Thesis (Ph. D.)--University of California, Riverside, 2009. / Includes abstract. Available via ProQuest Digital Dissertations. Title from first page of PDF file (viewed March 10, 2010). Includes bibliographical references. Also issued in print.

Thesis (PH. D.)--Michigan State University. Food Science, 2008. / Title from PDF t.p. (viewed on Sept. 2, 2009) Includes bibliographical references (p. 181-200). Also issued in print.

No description available.

Glucansucrases são enzimas que atuam em reações de síntese de polissacarídeos e oligossacarídeos. Para que esses biocatalisadores sejam aplicados em escala industrial, é desejável ótimas estabilidades térmica e operacional, o que pode ser alcançado com a imobilização de enzimas. Como alternativa aos suportes sólidos amplamente estudados, está a quitosana, polímero que não apresenta toxicidade e possui alta biocompatibilidade e alta afinidade com proteínas. Outra possibilidade promissora na imobilização de enzimas, é a síntese dos agregados enzimáticos entrecruzados (CLEAs), os quais apresentam alta atividade catalítica e alta estabilidade. Contudo, uma peculiaridade das glucansucrases quando produzidas em meio contendo sacarose é a camada de polímero que as envolve, e que bloqueia o acesso aos grupos reativos na superfície da proteína. No caso da expressão heteróloga das glucansucrases em Escherichia coli essa dificuldade pode ser contornada. Além disso, o uso da mutagênese sítio-dirigida pode proporcionar modificações de aminoácidos na superfície da enzima, tais como os resíduos Lys, Cys, His, com o intuito de que melhorias na imobilização sejam alcançadas. Sendo assim, na primeira etapa desse trabalho, uma extensa discussão é apresentada em relação às metodologias de imobilização de dextransucrase encontradas na literatura. A seguir, estudos referentes à imobilização da dextransucrase de Leuconostoc mesenteroides B-512 F em esferas de quitosana ativadas com glutaraldeído foram realizados. Esse imobilizado apresentou alta atividade catalítica (197 U/g) quando utilizada a carga de proteína de 400 mg/g de suporte. Além disso, observou-se que a imobilização covalente e os açúcares maltose e glicose promoveram proteção à enzima em temperaturas de 40 ºC e 50 ºC. Na etapa seguinte, a produção e a caracterização de CLEAs de dextransucrase de L. mesenteroides B-512 F foram investigados. Demonstrou-se que o tratamento com a dextranase foi essencial para a imobilização da glucansucrase e que o isopropanol foi o melhor agente precipitante. Os CLEAs apresentaram pH e temperatura ótimos de 3,0 e 60 ºC, respectivamente, enquanto que a dextransucrase imobilizada nas esferas de quitosana funcionalizada com glutaraldeído apresentaram os valores de 4,5 e 20 ºC. Ambas formas imobilizadas apresentaram boa estabilidade operacional na síntese de oligossacarídeos uma vez que após 10 ciclos, 40 % de atividade residual foi observada. Por fim, estão apresentados estudos sobre a modelagem das estruturas tridimensionais e a mutagênese sítio-dirigida das glucansucrases DSR-S vardel Δ4N and ASR C-APY del. Os modelos preditos demonstraram boa qualidade e a mutagênese sítio-dirigida não promoveu perdas significativas na atividade enzimática dos mutantes. Somente o mutante DSR_S326C mostrouse inativo. Os resultados obtidos sugerem que a imobilização da dextransucrase foi satisfatória e que cada técnica possibilita diferentes características ao imobilizado. Além disso, os imobilizados foram adequados para síntese de dextrana e oligossacarídeos. / Glucansucrases are enzymes that catalyze the synthesis of polysaccharides and oligosaccharides. In order to assure continuous processing and reuse of the biocatalyst in industrial applications, enzyme immobilization techniques are required to promote good thermal and operational stabilities. Among the several solid supports for enzyme immobilization, chitosan shows interesting properties because it is non-toxic, it is biocompatible, and it has high protein affinity. Other possibility is the production of cross-linked enzyme aggregates (CLEAs), which presents high catalytic activity and good stability. However, glucansucrases have a particularity when produced in sucrose medium, since a polymer layer surrounds the protein, blocking the access to reactive groups on the enzyme surface. To overcome this problem, it is possible to make the heterologous production of glucansucrases in Escherichia coli. Likewise, the site-directed mutagenesis may promote changes in the amino acids located on the surface to improve immobilization parameters. Therefore, this work aimed to discuss the several techniques applied for dextransucrase immobilization, and to design new immobilized biocatalysts. In a first step, it is presented a review about the distinct immobilization methodologies for dextransucrase. In a second study, an investigation about dextransucrase from Leuconostoc mesenteroides B-512 F immobilized on glutaraldehyde-activated chitosan particles was carried out. The novel immobilized biocatalyst showed 197 U/g (400 mg/g dried support) of catalytic activity. The covalent immobilization promoted protection against enzyme damages at 40 ºC and 50 ºC, whereas maltose and glucose acted as stabilizers. Furthermore, it was studied the production and characterization of CLEAs dextransucrase from L. mesenteroides B-512 F. It was demonstrated that dextranase treatment was crucial for immobilization. Isopropanol was chosen as the best precipitant agent. CLEAs presented optimal pH and temperature of 3.0 and 60 ºC, respectively, whereas it was found values of 4.5 e 20 ºC for dextransucrase immobilized on glutaraldehyde-activated chitosan particles. Both immobilized biocatalysts showed good operational stability in the oligosaccharides synthesis, exhibiting 40 % of residual activity after 10 cycles. Finally, the study concerning the homology modeling and site-directed mutagenesis of glucansucrases DSR-S vardel Δ4N and ASR C-APY del is presented. The predicted models showed good quality and it has been demonstrated that the site-directed mutagenesis did not promote significant losses in the variant enzyme activities. Only one mutant (DSR_S326C) had shown no dextransucrase activity. The results obtained in this work suggest that the immobilization of dextransucrase was satisfactory, also showing that each technique promotes different characteristics to the immobilized biocatalyst. Besides, these immobilized enzymes were feasible for the synthesis of dextran and oligosaccharides.

Glucansucrases são enzimas que atuam em reações de síntese de polissacarídeos e oligossacarídeos. Para que esses biocatalisadores sejam aplicados em escala industrial, é desejável ótimas estabilidades térmica e operacional, o que pode ser alcançado com a imobilização de enzimas. Como alternativa aos suportes sólidos amplamente estudados, está a quitosana, polímero que não apresenta toxicidade e possui alta biocompatibilidade e alta afinidade com proteínas. Outra possibilidade promissora na imobilização de enzimas, é a síntese dos agregados enzimáticos entrecruzados (CLEAs), os quais apresentam alta atividade catalítica e alta estabilidade. Contudo, uma peculiaridade das glucansucrases quando produzidas em meio contendo sacarose é a camada de polímero que as envolve, e que bloqueia o acesso aos grupos reativos na superfície da proteína. No caso da expressão heteróloga das glucansucrases em Escherichia coli essa dificuldade pode ser contornada. Além disso, o uso da mutagênese sítio-dirigida pode proporcionar modificações de aminoácidos na superfície da enzima, tais como os resíduos Lys, Cys, His, com o intuito de que melhorias na imobilização sejam alcançadas. Sendo assim, na primeira etapa desse trabalho, uma extensa discussão é apresentada em relação às metodologias de imobilização de dextransucrase encontradas na literatura. A seguir, estudos referentes à imobilização da dextransucrase de Leuconostoc mesenteroides B-512 F em esferas de quitosana ativadas com glutaraldeído foram realizados. Esse imobilizado apresentou alta atividade catalítica (197 U/g) quando utilizada a carga de proteína de 400 mg/g de suporte. Além disso, observou-se que a imobilização covalente e os açúcares maltose e glicose promoveram proteção à enzima em temperaturas de 40 ºC e 50 ºC. Na etapa seguinte, a produção e a caracterização de CLEAs de dextransucrase de L. mesenteroides B-512 F foram investigados. Demonstrou-se que o tratamento com a dextranase foi essencial para a imobilização da glucansucrase e que o isopropanol foi o melhor agente precipitante. Os CLEAs apresentaram pH e temperatura ótimos de 3,0 e 60 ºC, respectivamente, enquanto que a dextransucrase imobilizada nas esferas de quitosana funcionalizada com glutaraldeído apresentaram os valores de 4,5 e 20 ºC. Ambas formas imobilizadas apresentaram boa estabilidade operacional na síntese de oligossacarídeos uma vez que após 10 ciclos, 40 % de atividade residual foi observada. Por fim, estão apresentados estudos sobre a modelagem das estruturas tridimensionais e a mutagênese sítio-dirigida das glucansucrases DSR-S vardel Δ4N and ASR C-APY del. Os modelos preditos demonstraram boa qualidade e a mutagênese sítio-dirigida não promoveu perdas significativas na atividade enzimática dos mutantes. Somente o mutante DSR_S326C mostrouse inativo. Os resultados obtidos sugerem que a imobilização da dextransucrase foi satisfatória e que cada técnica possibilita diferentes características ao imobilizado. Além disso, os imobilizados foram adequados para síntese de dextrana e oligossacarídeos. / Glucansucrases are enzymes that catalyze the synthesis of polysaccharides and oligosaccharides. In order to assure continuous processing and reuse of the biocatalyst in industrial applications, enzyme immobilization techniques are required to promote good thermal and operational stabilities. Among the several solid supports for enzyme immobilization, chitosan shows interesting properties because it is non-toxic, it is biocompatible, and it has high protein affinity. Other possibility is the production of cross-linked enzyme aggregates (CLEAs), which presents high catalytic activity and good stability. However, glucansucrases have a particularity when produced in sucrose medium, since a polymer layer surrounds the protein, blocking the access to reactive groups on the enzyme surface. To overcome this problem, it is possible to make the heterologous production of glucansucrases in Escherichia coli. Likewise, the site-directed mutagenesis may promote changes in the amino acids located on the surface to improve immobilization parameters. Therefore, this work aimed to discuss the several techniques applied for dextransucrase immobilization, and to design new immobilized biocatalysts. In a first step, it is presented a review about the distinct immobilization methodologies for dextransucrase. In a second study, an investigation about dextransucrase from Leuconostoc mesenteroides B-512 F immobilized on glutaraldehyde-activated chitosan particles was carried out. The novel immobilized biocatalyst showed 197 U/g (400 mg/g dried support) of catalytic activity. The covalent immobilization promoted protection against enzyme damages at 40 ºC and 50 ºC, whereas maltose and glucose acted as stabilizers. Furthermore, it was studied the production and characterization of CLEAs dextransucrase from L. mesenteroides B-512 F. It was demonstrated that dextranase treatment was crucial for immobilization. Isopropanol was chosen as the best precipitant agent. CLEAs presented optimal pH and temperature of 3.0 and 60 ºC, respectively, whereas it was found values of 4.5 e 20 ºC for dextransucrase immobilized on glutaraldehyde-activated chitosan particles. Both immobilized biocatalysts showed good operational stability in the oligosaccharides synthesis, exhibiting 40 % of residual activity after 10 cycles. Finally, the study concerning the homology modeling and site-directed mutagenesis of glucansucrases DSR-S vardel Δ4N and ASR C-APY del is presented. The predicted models showed good quality and it has been demonstrated that the site-directed mutagenesis did not promote significant losses in the variant enzyme activities. Only one mutant (DSR_S326C) had shown no dextransucrase activity. The results obtained in this work suggest that the immobilization of dextransucrase was satisfactory, also showing that each technique promotes different characteristics to the immobilized biocatalyst. Besides, these immobilized enzymes were feasible for the synthesis of dextran and oligosaccharides.

Made available in DSpace on 2016-03-07T19:20:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-10-15. Added 1 bitstream(s) on 2016-03-07T19:24:20Z : No. of bitstreams: 1 000858042.pdf: 2321453 bytes, checksum: d56468b7b1d5643182c61f38e1a2372d (MD5) / Com o atual cenário de mudanças climáticas, observa-se a tendência a eventos de seca mais longos e recorrentes, desta forma a obtenção de plantas mais tolerantes à seca figura como um dos principais investimentos dentro da ciência e tecnologia nacional. Os oligossacarídeos da família das rafinoses (RFOs) desempenham múltiplas funções nas plantas e sabe-se que estes são acumulados nos tecidos vegetais em situações de déficit hídrico, garantindo a estabilidade das membranas celulares, consequentemente mantendo as funções vitais da planta. Por sua vez, a enzima galactinol sintase (GolS, EC 2.4.1.123), catalisa o primeiro passo na biossíntese dos oligossacarídeos dos RFOs desempenhando um importante papel regulador na partição do carbono entre sacarose e RFOs. Desta forma, o objetivo do presente trabalho foi introduzir em soja, via Agrobacterium tumefaciens, a construção gênica 35S:AtGolS2 e caracterizar molecularmente e fisiologicamente os eventos obtidos sob déficit hídrico. Para o processo de transformação, a cultivar convencional de soja BRS 184 foi utilizada e os eventos obtidos foram caracterizados quanto ao número de cópias através da técnica de qPCR. Para a análise da expressão gênica constitutiva o RNA total dos eventos, em condições bem irrigadas, foi extraído e a expressão determinada via RT-qPCR. A taxa de segregação foi calculada através do teste do X2 (p≤ 0.05). Com base nos resultados obtidos, dois eventos (2Ia1 e 2Ia4) foram selecionados para serem analisados quanto a respostas moleculares e fisiológicas sob déficit hídrico induzido em condições de casa de vegetação. Os resultados mostraram que nas plantas do evento 2Ia4 o maior acumulo de água associado a menor área foliar na condição controle levou a manutenção das trocas gasosas causado pela redução na transpiração foliar, maior acúmulo de água no substrato e acúmulo de transcritos de rafinose e galactinol nos tecidos / With the current scenario of climate change, there is a tendency to longer and recurrent drought events, thus obtaining more drought tolerant plants figure as a major investment in the national science and technology. Raffinose family oligosaccharides (RFOs) plays multiple functions in plants and it is known that these are accumulated in plant tissues in water deficit situations, guaranteeing the stability of cell membranes, thus maintaining the vital functions of the plant. In turn, galactinol synthase (GolS, EC 2.4.1.123) catalyzes the first step in the biosynthesis of RFOs plays an important regulatory role in carbon partitioning between sucrose and orphans. Thus, our objective was to introduce gene construction 35S:AtGolS2 via Agrobacterium tumefaciens in soybean plants and characterize molecularly and physiologically events obtained under water deficit. In this context, the conventional soybean BRS 184 was used in the transformation process and the soybean events were molecularly characterized in regard to the transgene copy number by qPCR technique. For the analysis of constitutive gene expression total RNA of events, well-watered conditions, was extracted and the expression determined by RT-qPCR. The segregation rate was calculated using the X2 test (p ≤ 0.05). Based on our results, two events (2Ia1 and 2Ia4) were selected to be analyzed for physiological responses under drought simulated under greenhouse conditions. The results showed that the plants 2Ia4 event the largest accumulation of water associated with lower leaf area in the control condition led to maintenance of gas exchange caused by the reduction in leaf transpiration, increased water accumulation in the substrate and accumulation of raffinose and galactinol transcripts in tissues. Thus, the increased levels of these carbohydrates would have made these act as osmoprotectors, enabling the recommendation of 2Ia4 plants breeding programs aimed at tolerance to drought

Orientadores: Gláucia Maria Pastore, Rosângela dos Santos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-21T19:39:00Z (GMT). No. of bitstreams: 1 Adami_AngelicaAparecidaVieira_M.pdf: 12653852 bytes, checksum: a5b3e75b4c45344be161ab51a7df7f0a (MD5) Previous issue date: 2013 / Resumo: Estudos sao realizados com oligossacarideos, probioticos e a combinação deles; sao varios os fatores que envolvem os beneficios que estes em combinacao podem agregar para a saude dos consumidores. Esta pesquisa teve como objetivo avaliar o comportamento simbiotico in vitro das linhagens probioticas Bifidobacterium animalis (Bb-12) e Lactobacillus acidophillus (LA-05), em substratos enriquecidos com oligossacarideos, ou seja, avaliar se esses potencializavam a capacidade probiotica das linhagens estudadas. Os oligossacarideos utilizados no estudo foram o galactooligossacarideo (GOS) sintetizado pela ?-galactosidase de Scopulariops sp., frutooligossacarideo (FOSOrafti) e extrato bruto de Yacon. Avaliou-se o efeito bifidogenico de diferentes concentracoes de GOS, FOS e Yacon utilizando as culturas probioticas e sua capacidade de acidificacao do meio. Foi avaliado o perfil hidrofobico e acido da membrana celular dos probioticos usando meio enriquecido com GOS, FOS e controle meio MRS (Man Rogosa e Sharpe) sem fonte de dextrose; pelo método MATH (aderencia microbiana de hidrocarbonetos). Realizou-se tambem a producao, extracao e quantificacao de exopolissacarideos (EPS) por Lactobacillus acidophillus (LA-05) em meios enriquecidos com FOS e GOS, a quantificacao foi pelo metodo fenol-sulfurico seguida de leitura de absorbancia a 490nm, os resultados foram submetidos a uma curva padrao de glicose. Verificou-se ainda o antagonismo das linhagens probioticas cultivadas em meios suplementados com GOS, FOS, Yacon, MRS sem fonte de dextrose e controle MRS adicionado de NaOH, sobre linhagens patogenicas; o antagonismo foi avaliado atraves do metodo de difusao em agar. Avaliou tambem a capacidade de producao de acido latico. Na avaliacao bifidogenica os resultados revelaram que os substratos estimularam o metabolismo dos probioticos estudados. A melhor atividade para as linhagem de Lactobacillus acidophillus (LA-05) foi com o substrato GOS com atividade de 11,56 LogUFC (na concentracao de 200mg de GOS, em 12 horas de incubacao), e para e Bifidobacterium animalis (Bb12) foi com o substrato FOS com atividade de 11,3 LogUFC (na concentracao de 100mg FOS, em 12 horas de incubacao), na medida que se aumentou a concentracao dos substratos, houve a reducao da atividade bifidogenica. Os resultados revelaram que os prebioticos GOS e FOS estimularam o perfil de adesao e acidez da membrana celular das linhagens probioticas estudadas, quando comparado com o controle meio MRS sem fonte de dextrose. Quanto a producao de EPS, o maior valor (13,53 \g.mL) foi obtido apos 48 horas de cultivo em caldo MRS suplementado com GOS (25% v/v), enquanto que o FOS (25% v/v) tambem estimulou a producao (7,68 \g.mL) em 12 horas, seguida da queda de producao apos este periodo. Verificou-se que os oligossacarideos estimularam a acao antagonica dos probioticos sobre os micro-organismos patogenicos, e que esta inibicao do crescimento pode estar ligada com a producao de acidos pelos probioticos em conjunto com os oligossacarideos. Em sintese, os resultados revelaram que os oligossacarideos estimularam o potencial probiotico das linhagens estudadas / Abstract: Several studies have been conducted with oligosaccharides in general and also with probiotics, or combination of them, due to factors involving the combined benefits they can add to the health of consumers. This research aimed to evaluate in vitro the behavior of symbiotic probiotic strains Bifidobacterium animalis (Bb-12) and Lactobacillus acidophilus (LA-05), on substrates enriched with oligosaccharides in the case to assess whether the oligosaccharides intensified the ability of probiotic strains studied. The oligosaccharides used in this study were synthesized by the galactooligosaccharide ?-galactosidase Scopulariops sp., fructooligosaccharide and crude extract of yacon. We evaluated the bifidogenic effect of different concentrations of GOS, FOS and Yacon using probiotic cultures and their ability to acidification of the medium. Was evaluated the profile acid and hydrophobic cellular membrane of probiotics using medium supplemented with GOS, FOS and control MRS medium without added dextrose; MATH method (microbial adherence to hydrocarbons). Was also conducted production, extraction and quantification of exopolysaccharides (EPS) by Lactobacillus acidophilus (LA-05) media supplemented with FOS and GOS, quantification was by the phenolsulfuric method followed by absorbance reading at 490nm, the results were subjected to a standard curve of glucose. It was further antagonism of probiotic strains grown in media supplemented with GOS, FOS, yacon, MRS without dextrose and source control MRS added NaOH on pathogenic strains; antagonism was assessed by the agar diffusion method; also evaluated the ability to produce lactic acid. In evaluating the results revealed that bifidogenic substrates stimulated metabolism studied probiotics, the best activity for the strain of Lactobacillus acidophilus (LA-05) was the substrate with GOS activity of 11.56 LogUFC (200mg in 12 hours incubation), and for and Bifidobacterium animalis (Bb-12) was with the FOS substrate with activity of 11.3 LogUFC (100mg in 12 hours incubation), as it increased the concentration of the substrates decreased the activity of microorganisms. The results revealed that prebiotic GOS and FOS stimulated acid profile of adhesion and cell membrane of probiotic strains studied, as compared to the MRS control medium without added dextrose. As for the highest production of EPS production (13.53 ?g.ml) was after 48 hours of culture in MRS broth supplemented with GOS, while also stimulated FOS production (7.68 ?g.ml) for 12 hours followed by production decrease thereafter. It was found that the oligosaccharides stimulated antagonistic action of probiotics on pathogenic microorganisms and that this growth inhibition is connected with acid production by probiotics together with the oligosaccharides. In summary, the results revealed that the oligosaccharides stimulated the potential probiotic strains studied / Mestrado / Ciência de Alimentos / Mestra em Ciência de Alimentos

This research examined a single bulb-bearing edible plant species, yellow glacier lily (Erythronium grandiflorum ). Three main approaches to the research were taken: 1) an ecological study, to determine the general habitat requirements of the species in western Canada, and to investigate the nature of vegetative reproduction in the species; 2) an ethnobotanical study, consisting of an extensive literature search for all recorded First Nations' uses of the species (in Canada and elsewhere), in addition to interviews with contemporary Interior Salish elders; 3) a nutritional study, examining in detail the nutritional characteristics of the bulbs, and particularly changes in the carbohydrate content over the course of the growing season and with different types of treatments. The ecological data indicate that E. grandiflorum is more abundant in meadow environments or sites with deciduous cover than in sites with coniferous forest cover. Flowering plants tended to be more abundant and robust at low elevation meadows, while seedlings and juveniles were disproportionately represented at high elevation meadows. Decreased juvenile success in the low-elevation meadows may be related to relatively high litter from shrubs and grasses. Experimental data indicate that appendages on the bulbs, which persist as remnants of previous years' bulbs, can act as vegetative propagules if mechanically separated. In addition, both bulbs and appendages were successfully transplanted over a two-year period from a subalpine meadow to a very different habitat type, 1500 m lower in elevation. The ethnobotanical review confirms that the species was traditionally a highly significant root resource for northern plateau peoples, particularly the Secwepemc and Nlaka'pamux peoples, for probably thou.sands of years. These peoples collected, stored, and traded large quantities of the bulbs, and the traditional processing strategies generally included drying and pit-cooking. People developed a detailed ecological understanding of the species, and practiced active resource management strategies. Nutritional results indicated a carbohydrate-rich food resource, with the main storage carbohydrate consisting of starch (not inulin or other fructan) through most of the growing season. There are significant quantities of sugars (including fructo-oligosaccharides) present at the beginning of the growing season, but starch increases rapidly and peaks (along with overall food value) in the early (green) fruit stage of growth. For bulbs at the fruiting stage, drying markedly increases sugars in the bulbs relative to starch, while pit-cooking the dried bulbs does not have significant effects on relative amounts of carbohydrates. However, pit-cooking has important qualitative effects on the appearance, taste, and possibly storage properties of the bulbs, as well as representing an efficient processing strategy. I argue that traditional harvesting and management strategies practiced by First Nations people (including tilling, thinning, replanting of appendages, and landscape burning) mean that the ecology and ethnobotany of the species cannot be considered in isolation. Based on previous ecological and ethnoecological work on this and similar species, it seems likely that yellow glacier lily is adapted to a periodic, moderate disturbance regime, which traditional practices may have mimicked or enhanced. / Graduate

No description available.

No description available.