chapter 5.2.2 --- Hypolipidemic and Anitatherosclerosis Effect of APPII (Exp. 5) --- p.113 / Chapter 5.2.3 --- Measurement of Atheroma Formation --- p.115 / Chapter 5.3 --- Results (Exp. 5) --- p.117 / Chapter 5.3.1 --- Food Intake and Growth --- p.117 / Chapter 5.3.2 --- Effect of APPII on Plasma AST and ALT --- p.117 / Chapter 5.3.3 --- "Effect of APPII on Plasma TC, LDL-C, HDL-C and TG" --- p.117 / Chapter 5.3.4 --- Effect of APPII on Hepatic and Heart Cholesterol --- p.125 / Chapter 5.3.5 --- Effect of APPII on Perirenal Adipose Tissue Triglycerige Composition --- p.125 / Chapter 5.3.6 --- Effect of APPII on the Formation of Atheroma --- p.125 / Chapter 5.4 --- Discussion (Exp. 5) --- p.130 / Chapter Chapter Six: --- Conclusion --- p.132 / References --- p.135