Made available in DSpace on 2014-10-09T12:36:04Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:59:22Z (GMT). No. of bitstreams: 0 / A prolactina humana (hPRL) é um hormônio polipeptídico secretado pela hipófise anterior sob regulação do hipotálamo, envolvido em uma variedade de processos biológicos como o desenvolvimento da glândula mamária e lactação. O produto recombinante é importante no diagnóstico médico e no tratamento de insuficiência da lactação. Este hormônio pode ocorrer sob a forma de proteína não glicosilada (NG-hPRL) e glicosilada (G-hPRL), com pesos moleculares de aproximadamente 23 e 25 kilodalton (kDa), respectivamente; possui um único sítio de N-glicosilação localizado na asparagina (Asn) posição 31, que é parcialmente ocupado, representando assim um modelo particularmente interessante de glicosilação. A atividade biológica da G-hPRL é muito menor comparada à NG-hPRL (~4 vezes) e sua função fisiológica ainda não é bem definida: a porção de carboidrato parece ter um importante papel na biossíntese, secreção, atividade biológica, e sobrevivência plasmática do hormônio. O objetivo principal desse trabalho foi comparar as estruturas dos N-glicanos presentes na prolactina glicosilada hipofisária (G-hPRL-NHPP) com a recombinante. Para obter a G-hPRL recombinante foi realizada uma produção em escala laboratorial a partir de células de ovário de hamster chinês (CHO) geneticamente modificadas e adaptadas ao crescimento em suspensão. Foi adicionada, ao meio de cultura cicloheximida (CHX), cujo efeito principal foi aumentar a relação G-hPRL/NGhPRL que passou de 5% para 38%, facilitando assim a purificação da G-hPRL. A G-hPRL foi purificada em duas etapas, uma troca catiônica seguida de purificação por cromatografia liquida de alta eficiência de fase reversa (RP-HPLC) que se demonstrou eficiente na separação das duas isoformas de hPRL. A G-hPRL recombinante IPEN foi assim analisada por diversas técnicas confirmando a sua pureza e atividade biológica, incluindo comparações com outras amostras de referências de origem hipofisária adquirida junto ao National Hormone & Peptide Program (NHPP-E.U.A.) . Foi realizada também a determinação inédita de Nglicanos presentes na G-hPRL produzida por células CHO e na G-hPRL nativa, produzida pela hipófise humana, possibilitando comparar as duas estruturas de carboidratos e alcançando assim uma das principais metas desse projeto. Entre as principais diferenças encontradas nas estruturas dos dois N-glicanos, destacam-se a baixa quantidade de ácido siálico (NeuAc), a alta porcentagem de glicanos sulfatos (74,0%) e com fucose (Fuc) (93,3%) presentes na amostra hipofisária e a tendência da preparação recombinante de apresentar glicanos com maior peso molecular e com uma menor variação nas isoformas. / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Orientador: Marcos Silveira Buckeridge / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T07:05:13Z (GMT). No. of bitstreams: 1 Cavalari_AlineAndreia_D.pdf: 2066715 bytes, checksum: 503395a4dfdc3832fc20cbf4193ea790 (MD5) Previous issue date: 2009 / Resumo: A parede celular é um componente particular dos tecidos vegetais e conhecer a composição dos polissacarídeos que a constituem e suas interações é essencial para compreender a textura dos alimentos e suas alterações pós-colheita, em especial em frutos climatérios, como é caso do mamão. A parede celular esta dividida por três domínios: o primeiro é formado por celulose e hemiceluloses, o segundo domínio é formado por pectinas e o terceiro um domínio composto por proteínas. As modificações dos polímeros e suas proporções nestes respectivos domínios são resultados de ações enzimáticas, que no caso dos frutos carnosos, leva ao amaciamento da polpa. Portanto, estudar as modificações nesses polímeros através da análise dos OXG obtidos por hidrolise com celulase, é um caminho importante para entender as alterações neste polissacarídeo ao longo do desenvolvimento de frutos. O presente trabalho teve como objetivo compreender as modificações da parede celular durante o desenvolvimento do fruto do mamoeiro. Foram utilizados frutos de Carica papaya L. cv. Sunrise solo, coletados diretamente do produtor (Caliman S/A- Unhares- ES). As amostras de frutos foram colhidas em intervalos de 30 dias, sendo os estádios analisados de 30 a 150 após a antese (dpa). Os resultados demonstram queda acentuada na proporção de parede celular em relação a outros compostos, como açúcares, por exemplo, o que é possivelmente uma indicação do processo de expansão celular e conseqüentemente uma alteração de textura da parede celular durante o desenvolvimento. Observou-se que o principal açúcar solúvel é a sacarose, sendo esta provavelmente a principal fonte energética para o desenvolvimento do fruto de mamão, uma vez que este não sintetiza amido. De maneira geral, a proporção de oligossacarídeos de xiloglucano menos ramificados diminuiu aos 120 dpa, enquanto que os de maior peso molecular e ou grau de ramificação (com fucose) aumentaram proporcionalmente. Estes resultados sugerem que xiloglucanos (ou parte das moléculas destes) pobremente ramificados com fucose, são retirados da parede celular, consequente enriquecimento de oligosasacarídeos fucosilados. Como estes últimos tornam o xiloglucano mais interativo com ele próprio e com a celulose, é possivel que estes sejam os principais efeitos que as transformações na parede promovam no fruto. As alterações na parede foram acompanhadas pelo aumento concomitante nas ativades de beta­galacosidase e beta-glucosidase, duas das principais hidrolases de xiloglucano. Concumitantemente, observou-se uma diminuição acentuada na proporção de celulose na parede. Com base nestas observações, sugere-se que as paredes celulares sofrem transformações importantes nos frutos do mamoeiro até os 120 dpa I sendo que a partir deste estádio a parede se torna mais acessível à hidrólise e denotando a preparação do fruto para o amadurecimento. / Abstract: The plant cell wall is a unique component of plant tissues and its polysaccharide composition essential to understand food texture and its changes during post-harvestingl especially of climateric fruits, as is the case of papaya. The plant cell wall is composed of three domains: the first is formed by cellulose and hemicelluloses, the second of pectins and the third of proteins. The changes in polymers and their proportions in these domains are a result of enzyme action, which in the case of fleshy fruits lead to the softening of the pulp. Hydrolysis of hemicelluloses such as xyloglucan can play important functions in cell expansion, cell growth and cell wall degradation. Therefore, studying the modifications in xyloglucan by looking at is fine structure (Le. oligosaccharide (OXG) pattern obtained after cellulase action) may be an important way to understand polysaccharide change during fruit development. The present work aimed at understanding the modifications in cell wall during the development of the papaya fruit. Fruits of Carica papaya L. Cv.Sunrise solol were collected directly by the producer (Calimanl SAI Unharesl Espirito Santol Brazil). Samples of fruits were harvested at intervals between 30 and 150 days after anthesis (daa). Our results showed that there were drastic changes in the cell wall of the mesocarp in relation to other compoundsl such as soluble sugars. This is probably an indication that cell expansion process is at least part of the cause of the changes in texture during development. We observed that the main soluble sugar found in fruits is sucrose, this being probably the principal source of energy for development of the organ, as no starch is synthesised in this fruit. In general, the proportion of less branched xyloglucan oligosaccharides decreased at 120 daa, whereas the OXG branched with fucose increased constantly during development up to the same stage. These results suggest that xyloglucans (or part of their molecules) that are poorely brached with fucose are retrieved from the cell wall. This seems to lead to enrichment of fucosylation of xyloglucan. As these OXG turn xyloglucan more interactive with itself and with cellulose, it is possible that these would be the principal effects that the cell walls provoke in the fruit. The changes in the wall were followed by a concomitant increase in activities of beta-galactosidase and betaglucosidase, both thought to be related to xyloglucan hydrolysis in vivo. At the same time, we observed a decrease in the proportion of cellulose in the walls during development. On the basis of these results, we suggest that the cell walls of papaya fruits undertake structural changes untill 120daa after which the wall becomes more accessible to hydrolases denoting the preparation of the papaya fruit for ripening. / Doutorado / Doutor em Biologia Vegetal

O presente estudo buscou avaliar os efeitos dos Mananoligossacarídeos (MOS) como aditivo alimentar no desempenho de primíparas suínas em final de gestação e em lactação, bem como no desempenho e na saúde dos leitões até os 65 dias de idade. O experimento foi realizado no Laboratório de Pesquisa em Suínos (VNP-FMVZ-USP) Pirassununga SP. O delineamento experimental foi inteiramente casualizado em arranjo fatorial de tratamentos, sendo um fator o fornecimento dos MOS para fêmeas e o segundo para os leitões. Foram utilizadas 17 primíparas prenhes, tratadas a partir de 81 &plusmn;1,36 dias de gestação com 0,2% de MOS na dieta. Aos 82 &plusmn; 1,36 dias de gestação as fêmeas foram submetidas à coleta de sangue e à vacinação contra rinite atrófica progressiva, sendo pesadas quinzenalmente até a transferência para a maternidade, aos 109 &plusmn; 1,36 dias de gestação. Na segunda quinzena as fêmeas tratadas apresentaram uma vantagem numérica no ganho de peso médio diário (P=0,063). Durante o parto foram colhidas amostras de sangue e colostro para titulação de anticorpos contra o antígeno vacinal, sendo consideradas positivas 66,67% das fêmeas MOS e 42,86% das fêmeas controle. Os leitões aleitados por fêmeas tratadas tiveram um maior ganho numérico de peso na primeira semana (p=0,0614), significativo na segunda semana (p=0,047). Na terceira semana foi introduzido o segundo fator através do oferecimento de alimento sólido (0,4% de MOS). No período total, do nascimento aos 21 dias, foi observada uma vantagem numérica (p=0,0989) no ganho de peso a favor dos leitões de fêmeas MOS. As fêmeas, contudo, não diferiram quanto à variação de peso ou consumo durante a fase de lactação. Aos 23 &plusmn; 1,91 dias de idade dos leitões foi realizado o desmame abrupto com a transferência dos leitões para unidade de creche, onde foram alojados em gaiolas para 4 animais. O peso ao desmame foi maior nos leitões aleitados por fêmeas tratadas (p<0,0001), bem como em todas as pesagens semanais até a quinta semana pós-desmame. O consumo na primeira semana sofreu influência da suplementação de MOS nas fêmeas e do maior peso à desmama se mostrando superior nesses animais (P=0,049) influenciando diretamente o ganho de peso, superior na segunda semana (p=0,002). O MOS para os leitões aumentou o consumo de alimento (p=0,0784), e a conversão alimentar na segunda semana (p=0,0103). Aos 14 dias pós-desmame foram colhidas amostras de sangue para hemograma e realizada a aplicação oral de 108 UFC de Salmonella Typhimurium. Foram observadas diariamente a consistência das fezes por 28 dias e aferidas as temperaturas retais por 9 dias. Leitões tratados com MOS e os leitões oriundos de fêmeas MOS tenderam a apresentar o pico de hipertermia mais cedo que os demais (p=0,0629 e p=0,0976, respectivamente) e os leitões alimentados com MOS tenderam a ter uma temperatura de pico mais baixa (p=0,0989). Na última semana os leitões de fêmeas MOS apresentaram um maior consumo (p=0,0007) e uma incidência de Salmonella numéricamente inferior em linfonodos mesentéricos e nas fezes colhidas aos 36 dias pós-desafio. Pode-se concluir que a suplementação de MOS para primíparas prenhes e lactentes pode melhorar o desempenho e a saúde entérica de seus leitões nas fases de aleitamento e creche. / The present study evaluated the effects of mananoligossacarides (MOS) as a feed additive on the performance of primiparous sows in late gestation and lactation, as well as on performance and health status of their progeny up to 65 days of age. The experiment was conducted in the Laboratory of Swine Research (FMVZ/USP) Pirassununga SP. For this purpose a completely random factorial design was used, factors which corresponded to (1) feeding sows with MOS and (2) feeding piglets with MOS, characterizing 4 treatments: MM Feeding MOS to both sows and piglets, MC Feeding MOS only to the sows, CM Feeding MOS only to piglets, CC Control diets for both sows and piglets. A total of seventeen pregnant gilts were used, and divided into 2 groups: MOS (n=9) and Control (n=8). MOS gilts had 0,1% MOS added to their diets from 81 &plusmn; 1,36 days of gestation onward. On day 82 &plusmn; 1,36 blood was collected and vaccination against Progressive Atrophic Rhinitis was conducted. Animals were weighted biweekly until 109 &plusmn; 1,36 days of gestation, when transference to the farrowing unit was conducted. Between the first and second weighing, MOS gilts show a numerical advantage in daily weight gain (p=0,063). At farrowing, blood and colostrum samples were collected for determination antibodies titles against vaccine antigen, being considered positive 66,67% and 42,86% of MOS and Control sows, respectively. Piglets nursed by MOS sows had a numerical advantage in weight gain in the first week (p=0,0614), with statistical significance in the second week (p=0,047). On the third week, the second factor was introduced by the offering of solid feed (0,4% MOS). During the suckling period, from birth to 21 days of age, a numerical advantage in weight gain was observed for MOS sows piglets (p=0,0989). The sows themselves did not differ in weight change or feed consumption. At 23 &plusmn; 1,91 days of age, weaning was conducted, as piglets were transferred to nursing facilities and allocated in pens of 4 animals. Weight of MOS sows piglets was higher at weaning (p<0,001) and until the 5th week port-weaning. Feed consumption was affected by MOS supplementation of sows and by the higher weaning weight (p=0,0049), directly influencing weight gain, which was superior in the 2nd week. Feeding MOS to piglets enhanced feed consumption (p=0,0784) and feed conversion (p=0,0103) on the 2nd week. At 14 days of age, blood samples were collected for hemogram analysis and an oral dose of 108 CFU of Salmonella Typhimurium administered. Fecal consistency and rectal temperature were evaluated for 28 and 9 days, respectively. Piglets treated with MOS and MOS sows\' piglets tended to show a peek of hyperthermia earlier (p=0,0629 e p=0,0976, respectively). Piglets fed with MOS tended to show a lower temperature peek (p=0.0989). In the last week, MOS sows\' piglets show a higher feed consumption (p=0.0007) and a numerically inferior Salmonella incidence in mesenteric lymph nodes and feces 36 days after the bacterial challenge. In conclusion, supplementing primiparous sows with MOS during gestation and lactation can enhance their body weight gain and the performance and intestinal health of the offspring during the suckling and nursing period.

Pathogenesis originating from mycobacterial invasion on host cells is prevalent and is a major challenge in efforts towards overcoming the burden of mycobacterial diseases. Complex architecture of mycobacterium cell wall includes an assortment of glycolipids, phospholipids, glycopeptidolipids (GPLs), peptidoglycans, arabinogalactans, lipoarabinomannans and mycolic acid. Aided by thick cell wall envelope, mycobacteria are known to survive in hostile environment. As most antibiotics target the log phase of the bacteria, bacterial survival is also largely dependent on its stationary phase. Mycobacteria have evolved colonization by means of biofilm formation in the stationary phase, so as to survive under stress and hostile conditions. Biofilms are the specialized form of phenotype which makes bacteria several fold resistant to antibiotics. Development of inhibitors against biofilms remains a challenge due to the poor permeability of molecules and coordination among cells. The first part of Chapter 1 of the thesis describes the details of formation of biofilm in the stationary phase of bacteria and understanding the molecular level details for making the strategies to overcome antidrug resistance of mycobacteria. Among the cyclic hosts, cyclodextrins are prominent. Due to their unique structural and physical properties, cyclodextrins can form inclusion complexes with a wide range of guest molecules. Although synthetic modifications of cyclodextrins through hydroxy groups are very common, modifications at backbone continue to be a challenge. Backbone modified cyclodextrins using different organic moieties were developed and their altered cavity properties were explored in many instances. Chemical synthesis of cyclic oligosaccharides is, in general, involved (i) a cyclo-oligomerization of linear oligosaccharide precursor and (ii) an one-pot polycondensation of appropriately designed monomer under suitable reaction conditions. The second part of Chapter 1 deals with a literature survey of skeletal modification of cyclodextrins, their synthesis and binding abilities with different guest molecules. In my research programme, synthesis and studies of oligosaccharide glycolipids relevant to mycobacterial cell wall were undertaken. Arabinofuranoside trisaccharide glycolipids, containing β-anomeric linkages at the non-reducing ends and double hexadecyloxy lipid moieties, interconnected to the sugar moiety through a glycerol core, were synthesized (Figure 1). Arabinan trisaccharides 1 with lipidic chain and 3 without lipidic chain comprise β-(1→2), β-(1→3) anomeric linkages at the non-reducing end, whereas in the case of arabinan trisaccharides 2 and 4, β-(1→2), β-(1→5) linkages are present between the furanoside units. In the scheme of synthesis of trisaccharide glycolipids, monosaccharide derivative and lipidic portions were individually prepared first and were assembled subsequently to secure the target glycolipids. Incorporation of β-arabinofuranoside linkages in trisaccharide arabinofuranosides 1-4 was achieved by low temperature activation of silyl group protected conformationally locked thioglycoside donor 5 (Figure 1), in the presence of N-iodosuccinimide (NIS) and silver trifluoromethanesulfonate (AgOTf). Figure 1. Molecular structures of trisaccharides 3, 4 and glycolipids 1, 2 with β-arabinofuranoside linkages at the non-reducing end and glycosyl donor 5. Following the synthesis, the efficacies of synthetic glycolipids to interact with surfactant protein A (SP-A) were assessed by using surface plasmon resonance (SPR) technique, from which association-dissociation rate constants and equilibrium binding constants were derived. SP-A, a lung innate immune system component, is known to bind with glycolipids present in the cell surface of a mycobacterial pathogen. From the analysis of SPR studies with glycolipids 1, 2 and SP-A, the association rate constants (ka) were found to be in the range of 0.3 to 0.85 M−1 s−1, whereas the dissociation rate constants (kd) were varied between 2.21 and 3.2×10−3 s−1. The equilibrium constants (Ka) values were in the range of 93 and 274 M−1. Trisaccharides 3 and 4, without lipidic chains, were also assessed for their efficacies to interact with SP-A. The association constants for 3 were found to be in the range of 2,470 to 9,430 M−1, whereas for the derivative 4, Ka values varied between 25,600 and 76,900 M−1. The association and equilibrium binding constants for 3 and 4 were found to be significantly higher when compared to glycolipids 1 and 2. In conjunction with our previous report, the present study shows that arabinofuranoside glycolipids, with β-anomeric linkages bind to SP-A with lesser extent as compared to α-anomers. Further, the studies of trisaccharides and glycolipids in mycobacterial growth and sliding motility assays were performed with model organism M. smegmatis and it was found that the synthetic compounds affected both growth and motility and the extent was lesser than that of α-anomeric glycosides and glycolipids. Chapter 2 of the thesis describes the details of synthesis, biophysical and biological studies of arabinan trisaccharide glycolipids, with β-anomeric linkages at the non-reducing end. Continuing the synthesis and studies of arabinan oligosaccharides, a linear arabinomannan pentasaccharide and heptasaccharide glycolipids 6 and 10, containing α-(1→2) and α-(1→3) linkages between core arabinofuranoside units, as well as, a branched arabinomannan pentasaccharide and heptasaccharide glycolipids 7 and 11, with α-(1→2) and α-(1→5) linkages between core arabinofuranoside units, were synthesized (Figures 2 and 3). Figure 2. Molecular structures of arabinomannan glycolipids 6 and 7 and the corresponding oligosaccharides 8 and 9. In addition to glycolipids, arabinomannan pentasaccharides without lipidic chain 8 and 9 and arabinomannan heptasaccharides without lipidic chain 12 and 13, were also synthesized. Synthesis was performed using trichloroacetimidate and thioglycosides as glycosyl donors. A block condensation methodology was adopted by which disaccharide donor and monosaccharide acceptor were chosen to assemble the pentasaccharide, by a two-fold glycosylation. Monosaccharide acceptors with and without lipidic chain were used in the glycosylations for the synthesis of glycolipids and pentasaccharides, respectively. Similarly, a trisaccharide thioglycoside donor and monosaccharide acceptors were chosen for the double glycosylation to synthesize heptasaccharides in the presence of NIS and AgOTf. Figure 3. Molecular structures of arabinomannan heptasaccharide glycolipids 10, 11 and corresponding heptasaccharides 12 and 13. Subsequent to synthesis, activities of pentasaccharide glycolipids were assayed on M. smegmatis bacterial growth, sliding motilities and also the effects on mycobacterial biofilms. Profound effects were observed with the synthetic compounds, to reduce the mycobacterial growth, sliding motilities and biofilm structures. Whereas reduction up to ~50% occurred on mycobacterial growth, as much as, 70% reduction in the motilities of the bacteria was observed in the presence of the synthetic glycolipids, at 100 µg mL-1 concentration. At the same concentration, 80–85% reduction in the biofilm was observed. These effects were more pronounced with branched glycolipids than linear analogues. Chapter 3 of the thesis presents the synthesis of linear and branched arabinomannan penta- and heptasaccharide glycolipids and biological studies of arabinomannan pentasaccharide glycolipids with M. smegmatis. Cyclodextrins, the most abundant naturally-occurring cyclic oligosaccharides, are valuable synthetic hosts, primarily as a result of their properties to form inclusion complexes with guest molecules. In spite of voluminous literature on the application of cyclodextrins, through modifications of hydroxy groups, modifications at the backbone continue to be a challenge. Skeletal modifications using aromatic, triazole, diyne, thioether and disulfide moieties were developed, that helped to alter the cavity properties of cyclodextrins. A programme was undertaken to synthesize backbone modified cyclic oligosaccharide, which was achieved using a monomer wherein a one carbon insertion is conducted at C4 of a pyranose, such that the hydroxy moiety at C4 is replaced with a hydroxymethyl moiety. In an approach, a linear trisaccharide monomer was anticipated to provide cyclic oligosaccharides in multiples of such a monomer. In the event, a trisaccharide linear monomer 14 was found to afford a cyclic trisaccharide macrocycle 15, as the major cyclo-oligomer (Scheme 1). Subsequent solid state structural studies show that the molecule confers a perfect trigonal symmetry in the P3 space group, in a narrow cone shape and a brick-wall type arrangement of molecules, such a geometry is hither-to unknown to a cyclic oligosaccharide (Figure 4). Furthermore, binding abilities of cyclic trisaccharide with few organic bases, such as 1-aminoadamantane and hexamethylenetetramine, was evaluated by the means of isothermal titration calorimetry and it was found that such a cyclic trisaccharide exhibits strong binding affinities towards 1-aminoadamantane in aqueous solutions, as compared to the same with naturally-occurring β-cyclodextrin. Scheme 1 Apart from cyclic trisaccharide, synthesis of cyclic tetrasaccharide 17, containing alternative anomeric α-(1→4) and β-(1→4) linkages was also undertaken by one-pot cyclo-oligomerization in the suitable reaction condition, from an activated disaccharide thioglycoside monomer 16, having β-(1→4) linkage at the non-reducing end (Scheme 2). Chapter 4 describes the synthesis of cyclic oligosaccharides 15 and 17, as well as, the details of solid state structure and binding studies of cyclic trisaccharide 15. Scheme 2 Figure 4. (a) Stick model of the crystal structure of 15, as viewed along the crystallographic c-axis; (b) trigonal view from crystal packing; (c) packing diagram crystal lattice, as viewed along the crystallographic b-axis, and without solvent inclusion and (d) packing diagram included with methanol (grey) and water (red) solvents, as viewed along the crystallographic c-axis. Hydrogen atoms are omitted for clarity in (c and d). In summary, the thesis presents (i) synthesis, biophysical and biological studies of synthetic arabinan and arabinomannan glycolipids, and (ii) synthesis, solid-state structural analysis and binding studies of glycosidic bond expanded cyclic oligosaccharides. Synthetic trisaccharide arabinofuranoside glycolipids containing β-anomeric linkages at the non-reducing end showed binding affinity towards pulmonary surfactant protein A, as assessed by surface plasmon resonance technique, with comparatively lower extent as compared to synthetic glycolipids having α-anomeric linkages. Linear and branched arabinomannan penta- and heptasaccharide glycolipids, having α-anomeric linkages were synthesized and biological studies with non-pathogenic strain M. smegmatis were conducted with pentasaccharide glycolipids. It was found that arabinomannan glycolipids inhibited the growth and sliding motility of mycobacteria. Importantly, disruption of biofilm and significant reduction in biofilm formation was observed in the presence of the synthetic glycolipids. Glycosidic bond expanded cyclic trisaccharide with anomeric α-(1→4) linkages and cyclic tetrasaccharide with alternative anomeric α-(1→4) and β-(1→4) linkages were prepared from suitably designed trisaccharide and disaccharide monomer, respectively, by cyclo-oligomerization. Solid-state structural analysis and binding studies of cyclic trisaccharide in solution by isothermal titration calorimetry were also conducted. Cyclic trisaccharide possessed a bowl shape and brick-wall type of arrangement in the solid-state structure, whereas it exhibited stronger binding affinity towards 1-aminoadamantane as compared to β-cyclodextrin in aqueous solution. Overall, the results presented in the thesis provide a possibility to develop new types of synthetic glycolipids that can act as inhibitors of biofilm formation of mycobacteria, as well as, to develop newer types of cyclic oligosaccharide synthetic hosts that can modify binding abilities towards various guest compounds.

No description available.

There is a growing attention in the synthesis of fructo-oligosaccharides (FOS) due to their excellent bio-functional and health-promoting properties. The current production processes are limited to chemical hydrolysis reactions of plant extracts, which are often associated with several drawbacks. In this study, fructosyltransferase (FTase) and polygalacturonase (PGase) activities, present in a commercial enzyme preparation (Pectinex® Ultra SP-L) sourced from Aspergillus aculeatus, have been separated and fully purified by anion-exchange and sizeexclusion chromatography. The FTase possesses fructosyl transfer activity for FOS synthesis and the PGase has pectin hydrolytic activity. Fructosyltransferase is a single-band protein with a molecular weight of 85 kDa, whereas PGase is a distinct protein of 40 kDa. The temperature and pH optima of FTase were 60 ºC and 6.0, with a half-life of 8 h; while that for PGase were 40 ºC and 6.0, respectively. FTase was slightly inhibited in the presence of Ni²⁺, Mg²⁺ and urea; but PGase was more susceptible to divalent ions such as Ca²⁺, Mg²⁺ and Mn²⁺. The kinetic parameters (Km and Vmax) of FTase for the hydrolysis of β-(2→1) linkages from sucrose were 752.3 mM and 120.5 μmol.min⁻¹.mL⁻¹, respectively; whereas the same parameters for pectin hydrolysis by PGase were 13.0 mg.mL⁻¹ and 263 μmol.min-1.mL⁻¹, respectively. The purified FTase was able to transfer fructosyl residues from sucrose, synthesizing the corresponding chains of FOS. PGase was relatively stable at 40 ºC (t½ > 3 h), depolymerizing the pectin backbone while releasing the inulins from within the chicory roots. Analysis of various mixtures of FOS by mass spectrometry, HPLC and ¹H-NMR was undertaken. Results indicated that MS with electrospray ionization and ¹H-NMR are capable of providing relative quantitative data of the FOS present in the mixtures. The pharmaceutical effects of various sc-FOS (0.5%, v/v) and SCFA (0.3%, v/v) on certain bacterial enzymes (β-glucuronidase, urease and β-glucosidase) associated with the formation of carcinogens were also studied. These enzyme activities were not directly influenced by the sc-FOS, but were found to be remarkably decreased by SCFA, pointing toward the prebiotic effect of FOS in intestinal microflora modulation.

Orientador: Helena Maria André Bolini / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-22T07:57:23Z (GMT). No. of bitstreams: 1 Ferraz_JulianaPereira_M.pdf: 2510030 bytes, checksum: a28d462816bf1afe8b5665a26f281aea (MD5) Previous issue date: 2013 / Resumo: O sorvete é um alimento altamente consumido no mundo e a cada ano nota - se que o consumo no Brasil cresce cada vez mais iniciando um processo de transição de um alimento sazonal para um alimento consumido durante o ano todo. Este produto possui uma matriz energética e nutricional altamente complexa, pois trata - se de um produto que contém em sua formulação proteínas, gorduras, açucares, ar e minerais. O FOS atuou agregando um valor funcional ao produto juntamente com os edulcorantes que tiveram a finalidade de reduzir o valor calórico do produto tornando - o mais saudável e permitindo que portadores de doenças crônicas como os diabéticos e obesos possam consumi - lo. Este trabalho teve como objetivo realizar análises a fim de se desenvolver um sorvete de creme de acordo com as preferências dos consumidores, foi avaliado a partir da análise descritiva quantitativa, análise múltipla de tempo intensidade, análise de aceitação e análises físico - químicas, sendo estas overrun, velocidade de derretimento, pH, textura e cor, para que as características do sorvete de creme fossem preservadas com a adição do prebiótico e dos edulcorantes. Os dados obtidos foram submetidos à análise de variância (ANOVA) e testes de médias de Tukey, utilizando o programa estatístico SAS. Com os dados de aceitação foi construído o Mapa de Preferência Interno através do programa XLStat. A análise de aceitação mostrou que todas as amostras foram bem aceitas pelos consumidores, exceto a amostra com estévia em sua formulação que recebeu notas inferiores a seis. Na ADQ® as amostras foram diferenciadas pelo gosto amargo, doce residual e gosto doce devido à presença dos edulcorantes na formulação. Na análise Múltipla de Tempo Intensidade foi possível verificar as semelhanças das amostras em relação à intensidade de dulçor e amargor diferenciando - se somente a amostra com estévia que obteve intensidades altas tanto para amargor quanto para dulçor. Podendo - se concluir que a adição do FOS e dos edulcorantes, aspartame, sucralose, neotame e ciclamato/ sacarina na proporção de 2:1 no sorvete de creme não afetou a aceitação das amostras perante aos consumidores, já a amostra com FOS e estévia em sua formulação foi rejeitada pelos consumidores, estes resultados auxilia a indústria de sorvetes, que poderá desenvolver um sorvete com apelo funcional e redução calórica sem influenciar nas características sensoriais e físico - químicas do sorvete de creme atingindo um novo público consumidor / Abstract: The ice cream is a highly consumed aliment in all around the world and year by year we can notice that brazilian ice cream consumption increases starting a transition process of a seasonal food to a food that is consumed throughout the year. This product has an energetic and nutritional load that is highly complex, because it formulation contains proteins, fats, sugars, minerals and air. The FOS had operated adding a functional value to the product with the sweeteners that had the finality or reduce the product's calories that becomes more healthy and allowing that patients with chronic diseases, like diabetes, can consume the product. This research had as purpose perform analysis in order to develop a product according to the consumers preferences, it had been valued from qualitative descriptive analysis, multiple time-intensity analysis, acceptance analysis and physical - chemical, these being overrun, melting rate, pH, texture and color, in order that the vanilla ice cream characteristics were still the same with the addition of the prebiotic and sweetener. The data were submitted for analysis of variance (ANOVA) and measurements Tukey tests using static programs. The acceptance analysis showed that all the samples were well accepted except the sample with estevia in its formula. In ADQ the samples were differentiated by the bitter or sweet taste. In the analyses multiple of time intensity was possible verifying the sample similarities in relation to sweetness and bitterness. We concluded that the addition of FOS, sweeteners, aspartame, sucralose, neotame and cyclamate / saccharin in a 2:1 ratio in the ice cream did not affect the acceptance of samples before consumers, on the other hand the sample with estevia was rejected by the consumers, this results helps the ice cream industry, wich may develop an ice cream with functional appeal and caloric reduction without affecting the sensory characteristics and physical - chemical ice cream reaching a new consumer audience / Mestrado / Consumo e Qualidade de Alimentos / Mestra em Alimentos e Nutrição

The impact of yeasts on food production, quality and safety is closely linked with their ecology and biological activities. Recently, as a consequence of the relationship between diet and health, yeasts are becoming relevant as new probiotics or for the production of bioactive compounds. In dairy products, yeasts play a key role in proteolysis, lipolysis and lactose fermentation during cheese ripening, promoting the development of sensory properties, particularly aroma. This thesis focuses on the yeast diversity in artisanal cheeses produced in the Natural Park Serra d¿Espadà (Castelló) from ewes¿ and goats¿ raw milk. Different molecular techniques have been employed in order to characterize yeast isolates. Moreover, the succession of species along the cheese ripening process was studied. The intraspecific variability of the most abundant identified species Debaryomyces hansenii and Kluyveromyces lactis was also assessed. Additionally, the potential of Kluyveromyces marxianus and K. lactis ß-galactosidases to synthetize prebiotic oligosaccharides from lactose and lactulose was tested. Finally, Kluyveromyces and Debaryomyces isolates were investigated for the production of cheese aromatic compounds. / Padilla López, B. (2014). Yeast diversity in artisanal cheeses: biotechnological applications [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/36065 / TESIS

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Presented diploma thesis was focused on study of biologically active compounds, especially some sugars in cereal products. In experimental part the total of 29 different cereal materials, food industry waste products and natural complex matrixes (microbial polysaccharide, honey, beer) were used. As a part of this work optimization of analytical methods suitable for analysis of simple sugars, oligo- and polysaccharides was done. In cereal samples reduced and neutral sugars were analyzed spectrophotometrically and individual sugars were detected by chromatography. For HPLC/RI analysis optimal mobile phase composition and chromatography conditions were proposed. For mono- a oligosaccharides C18-NH2 sorbent, mixture acetonitril:water 75:25 as mobile phase and flow 1 ml/min were verified as suitable separation parameters. Thin layer chromatography of mono- and oligosaccharides was optimized too. Introduced chromatography and spectrophotometry methods were then applied to analysis of cereal samples. As a model sugar natural microbial polysaccharide pullulan was used to analytical method testing. In cereal products and food matrixes total neutral and reduced sugars as well as products of their acid and enzyme hydrolysis were measured. Detailed analysis of some glycoside composition was tested too. As the most usable method for both qualitative and quantitative analysis of cereal sugars HPLC/RI method was found. To detailed identification of malto-oligosacharides tandem LC/MS/MS technique using derivatization by 1-phenyl-3-methyl-5-pyrazolon was tested too.