CAPES / O mercado mundial de polissacarídeos tem atraído grandes companhias industriais interessadas em conquistar novos e rentáveis campos de atuação. Polissacarídeos com propriedades tecnológicas e biológicas podem ser obtidos a partir de plantas, algas e de microrganismos. Dentre os polissacarídeos com propriedades biológicas, as glucanas tem se destacado por apresentarem atividade imunoestimulante e potencialidades no tratamento de doenças como câncer, hipercolesterolemia, diabetes, esclerose múltipla e doenças cardiovasculares. Recentes estudos demonstram a produção extracelular de β-glucanas por fungos filamentosos em cultivos submersos. A modificação na estrutura química das glucanas por carboximetilação é considerada uma importante rota para melhorar suas propriedades, podendo contribuir para o aumento da solubilidade da molécula, bem como atividades biológicas, especialmente aquelas associadas a mecanismos de ação antioxidante e antiproliferativa. Nesse sentido, o presente trabalho teve como objetivo a produção de β-1,6-D-glucana (lasiodiplodana) pelo fungo Lasiodiplodia theobromae MMPI em cultivo submerso, bem como a carboximetilação da molécula, caracterização e avaliação da citotoxicidade e atividade antioxidante. A carboximetilação da molécula foi confirmada através da verificação de sinais químicos específicos identificados por espectroscopia de FT-IR e RMN 13C e a molécula carboximetilada apresentou grau de substituição (DS) de 1,27. A análise térmica (TG/DTA) indicou que a amostra bruta e carboximetilada apresentaram quatro estágios de perda de massa. O primeiro estágio ocorreu em 125ºC (perda de água) e houve dois eventos consecutivos de perda de massa (200ºC-400ºC) atribuídos à degradação da molécula. O quarto estágio ocorreu entre 425ºC e 620ºC (decomposição final) com pico exotérmico em 510ºC. Análise por MEV indicou que a lasiodiplodana bruta apresenta estruturas granulares que se rompem após carboximetilação. Análise de DRX demonstrou que o polímero bruto e carboximetilado apresentam estrutura não cristalina. A carboximetilação contribuiu para melhorar a hidrossolubulidade da molécula (aumento de 60%) e para melhorar a atividade antioxidante avaliada pela capacidade de captura dos radicais ABTS, DPPH e poder redutor do íon férrico (FRAP). Não foi verificado efeito citotóxico da lasiodiplodana bruta e modificada sobre hemácias. Os resultados obtidos sugerem que a carboximetilação da lasiodiplodana pode contribuir para melhoria das propriedades biológicas e para o potencial de uso biotecnológico da molécula. / The world market of polysaccharides has attracted large industrial companies interested in gaining new and profitable fields. Polysaccharides with technological and biological properties can be obtained from plants, algae and microorganisms. Among the polysaccharides with biological properties, glucans have been highlighted by demonstrate immunostimulatory activity and potential for treating diseases such as cancer, hypercholesterolemia, diabetes, multiple sclerosis and cardiovascular diseases. Recent studies demonstrate the production of exocellular β-glucans by filamentous fungi in submerged cultivations. Modifications in the chemical structure of glucans by carboxymethylation is considered an important route to improve its properties, may contribute to the increased solubility of the molecule as well as biological activities, especially those associated with antioxidant and antiproliferative mechanisms of action. Therefore, the present work aimed the production of β-1,6-D glucan (lasiodiplodana) by the Lasiodiplodia theobromae MMPI fungus in submerged cultivation and carboxymethylation of the molecule, characterization and evaluation of cytotoxicity and antioxidant activity. The carboxymethylation of the molecule was confirmed by checking specific chemical signals identified by FT-IR and NMR and 13C spectroscopy. Carboxymethylated molecule presented degree of substitution (DS) of 1.27. Thermal analysis (TG / DTA) indicated that native and carboxymethylated samples had four stages of mass loss. The first stage was at 125 ºC (loss of water) and there were two consecutive events of weight loss (200 ºC - 400 ºC) attributed to the degradation of the molecule. The fourth stage occurred between 425 ºC and 620 ºC (final decomposition) with exothermic peak at 510 ºC. SEM analysis indicated that the raw lasiodiplodan presents granular structures which are broken after carboxymetylation. XRD analysis showed that native and carboxymethylated biopolymers have no crystalline structure. Carboxymethylation aided to improve water solubility the molecule (60% increase) and to improve antioxidant activity assessed by ability to capture the ABTS, DPPH radical scavenging and the ferric ion reducing power (FRAP). There was no cytotoxic effect of raw lasiodiplodana and modified on the erythrocytes. The results suggest that the carboxymethylation of lasiodiplodan can contribute to improved biological properties and the potential biotechnological use of the molecule.

Glucans are microbial cell wall carbohydrates that are shed into the circulation of patients with infections. Glucans are immunomodulatory and have structures that are influenced by bacterial or fungal species and growth conditions. We developed a method to covalently label carbohydrates with a fluorophore on the reducing terminus, and used the method to study the pharmacokinetics following intravenous administration of three highly purified and characterized glucans (glucan phosphate, laminarin and scleroglucan) that varied according to molecular size, branching frequency and solution conformation. Elimination half-life was longer (3.8±0.8 vs. 2.6±0.2 and 3.1±0.6 h) and volume of distribution lower (350±88 ml/kg vs. 540±146 and 612±154 ml/kg) for glucan phosphate than for laminarin and scleroglucan. Clearance was lower for glucan phosphate (42±6 ml/kg h) than for laminarin (103±17 ml/kg h) and scleroglucan (117±19 ml/kg h). Since plasma levels at steady state are inversely related to clearance, these differences suggest that pharmacokinetics could favor higher blood levels of glucans with certain physicochemical properties.

Zymosan is a β-glucan- and mannan-rich particle that is widely used as a cellular activator for examining the numerous responses effected by phagocytes. The macrophage mannose receptor (MR) and complement receptor 3 (CR3) have historically been considered the major macrophage lectins involved in the nonopsonic recognition of these yeast-derived particles. Using specific carbohydrate inhibitors, we show that a β-glucan receptor, but not the MR, is a predominant receptor involved in this process. Furthermore, nonopsonic zymosan binding was unaffected by genetic CD11b deficiency or a blocking monoclonal antibody (mAb) against CR3, demonstrating that CR3 was not the β-glucan receptor mediating this activity. To address the role of the recently described β-glucan receptor, Dectin-1, we generated a novel anti-Dectin-1 mAb, 2A11. Using this mAb, we show here that Dectin-1 was almost exclusively responsible for the β-glucan-dependent, nonopsonic recognition of zymosan by primary macrophages. These findings define Dectin-1 as the leukocyte β-glucan receptor, first described over 50 years ago, and resolves the long-standing controversy regarding the identity of this important molecule. Furthermore, these results identify Dectin-1 as a new target for examining the immunomodulatory properties of β-glucans for therapeutic drug design.

No description available.

Lee, Ying. / On t.p. "beta" appears as the Greek letter. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 171-196). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.i / Acknowledgement --- p.ii / Abstract --- p.iii / 摘要 --- p.v / List of Tables --- p.vii / List of Figures --- p.x / List of Abbreviations --- p.xvii / Content --- p.xviii / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Probiotics and Prebiotics --- p.1 / Chapter 1.1.1 --- Definitions --- p.1 / Chapter 1.1.2 --- Previous studies --- p.2 / Chapter 1.1.3 --- Properties of enhanced prebiotics --- p.6 / Chapter 1.1.4 --- Synbiotics --- p.7 / Chapter 1.2 --- Colonic fermentation --- p.10 / Chapter 1.2.1 --- Major substrates and metabolites of colonic fermentation --- p.10 / Chapter 1.2.2 --- Health-related effects of Short-Chain Fatty Acids (SCFAs) --- p.12 / Chapter 1.3 --- Bifidogenic effect --- p.14 / Chapter 1.3.1 --- Definition of bifidogenic factor and its health benefits --- p.14 / Chapter 1.3.2 --- Carbohydrate metabolism by related enzymes of bifidobacteria --- p.16 / Chapter 1.3.3 --- Previous studies on bifidogenic effects of carbohydrates --- p.18 / Chapter 1.4 --- Barley β-glucan --- p.18 / Chapter 1.4.1 --- Cereal fibres as prebiotics --- p.18 / Chapter 1.4.2 --- Chemical and physical properties and related health impacts of barley β-glucan --- p.19 / Chapter 1.4.3 --- Impacts on intestinal microecology --- p.21 / Chapter 1.4.4 --- Previous studies on bifidogenic effects of barley β-glucan --- p.21 / Chapter 1.5 --- Methodology for evaluating prebiotic and bifidogenic effect --- p.22 / Chapter 1.5.1 --- In vivo animal models --- p.23 / Chapter 1.5.2 --- Human clinical study --- p.23 / Chapter 1.5.3 --- In vitro fermentation study --- p.24 / Chapter 1.5.3.1 --- Pure culture --- p.24 / Chapter 1.5.3.2 --- Mixed culture bacterial fermenters --- p.25 / Chapter 1.5.3.3 --- Continuous culture systems as in vitro gut models --- p.25 / Chapter 1.5.4 --- Advanced molecular techniques in quantifying intestinal bacteria --- p.26 / Chapter 1.6 --- Factors affecting bifidogenic effect --- p.30 / Chapter 1.6.1 --- Molecular weight --- p.30 / Chapter 1.6.2 --- Species difference --- p.31 / Chapter 1.7 --- Enzymatic activities involved in fermentation of β-glucan --- p.32 / Chapter 1.7.1 --- "Endo-1,3-1,4-(3-glucanase (Lichenase)" --- p.32 / Chapter 1.7.2 --- "Endo-l,4-β-Glucanase (Cellulase)" --- p.33 / Chapter 1.7.3 --- Enzymatic assays --- p.33 / Chapter 1.8 --- Project objectives --- p.36 / Chapter Chapter 2. --- Materials and Methods --- p.37 / Chapter 2.1 --- Materials --- p.37 / Chapter 2.1.1 --- "Trehalose, chitin and lactulose" --- p.37 / Chapter 2.1.2 --- Barley β-glucan --- p.37 / Chapter 2.1.3 --- Pure Bifidobacterium species of human origin --- p.39 / Chapter 2.2 --- Static batch culture fermentation using fecal inoculums --- p.39 / Chapter 2.2.1 --- Substrate preparation --- p.39 / Chapter 2.2.2 --- Human fecal inoculum preparation --- p.41 / Chapter 2.2.3 --- Inoculation of human fecal inoculums --- p.41 / Chapter 2.3 --- Static batch culture fermentation using pure culture of bifidobacteria --- p.42 / Chapter 2.3.1 --- Substrate preparation --- p.42 / Chapter 2.3.2 --- Cultivation of pure bifidobacterium cultures --- p.43 / Chapter 2.3.3 --- Inoculation of bifidobacterium culture --- p.44 / Chapter 2.3.4 --- Growth curve of Bifidobacterium species --- p.44 / Chapter 2.4 --- Dry matter and organic matter disappearance in batch fermentation --- p.47 / Chapter 2.5 --- Gas chromatographic (GC) determination of short-chain fatty acids (SCFAs) --- p.48 / Chapter 2.6 --- MTT assay --- p.51 / Chapter 2.7 --- Microbial identification and enumeration --- p.53 / Chapter 2.7.1 --- Fluorescent in situ hybridization --- p.53 / Chapter 2.7.1.1 --- Oligonucleotide probes for fluorescent in situ hybridization --- p.53 / Chapter 2.7.1.2 --- Cell fixation --- p.54 / Chapter 2.7.1.3 --- In situ hybridization --- p.55 / Chapter 2.7.1.4 --- Automated image analysis --- p.55 / Chapter 2.7.1.5 --- Quantification of bacteria --- p.57 / Chapter 2.7.2 --- Optical Density (OD) measurement --- p.58 / Chapter 2.7.3 --- Direct microscopic count --- p.59 / Chapter 2.8 --- Enzyme assays --- p.60 / Chapter 2.8.1 --- Enzyme extraction --- p.60 / Chapter 2.8.2 --- "Endo-1, 3:1, 4-β-glucanase (Lichenase)" --- p.61 / Chapter 2.8.2.1 --- Principle --- p.61 / Chapter 2.8.2.2 --- Preparation of substrate and assay solutions --- p.63 / Chapter 2.8.2.3 --- Enzyme assay procedures --- p.64 / Chapter 2.8.3 --- "Endo-l,4-β-Glucanase (Cellulase)" --- p.65 / Chapter 2.8.3.1 --- Principle --- p.65 / Chapter 2.8.3.2 --- Dissolution of substrate and preparation of assay solutions --- p.65 / Chapter 2.8.3.3 --- Enzyme assay procedures --- p.66 / Chapter 2.8.4 --- API@ ZYM kit --- p.67 / Chapter 2.8.4.1 --- Principle --- p.67 / Chapter 2.8.4.2 --- Specimen preparation --- p.68 / Chapter 2.8.4.3 --- "Preparation, inoculation and reading of the strips" --- p.70 / Chapter 2.9 --- Statistical analysis --- p.71 / Chapter Chapter 3 --- Results and Discussions --- p.72 / Chapter 3.1 --- Growth curves of Bifidobacterium species --- p.72 / Chapter 3.2 --- Batch in vitro fermentation using human fecal inoculum --- p.79 / Chapter 3.2.1 --- Dry matter and organic matter disappearance --- p.79 / Chapter 3.2.2 --- Colonic bacterial profile evaluated by FISH with CellC software --- p.81 / Chapter 3.2.2.1 --- Total colonic bacteria --- p.81 / Chapter 3.2.2.2 --- Bifidobacterial growth --- p.82 / Chapter 3.2.3 --- SCFA production --- p.86 / Chapter 3.2.3.1 --- Acetate --- p.88 / Chapter 3.2.3.2 --- Propionate --- p.89 / Chapter 3.2.3.3 --- Butyrate --- p.89 / Chapter 3.2.3.4 --- Total SCFA production --- p.90 / Chapter 3.2.3.5 --- Molar ratio of SCFAs --- p.92 / Chapter 3.3 --- In vitro fermentation of barley β-glucans with different molecular weight using pure culture of Bifidobacterium species --- p.95 / Chapter 3.3.1 --- Dry matter and organic matter disappearance --- p.96 / Chapter 3.3.2 --- Evaluation of bifidobacterial growth by optical density (OD) --- p.100 / Chapter 3.3.3 --- Time course study of SCFAs production --- p.109 / Chapter 3.3.3.1 --- "Total and individual SCFAs (Acetate, Propionate and Butyrate) production" --- p.109 / Chapter 3.3.4 --- Correlation between various parameters related to fermentation --- p.124 / Chapter 3.4 --- Enzymatic activities in 2 selected Bifidobacterium species during fermentation --- p.125 / Chapter 3.4.1 --- Dry matter and organic matter disappearance --- p.126 / Chapter 3.4.2 --- Bifidobacterial growth evaluated by direct microscopic count --- p.128 / Chapter 3.4.3 --- Time course study of SCFAs production --- p.131 / Chapter 3.4.3.1 --- "Total and individual SCFAs production (Acetate, Propionate and Butyrate)" --- p.131 / Chapter 3.4.3.2 --- MTT assay --- p.137 / Chapter 3.4.3.2.1 --- Effect of metabolites in the fermentation medium on the proliferation ofSW620 --- p.137 / Chapter 3.4.3.2.2 --- Effect of metabolites in the fermentation medium on the proliferation of Caco-2 --- p.145 / Chapter 3.4.4 --- Enzyme assays using commercial kits --- p.153 / Chapter 3.4.4.1 --- API @ZYM assay --- p.153 / Chapter 3.4.4.2 --- Efficiency of intra-cellular enzyme extraction using labiase --- p.156 / Chapter 3.4.5 --- Time course enzyme assays --- p.157 / Chapter 3.4.5.1 --- Lichenase activity assay --- p.157 / Chapter 3.4.5.2 --- Cellulase activity assay --- p.160 / Chapter Chapter 4. --- Conclusions and Future work --- p.168 / References --- p.171

No description available.

CWH41 is required for beta-1,6-glucan biosynthesis and encodes glucosidase I, an enzyme involved in protein N-chain glucose processing. Therefore, the effects of N-chain glucosylation and processing on beta-1,6-glucan biosynthesis were examined, and it was shown that incomplete N-chain glucose processing results in loss of beta-1,6-glucan. To explore the involvement of other N-chain-dependent events with beta-1,6-glucan synthesis, the S. cerevisiae KRE5 and CNE1 genes were investigated, which encode homologs of the 'quality control' components UDP-Glc:glycoprotein glucosyltransferase and calnexin, respectively. The essential activity of Kre5p was found to be separate from its possible role as a UDP-Glc:glycoprotein glucosyltransferase. A ∼30% decrease in beta-1,6-glucan was observed upon disruption of CNE1, a phenotype which is additive with other beta-1,6-glucan synthetic mutants. Analysis of the cell wall anchorage of alpha-agglutinin suggests the existence of two beta-1,6-glucan biosynthetic pathways, one N-chain dependent, the other involving protein glycosylphosphatidylinositol modification. / Fks1p and Fks2p are related proteins thought to be catalytic subunits of the beta-1,3-glucan synthase. The fks1Delta mutant was partial K1 killer toxin resistant and showed a 30% reduction in alkali-soluble beta-1,3-glucan that was accompanied by a modest reduction in beta-1,6-glucan. The gas1Delta mutant lacking a 1,3-beta-glucanosyltransferase displayed a similar reduction in alkali-soluble beta-1,3-glucan but did not share the beta-1,6-glucan defect, indicating that beta-1,6-glucan reduction is not a general phenotype among beta-1,3-glucan biosynthetic mutants. FKS2 overexpression suppressed the killer toxin phenotype of fks1Delta mutants, implicating Fks2p in the biosynthesis of the residual beta-1,6-glucan present in fks1Delta cells. Eight out of twelve fks1tsfks2Delta mutants had altered beta-glucan levels at the permissive temperature: the FKS1F1258Y N1520D allele was severely affected in both polymers and displayed a 55% reduction in beta-1,6-glucan, while the in vitro hyperactive FKS1T6051 M761T allele increased both beta-glucan levels. These beta-1,6-glucan phenotypes may be due to altered availability of, and structural changes in, the beta-1,3-glucan polymer, which might serve as a beta-1,6-glucan acceptor at the cell surface. Alternatively, Fks1p and Fks2p could actively participate in the biosynthesis of both polymers as beta-glucan transporters. beta-1,6-Glucan deficient mutants had reduced in vitro glucan synthase activity and mislocalized Fks1p and Fks2p, possibly contributing to the observed beta-1,6-glucan defects.

Thesis (M.S.)--Ohio University, March, 2010. / Title from PDF t.p. Includes bibliographical references.

Thesis (M. Sc.)--Memorial University of Newfoundland, 1997. / Bibliography: leaves 72-87.

O teor de β-glucanas interfere diretamente nos parâmetros de qualidade do malte para a produção de cerveja, principalmente na etapa de filtração. Elevadas concentrações de β-glucanas na cevada exigem maior tempo e temperatura na maceração e germinação. A aplicação de β-glucanase comercial é uma alternativa para reduzir o conteúdo de β-glucanas. Este trabalho teve como objetivo analisar o efeito da adição da β-glucanase no malte produzido a partir das cultivares de cevada BRS - Cauê e Elis (safra 2013/2014) com um tempo reduzido de germinação. As cultivares foram submetidas a micromalteações com 96 horas (convencional) e 64 horas (tempo reduzido) de germinação. As análises de β-glucanas e de qualidade da cevada, do malte e do mosto foram realizadas segundo a metodologia analítica EBC (European Brewery Convention). As cultivares BRS - Cauê e Elis, germinadas com 96 h apresentaram respectivamente os teores de 90,7 e 64,3 mg/L de β-glucanas. O processo com 64 h de germinação resultou em teores acima do limite máximo recomendado pela EBC (178 mg/L), sendo 320,0 e 370,7 mg/L para os maltes das cultivares BRS-Cauê e Elis respectivamente. A aplicação de 100 mg/kg de β- glucanase no malte produzido com 64 h de germinação reduziu os teores de β- glucanas para 74,7 (BRS-Cauê) e 81,7 mg/L (BRS-Elis). Houve uma redução no teor de β-glucanas de 76,67% para BRS - Cauê e 77,96% para BRS - Elis. Sendo que para o malte da cultivar BRS - Cauê a aplicação de 25 mg/kg da enzima e para a BRS - Elis a aplicação de 50 mg/kg foram suficientes para a obtenção de maltes com teores de acordo com o recomendado. A redução nos valores de viscosidade também foi observada. A aplicação da β-glucanase comercial reduziu o teor de β- glucanas no malte produzido em um tempo menor de germinação permitindo a otimização do tempo no processo de malteação. / The β-glucan content interferes directly in the malt quality parameters for the production of beer, especially in the filtration step. High β-glucans concentrations in barley require more time and temperature in the steeping and germination. The application of a commercial β-glucanase is an alternative to reduce the content of β- glucans. This study aimed to analyze the effect of the addition of β-glucanase in malt produced from barley cultivars BRS - Cauê and Elis (season 2013/2014) with a reduced germination time. The cultivars were subjected to micromalteações 96 hours (conventional) and 64 hours (reduced time) germination. The analyzes of β-glucans and quality of barley, malt and wort were performed according to the analytical methodology EBC (European Brewery Convention). The BRS - Cauê and Elis, germinated after 96 hours respectively showed the levels of 90.7 and 64.3 mg / L of β-glucans. The process with 64 h of germination resulted in levels over the maximum limit recommended by the EBC (178 mg / L), with 320.0 and 370.7 mg / L for the malts of BRS-Cauê and Elis cultivars respectively. The application of 100 mg / kg of malt β-glucanase produced in 64 h germination lowered β-glucan content to 74.7 (BRS-Cauê) and 81.7 mg / L (BRS-Elis). There was a reduction in β-glucan content of 76.67% for BRS - Cauê and 77.96% for BRS - Elis. Since for malt cultivar BRS - Cauê application of 25 mg / kg of the enzyme and the BRS - Elis application of 50 mg / kg was sufficient to obtain malt content according to recommended. The reduction in viscosity values were also observed. The application of commercial β-glucanase reduced the β-glucan content in malt produced in a shorter germination allowing the optimization of time in the malting process.