Orientador: Rosiane Lopes da Cunha / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-05T21:42:51Z (GMT). No. of bitstreams: 1 Braga_AnaLuizaMattos_D.pdf: 3362536 bytes, checksum: 58d82bf888ded45f62114695be4bbf6e (MD5) Previous issue date: 2006 / Resumo: A utilização de diversos biopolímeros é uma prática comum nas indústrias de alimentos, especialmente em produtos lácticos, embutidos e a base de soja. O objetivo geral deste trabalho foi estudar as interações entre proteínas e polissacarídeos em sistemas contendo, ou não, co-soluto, em solução aquosa (pH 7,0) ou géis acidificados. Dentre as proteínas e os polissacarídeos utilizados na indústria de alimentos, foram estudados mais profundamente a goma xantana, o caseinato de sódio e o isolado protéico de soja (tanto em solução quanto em géis). Os polissacarídeos jataí (LBG), gelana, Na-alginato e k-carragena foram estudados apenas em soluções. As interações entre os componentes foram estudadas 1) por microscopias confocal, de força atômica e de contraste de fases e rheo-SALS (¿small angle light scattering¿); 2) por reologia a baixas e altas deformações, em cisalhamento ou compressão; 3) por análises químicas para determinar o tipo de força das interações. Os estudos sobre as propriedades físicas de soluções puras de biopolímeros mostraram que soluções de proteína seguem um comportamento Newtoniano, enquanto que os polissacarídeos apresentam comportamento Newtoniano a baixa concentração e pseudoplástico com o aumento desta. O tratamento térmico de soluções de xantana bem como a adição de sacarose reduziram a elasticidade da solução, sendo que a adição de sacarose afetou as propriedades reológicas apenas de soluções anisotrópicas ou bifásicas (anisotropia e isotropia simultâneas). Um novo modelo baseado na equação de BST (Blatz, Sharda, Tschoegl, 1974) foi proposto para predizer um maior número de propriedades mecânicas dos géis biopoliméricos tendo sido observado um bom ajuste dos dados. Quanto mais lenta a acidificação promovida por GDL, mais interconectada e forte foi a rede protéica em géis puros de Na-caseinato e SPI. O aumento da tensão e deformação de ruptura de géis formados com GDL foi obtida pelo aumento da concentração de proteína em géis puros de SPI, pela adição de xantana ou pela redução do conteúdo de proteína em géis contendo xantana. Por outro lado, a adição de xantana enfraqueceu os géis térmicos de SPI, provavelmente devido a ligação deste polissacarídeo com a sub-unidade b-7S. Em soluções aquosas pH 7,0, a k-carragena foi mais compatível com o Na-caseinato e com SPI do que o Na-alginato. No entanto, os resultados de ¿rheo-SALS¿ e CLSM mostraram que misturas de caseinato e alginato apresentaram maior capacidade de formar emulsões do tipo água-água do que sistemas com carragena. Além disto, o sistema com SPI e Na-alginato também formou emulsão, mas com a fase dispersa de proteína gelificada. Misturas com gelana apresentaram-se homogêneas devido a baixa tensão interfacial do sistema. Palavras-chave: proteína, polissacarídeo, interação, gel, emulsão, biopolímero, solução, reologia, separação de fases, CLSM, ¿rheo-SALS¿ / Abstract: Biopolymers are widely used in dairy products, canned foods, bakery products, salad dressings, beverages, sauces, soups and other processed foodstuffs to improve textural characteristics, flavour and shelf life. The aim of the present Ph.D. thesis was to study the protein-polysaccharide interactions in aqueous systems (pH 7.0) or acidified gels. It was studied seven different biopolymers used in the food industry, being two proteins and five polysaccharides. Soy protein isolate (SPI), Na-caseinate and xanthan were studied in aqueous solutions and gels systems, while locust bean gum (LBG), gellan, Na-alginate and k-carrageenan were studied only in aqueous solutions. The protein-polysaccharide interactions were evaluated as following: 1) microstructures ¿ by using confocal (CLSM), atomic force and phase contrast microscopes and rheo-SALS (small angle light scattering); 2) macrostructures, evaluated trough small- and large-strain rheology under shear or compression; 3) chemical analysis - in order to determine the kind of interaction forces that maintained the gels structure. The results on the physical properties of pure biopolymers solutions revealed that the proteins showed Newtonian behaviour, while the polysaccharides showed either Newtonian or non-Newtonian behaviour depending on its type and solution concentration. The annealing temperature of xanthan solutions and the addition of sucrose caused a reduction on the solution elasticity. It was observed that sucrose affected the rheological properties over all frequency range studied for initially anisotropic or biphasic xanthan solutions. It was proposed a new model based on BST (Blatz, Sharda, Tschoegl, 1974) equation to predict a great number of mechanical properties, which revealed a very good fit to the data. The slower the acidification by GDL (glucone-delta-lactone) it was observed a more interconnected and harder network in Na caseinate and SPI pure gels. The increase of the breaking stress or strain was obtained by increasing the SPI concentration in pure gels, by adding xanthan or by increasing the protein concentration in gels made with xanthan. In another hand, the addtion of xanthan let to weaker heat-induced SPI gels, which was attributed to the electrostatic interactions between xanthan and the b-7S subunit of SPI. In mixed protein-polysaccharide solutions, the results indicated that k-carrageenan was more compatible with both soy protein and Nacaseinate than alginate. However, the Na-caseinate ¿ Na-alginate mixture showed a greater capacity to form water-in-water emulsions than Na-caseinate - carrageenan system as observed by rheo-SALS and CLSM. In addition, the system with SPI and Na-alginate also formed emulsions but with a gelified protein dispersed phase. The interfacial tension of systems containing gellan was very low resulting in homogeneous mixtures. Keywords: proteins, polysaccharides, interaction, gel, emulsion, biopolymer solution, rheology, phase separation, CLSM, rheo-SALS / Doutorado / Doutor em Engenharia de Alimentos

Orientador: Carlos Eduardo Pinheiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-07-22T12:43:12Z (GMT). No. of bitstreams: 1 Silva_AlmenaradeSouzaFonseca_M.pdf: 2997804 bytes, checksum: a38c6684f77243ec7d052c713f98685d (MD5) Previous issue date: 1997 / Resumo: Este trabalho teve como objetivo verificar a influência de carboidratos freqüentemente consumidos na dieta, como glicose, sacarose, frutose e dextrina, na fermentação e na síntese de polis sacarídeos extracelulares insolúveis pela placa dentária in vitro e a inibição destas atividades metabólicas promovida pelo íon citrato. Para tal, foi utilizada uma suspensão de placa dentária (10 mg / ml de tampão fosfato 0,01 pH 7,0), que foi incubada na presença dos substratos a 37°C, durante 2 horas para fermentação e 18 horas para a síntese de polissacarídeos extracelulares insolúveis (PEI). A fermentação foi medida por titulação com solução de NaOH 0,05N. A avaliação da síntese de PEI foi realizada pela dosagem de carboidratos totais. O citrato de sódio foi acrescentado aos meios de incubação, nas concentrações de 50 e 100 mM. Os resultados demonstraram que a glicose produziu a maior quantidade de ácidos e a sacarose sintetizou a maior quantidade de polissacarídeos extracelulares insolúveis, quando omparados com outros carboidratos. O capaz de inibir significativamente a polissacarídeos os extracelulares insolúveis, quando comparados com outros carboidratos. O capaz de inibir significativamente a polissacarídeos os extracelulares insolúveis / Abstract: The purpose of this work was to evaluate the influence of carbohydrates often consumed in diet, as glucose, sucrose, fructose and dextrio, on fermentation and synthesis of extracellular insoluble polysaccarides of dental paque, in vitro and the inhibition of these metabolic activities produced by citrate. So, it was used a suspension of dental paque (10 mg/ml of phosphate buffer 0,01M pH 7,0 ), that was incubated at presence of substrates at 37 'DEGREE' C, for 2 hours for fermentation and 18 hours for synthesis of insoluble, palysaccarides. The fermentation was measured by titration with 0.05N NaOH solution. The estimation of the synthesis of insoluble polysaccarides was achieved by dosage of total carbohydrates. The sodium citrate was increased in an incubation medium on 50 and 100 mM of concentration. The results indicated that the glucose showed the largest acid production activity and sucrose promoted the greatest synthesis of insoluble polysaccarides, when compared with the other carbohydrates. The citrate was able to inhibit the carbohydrate fermentation and showed itself a great inhibitor of synthesis of extracellular insoluble polysaccarides / Mestrado / Fisiologia e Biofisica do Sistema Estomatognatico / Mestre em Odontologia

Orientador: Marcos Silveira Buckeridge / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T18:38:24Z (GMT). No. of bitstreams: 1 Lisboa_CesarGustavoSerafim_D.pdf: 1945488 bytes, checksum: 831197d663c65044a02bdfd7ba13598f (MD5) Previous issue date: 2008 / Resumo: Aplicações biotecnológicas de polissacarídeos de plantas: uma estratégia sustentável para a produção em demanda. Atualmente, a busca por novas fontes de materiais pode levar ao depósito de patentes. Isto é importante para que novos produtos, que apresentem características inovadoras, potencial de redução de custos, rotas de produção ambientalmente corretas, partição de benefícios e, finalmente, possibilitem a exploração de novos mercados de maneira sustentável. Nesse contexto, o presente trabalho teve como objetivo avaliar o potencial de uso de polissacarídeos de plantas de diferentes fontes nos setores de alimentos, farmacosméticos e papel. Os resultados são apresentados na forma de um artigo e 4 patentes. Os xiloglucanos de sementes de espécies nativas de plantas (Hymenaea courbaril) e de plântulas de eucalipto foram aplicados no processo de confecção do papel, demonstrando que suas propriedades mecânicas podem ser melhoradas. Resultados similares foram obtidos com galactomananos de sementes de Sesbania virgata e Dimorphandra mollis. O xiloglucano de H. courbaril foi estudado mais profundamente e demonstrou-se que pode ser também aplicado em cosméticos, pois não é citotóxico, é capaz de espessar formulações e interfere na produção de colágeno pela pele. As propriedades reológicas do galactomanano de D. mollis foram estudadas em comparação a outros galactomananos já utilizados na indústria. Demonstrou-se que ele é apropriado para várias aplicações em alimentos. Como um todo, nossos resultados indicam que tanto os polissacarídeos de sementes de espécies nativas como o de eucalipto podem ser utilizados em várias aplicações de tal forma que possam fazer parte de programas de uso sustentável da biodiversidade, levando a benefícios tecnológicos e também sociais. / Abstract: Biotechnological applications of plant polysaccharides: a sutainable strategy for production and demand. Nowdays, the search for new sources of materials can lead to new patent deposits. This is important because new products that display innovative features, cost reduction potential, environmentally clean production routes, partition of benefits and finally, may turn possible to explore differential market shares. These may be excellent socio-environmental and market shares for biotechnology companies. In this context, the present work aimed at evaluating the potential of use of plant polysaccharides from different sources in the food, farmacosmetic and paper industrial sectors. In the present work, we present studies that focus on the use of seed polysaccharides on applications on paper, cosmetics and food industries. The results are presented in the form of one article and 4 patents. The xyloglucan from seeds of native tropical tree species (Hymenaea courbaril - jatobá, Copaifera langsdorffii - copaiba) and from seedlings of Eucalyptus grandis (eucalypt) were applied to paper making and we demonstrated that several properties of paper can be improved by using these xyloglucans during preparation. Similar results were obtained with galactomann ans from Sesbania virgata and Dimorphandra mollis, two fast-growing legume trees. The polymers increased paper resistance to mechanical stress so that they can be applied for applications where more resistant papers (such as rapping paper) are used. The xyloglucan from H. courbaril was studied more deeply, demonstrating that it can be applied in cosmetics as it is not cytotoxic and can be used to improve skin biochemistry such as collagen production. Galactomannan from D. mollis was studied from the rheological point of view and its properties were compared with other commercially used galactomannans. Our results showed that D. mollis galactomannan is appropriate to several applications in food industries. As a whole, our results indicate that both, native seeds and seeds of eucalyptus, might be used in several industrial applications so that these might be used as part of programs of sustainable use of biodiversity, leading to social and technological benefits to society. / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Em muitas espécies vegetais alguns órgãos desempenham mais do que uma função em certos estágios da vida. Raízes, caules ou folhas começam a acumular substâncias de reserva e, dependendo da sua origem, podem ser transformados em órgão de reserva, como tubérculos, bulbos, rizóforos e raízes tuberosas. Entre os compostos de reversa, os carboidratos são responsáveis por diversas funções, tais como fonte de energia, proteção contra a seca e temperaturas extremas. Os campos rupestres são caracterizados por um clima mesotérmico, com três a cinco meses de seca, correspondendo ao inverno, e seis a oito meses de chuvas, que corresponde ao verão. Os solos são rasos, salinos e com afloramentos rochosos. A flora possui um alto grau de endemismo, sugerindo a existência de estratégias adaptativas metabólicas para sobreviver aos estresses ambientais. O objetivo do presente trabalho foi realizar uma triagem dos carboidratos não estruturais em diferentes órgãos de espécies herbáceas predominantes destas regiões e a análise anatômica do sistema subterrâneo de quatro espécies de Asteraceae para visualização e localização dos cristais de inulina. Foram coletadas 26 espécies em três regiões distintas: 14 na Serra do Cipó e, cinco na Serra de Itacambira (estado de Minas Gerais), e sete na Serra Dourada (estado de Góias), representantes das famílias Amaranthaceae, Orchidaceae, Eriocaulaceae, Velloziaceae, Apiaceae, Apocynaceae e Asteraceae, sendo a última a mais representativa em números de espécies. Carboidratos solúveis foram quantificados colorimetricamente e analisados cromatograficamente por CCD e HPAEC/PAD. Amido foi quantificado por método enzimático e cristais de inulina foram visualizados sob luz polarizada. Frutanos foram detectados nos órgão subterrâneos de reserva de todas as espécies de Asteraceae e Amaranthaceae. A maior concentração de frutose total foi encontrada em Gomphrena marginata (Amaranthaceae), compreendendo 30% da massa seca de seus órgãos subterrâneos. Lessingianthus psilophyllus e Richterago polymorpha (Asteraceae) também contêm altas porcentagens de açúcares solúveis (34% e 33%, respectivamente), dos quais 26 e 27% correspondem aos frutanos. Todas as Asteraceae apresentaram frutanos da série homóloga da inulina com alto grau de polimerização. Gomphrena agrestis e Gomphrena marginata (Amaranthaceae) apresentaram frutanos da série dos levanos. Vellozia mínima e Barbacenia plantaginea (Velloziaceae) apresentaram os oligossacarídeos da série da rafinose. De todas as espécies estudadas, somente Habenaria caldensis, Oncidium hidrophylum (Orchidaceae), Mandevilla tenuifolia (Apocynaceae) and Klotzschia brasiliensis (Apiaceae) acumulam amido como principal polissacarídeo de reserva em seus órgãos subterrâneos, enquantoem Leiothrix curvifolia (Eriocaulaceae) o amido foi detectado nos caules. Cristais de inulina foram visualizados n as quatro Asteraceae analisadas e se e localizam principalmente no cilindro vascular. Foi observada também, a ocorrência de estruturas secretoras em Chresta curumbensis e Strophopappus glomeratus. Este trabalho fornece informações úteis para expandir o conhecimento de estratégias fisiológicas das plantas para sobreviverem a condições ambientais adversas, como ocorre nos campos rupestres, e contribuir para estabelecer estratégias de conservação para a biodiversidade tropical. / In many plant species some organs perform more than one function at certain stages of the life cycle. Roots, stems or leaves begin to accumulate reserve substances and depending on the origin may be transformed into storage organs like tubers, bulbs, rhizophores and tuberous roots. Among other storage compounds, carbohydrates are assigned several functions such as source of energy and protection against drought and extreme temperatures. Rocky fields are characterized by mesothermal climate, with three to five months of dry season in winter, and seven to eight months of humidity in summer. The soils are shallow, sandy and with rocky outcrops. The flora has a high degree of endemism suggesting the existence of metabolic adaptive strategies to overcome environmental stresses. The aim of this work was to carry out a screening of reserve compounds accumulated in different organs of predominant herbaceous species, and to analyze the localization of inulin crystals in the underground system in four Asteraceae species.Twenty-six species of the following families, Amaranthaceae, Orchidaceae, Eriocaulaceae, Velloziaceae, Apiaceae, Apocynaceae and Asteraceae were collected in three regions: 14 at \"Serra do Cipó\" and five at \"Serra de Itacambira\" (state of Minas Gerais), and seven at \"Serra Dourada\" (state of Goiás). The Asteraceae was the most significant in species number. Soluble carbohydrates were quantified colorimetrically and analyzed chromatographically by TLC and HPAEC/PAD, and starch was quantified by enzymatic assay. Inulin crystals were visualized under polarized light. Fructans were detected in underground reserve organs of all the Asteraceae and Amaranthaceae species. The highest concentration of total fructose was found in Gomphrena marginata (Amaranthaceae) comprising 30% of the underground organ dry mass. Lessingianthus psilophyllus and Richterago polymorpha (Asteraceae) also contained high percentages of soluble carbohydrates on a dry mass basis (34% and 33%, respectively), from which 26% and 27% corresponded to fructans. All the Asteraceae analyzed presented the inulin homologous series with a high degree of polymerization while Gomphrena agrestis and G. marginata (Amaranthaceae) presented the levan series. Vellozia minina and Barbacenia plantaginea presented the raffinose family oligosaccharides. Of all the analyzed species, only Habenaria caldensis, Oncidium hidrophylum (Orchidaceae), Mandevilla tenuifolia (Apocynaceae) and Klotzschia brasiliensis (Apiaceae) accumulate starch as the main reserve carbohydrate in the underground organs while in Leiothrix curvifolia (Eriocaulaceae) starch is accumulated in stems. Inulin crystals were visualized mainly in the vascular cilynder. in the four Asteraceae analyzed. Secretory structures were identified in Strophopappus glomerathus and Chresta curumbensis This work provides information to enhance the knowledge on physiological strategies used by plants to survive adverse environmental conditions such as those predominating in rocky fields, and may contribute for the establishment of conservation strategies of tropical biodiversity.

Orientador: Marisa Masumi Beppu / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-24T04:07:39Z (GMT). No. of bitstreams: 1 Genevro_GiovanaMaria_M.pdf: 2115120 bytes, checksum: 180ca0fdf6523bd07a517e41100339f0 (MD5) Previous issue date: 2013 / Resumo: A busca incansável por qualidade de vida e longevidade torna necessário o desenvolvimento de novos materiais e tecnologias para uso médico. A engenharia tecidual é uma área de pesquisa recente que tem a finalidade de desenvolver materiais para substituir ou regenerar tecidos. Esses materiais devem apresentar uma série de características, incluindo biocompatibilidade, biodegradabilidade, resistência mecânica e porosidade. Dentre os materiais que vem sendo estudados para essa finalidade se destacam os polímeros naturais, em especial alguns polissacarídeos. Portanto, nesse estudo utilizou-se o polissacarídeo glucomanana de konjac (KGM), que é um biopolímero promissor, porém ainda pouco estudado para aplicação como scaffold, para a engenharia tecidual. A técnica de criogelificação é uma técnica interessante para modificar a morfologia dos hidrogéis, assim como aumentar a resistência mecânica dos mesmos. Para o preparo do hidrogel foram verificados os efeitos das variáveis, temperatura, concentração do polímero e concentração do agente alcalino. Os hidrogéis foram congelados a diferentes taxas de resfriamento e a temperaturas diferentes, sendo que a taxa é o fator mais relevante. O hidrogel com composição de 2 % de KGM, concentração da solução de Ca(OH)2 de 0,2 mol/L e temperatura de preparo de 65 °C apresentou consistência e propriedades próximas das quais se buscava. O congelamento lento apresentou estrutura com poros maiores, o que é interessante para a aplicação como biomaterial, pois as células precisam de espaço suficientemente grande para se desenvolverem. Os hidrogéis submetidos ao congelamento são cerca de quatro vezes mais resistentes à compressão do que os hidrogéis não congelados. Além disso, notou-se que a gelificação da KGM é lenta, sendo possível reduzir esse tempo com o processo de criogelificação / Abstract: The relentless pursuit of quality of life and longevity makes necessary the development of new materials and technologies for medical use. Tissue engineering is a recent area of research that aims to develop materials to replace or regenerate tissues. These materials should present a range of characteristics, including biocompatibility, biodegradability, mechanical strength and porosity. Among the materials that have been studied for this purpose we highlight the natural polymers, especially some polysaccharides. Therefore, in this study we used the polysaccharide konjac glucomannan (KGM) which is a promising biopolymer, but poorly studied for use as a scaffold for tissue engineering. For the preparation of the hydrogel, the effects of the variables, temperature, polymer concentration and concentration of alkali were observed. The hydrogel composition of 2% KGM, the solution concentration of Ca(OH)2 0,2 mol/L and preparation temperature of 65 °C showed consistency and properties of which are close sought. The cryogelation technique is an interesting technique to modify the morphology of the hydrogels, as well as increase the mechanical strength. The hydrogels were frozen at different cooling rates and different temperatures and the rate is the most relevant factor. The slow freezing showed structure with larger pores, which is interesting for application as biomaterial because the cells need space large enough to develop. The hydrogels subjected to freezing were about four times more resistant to compression. In addition, it was noted that gelation of KGM is slow, then it is possible to reduce this time using the cryogelation process / Mestrado / Engenharia de Processos / Mestra em Engenharia Química

Radix Astragali (the dried root of Astragalus membranaceous (Fisch) Bge.) and Dendrobii Officinalis Caulis (the dried stem of Dendrobium officinale Kimura et Migo) are two traditional Chinese tonic herbs. They are commonly used in the formula with other Chinese herbs for tonifying Qi, nourishing Yin, and treating various kinds of diseases, such as cancer, diabetes, inflammation, etc. The polysaccharides are considered the majority of the chemical components of decoction boiled from a formula including these two medicinal herbs. The previous study showed that the polysaccharides isolated from Radix Astragali (named RAP) and Dendrobii Officinalis Caulis (named DOP) have various pharmacological activities and most of their activities are closely related to their immunomodulating effects. Nonetheless, the exact mechanism of their immunomodulating effects, especially on macrophages is not known clearly. In the current study, we have conducted a comprehensive investigation of the bioactive properties and molecular mechanism of immunomodulating activities of DOP and RAP. We aimed to clarify the molecular immunomodulating mechanism of RAP on macrophages and the actual anti-fatigue activity of DOP in vivo. Results can be summarized as follows: RAP itself did not have any cytotoxic effect on mouse mammary carcinoma 4T1 cells, but it significantly enhanced cytotoxicity of the supernatant of RAW264.7cells on 4T1 cells. Furthermore, RAP enhanced the production of NO and cytokines in RAW264.7 cells, and significantly up-regulated gene expressions of TNF-α, IL-6, iNOS. All these bioactivities were blocked by the inhibitor of TLR4 (Toll-like receptor 4), suggesting that TLR4 is a receptor of RAP and mediates its immunomodulating activity. Further analyses demonstrated that RAP rapidly activated TLR4-related MAPKs, including phosphorylated ERK, phosphorylated JNK, and phosphorylated p38, and induced translocation of NF-κB as well as degradation of IκB-α. In addition, RAP induced higher gene expression of M1 marker, including iNOS, IL-6, TNF-a, CXCL10, compared with those of control group. RAP-induced BMDMs were polarized from M2 to M1 phenotypes. RAP stimulated RAW264.7 cells to express Notch1, Notch2, Jaddge1, Dll1 and SOCS3. Notch signaling pathway played an important role in the RAP-induced polarization of M1 phenotype macrophages. The RAP-induced BMDMs exhibited anti-cancer effect when they were transplanted with 4T1 cells together in vivo and it decreased tumor volume and tumor weight. DOP, the authentication marker of Dendrobii Officinalis Caulis, has immunomodulating activity in macrophage cell line RAW 264.7. DOP enhanced cell proliferation, TNF-α secretion, and phagocytosis in a dose-dependent manner. It induced the proliferation of lymphocytes alone and with mitogens. For further study the anti-fatigue effect of DOP in vivo, the weight-loaded swimming test was used, because it is an effective method for evaluation of the extent of fatigue. The results indicated that DOP treatment significantly increased the swimming endurance time, body weight, and food intake, compared to the positive control Rhodiola rosea extract. Moreover, the weight-loaded swimming test decreased the levels of glycogen in gastrocnemius muscle, SOD, GSH-Px in serum, and increased the levels of LDH, BUN, MDA, CK, TG, and LD in serum. All of these indicators of fatigue were inhibited to a certain extent by both DOP and Rhodiola rosea extract, and DOP's effects are stronger. Furthermore, DOP-feeding mice showed significantly increased cell variability of T lymphocytes and B lymphocytes, compared with control mice. In conclusion, RAP may induce cytokine production of RAW264.7 cells through TLR4-mediated activation of MAPKs and NF-κB. RAP-induced BMDMs were polarized from M2 to M1 phenotypes through Notch signaling pathway. The unique and dominant polysaccharide DOP is proven to be major, active polysaccharide markers of D. officinale, and showed stronger anti-fatigue activity than Rhodiola rosea extract. As such, DOP has promising potential for pharmaceutical development into anti-fatigue health product.

>Magister Scientiae - MSc / Marine macroalgae are known to carry diverse bacterial communities which interact with their hosts in both harmful and beneficial ways. Algae hosts provide the bacteria with a rich source of carbon in the form of carbohydrate polysaccharides such as fucoidan, agar and alginate, which the bacteria enzymatically degrade. Splachnidium rugosum is a brown alga (Phylum: Phaeophyta) that grows exclusively in the Southern Hemisphere along the temperate shores of South Africa, New Zealand and Australia. While several studies have investigated S. rugosum distribution and fucoidan production, the microbiome of S. rugosum remains largely uncharacterized. Thus, the major objective of the present study was to isolate, identify and characterize epiphytic bacterial communities associated with S. rugosum. Algae were sourced from Rooi Els (Western Cape, South Africa) during winter 2012. Culture based methods relied on a range of selective marine media including marine agar, nutrient sea water agar, nutrient agar and thiosulfate-citrate-bile-salts-sucrose agar to determine the composition and uniqueness of bacterial communities associated with S. rugosum. Epiphytic isolates were identified to species level by 16S rRNA gene sequence analysis and encompassed 39 Gram-negative and 2 Grampositive bacterial taxa. Isolates were classified into four phylogenetic groups, Gamma - Proteobacteria, Alpha-Proteobacteria, Firmicutes and Bacteriodetes. Bacteria belonging to the phylum Gamma-Proteobacteria were the most abundant, with Vibrio and Pseudoalteromonas being the dominant genera. Three isolates with low sequence identity (˂97%) to their closest relatives could possibly represent novel species. These isolates were grouped into the genera Shewanella, Sphingomonas and Sulfitobacter. All bacterial isolates (41) were screened for antimicrobial activity against the following test strains: Escherichia coli, Bacillus cereus, Staphylococcus epidermidis, Mycobacterium smegmatis Micrococcus luteus and Pseudomonas putida. Fifteen isolates (36%) displayed antimicrobial activity against one or more of the test strains, while one isolate (Pseudomonas species) showed broad spectrum antimicrobial activity against all the test strains except for E. coli. This study provides the first account of the diversity and composition of bacterial populations on the surface of S. rugosum, and demonstrates the ability of these bacteria to produce antimicrobial compounds. Despite recent advances in metagenomics, this study highlights the fact that traditional culturing technologies remain a valuable tool for the discovery of novel bioactive compounds of bacterial origin.

This study investigated the possible use of algal extracellular polysaccharide as a biosorbent for removal of heavy metals (copper and lead) from aqueous solutions as a means of bioremediation for metal containing effluents. This biopolymer has good biosorbent properties and a potential to provide a cost effective, selective and efficient purification system. A variety of environmental conditions induce the production of extracellular polysaccharides in algae. The production of exopolysaccharides by Dunaliella cultures was induced by nitrogen deficient conditions. A high ratio of carbon to nitrogen source considerably enhanced the polysaccharide release. Purified extracellular polysaccharide samples exhibited a monosaccharide composition consisting of the following sugars: xylose, arabinose, 2-0-methyl mannose, mannose, glucose and galactose. The relative abundance (%) of these sugars were calculated relative to xylose. The major sugar constituent was 2-0-methyl mannose, which was present at approximately 160% relative to xylose. The percentage relative abundance of other sugars was as follows: 18.8; 86.8; 85.3 and 22.3% for arabinose; mannose; glucose and galactose respectively. The identity of the various constituents were confirmed by mass spectrometry. The ability of Dunaliella exopolysaccharides to accumulate metals was investigated. The following parameters were studied because they affect metal uptake: solution pH, biomass concentration, temperature, time and metal concentration. The uptake of both copper and lead were pH dependent. However, metal uptake was not significantly affected by temperature. Kinetic studies showed that Dunaliella extracellular polysaccharides exhibit good bioremediation properties. Metal uptake was rapid. In addition, the exopolysaccharide has good metal binding capacity with an uptake capacity for lead of 80 mg/g from a solution containing initial lead concentration of approximately 40 mg/l. Competition studies revealed that the presence of a second metal in solution inhibits uptake of the other metal compared to uptake in single metal solution of that particular metal. The presence of lead inhibited the uptake of copper from approximately 65% in single metal solution to 10% in binary metal solution. The presence of copper also inhibited lead uptake, though not to the same extent. Higher concentrations of lead could not completely prevent removal of copper from solution and visa versa. The same was true for lead which could not be displaced by a four-fold concentration of copper. Instead, a certain percentage of copper was always removed showing that lead did not compete with copper for these binding sites. In conclusion it appears that, copper and lead bind to different sites on Dunaliella exopolysaccharides and that they exhibit selective or preferential removal of lead.

Cellulose and hemicellulose account for a large portion of the world‘s plant biomass. In nature, these polysaccharides are intertwined forming complex materials that require multiple enzymes to degrade them. Multi-enzyme complexes (MECs) consist of a number of enzymes working in close proximity and synergistically to degrade complex substrates with higher efficiency than individual enzymes. The cellulosome is a cellulolytic MEC produced by anaerobic bacteria that has been studied extensively since its discovery in 1983. The aim of this study was to purify a cellulolytic and/or hemicellulolytic MEC from an aerobic bacterium of the Bacillus genus. Several bacterial isolates were identified using morphological characteristics and 16S rDNA sequencing, and screened for their ability to degrade cellulose and xylan using a MEC. The isolate that produced a high molecular weight protein fraction with the greatest ability to degrade Avicel®, carboxymethyl cellulose (CMC) and birchwood xylan was identified as Bacillus subtilis SJ01. An optimised growth medium, consisting of vitamins, trace elements, birchwood xylan (as the carbon source), and yeast and ammonium sulphate (as the nitrogen sources), increased the production of CMCase and xylanase enzymes from this bacterium. The removal of a competing bacterial strain from the culture and the inhibition of proteases also increased enzyme activities. A growth curve of B. subtilis SJ01 indicated that xylanase production was highest in early stationary growth phase and thus 84 hours was chosen as the best cell harvesting time. To purify the MECs produced by B. subtilis SJ01 size-exclusion chromatography on a Sephacryl S-400 column was used. It was concluded that (for the purposes of this study) the best method of concentrating the culture supernatant prior to loading onto Sephacryl S-400 was the use of ultrafiltration with a 50 kDa cut-off membrane. Two MECs, named C1 and C2 of 371 and 267 kDa, respectively, were purified from the culture supernatant of B. subtilis SJ01. Electrophoretic analysis revealed that these MECs consisted of 16 and 18 subunits, respectively, 4 of which degraded birchwood xylan and 5 of which degraded oat spelt xylan. The MECs degraded xylan substrates (C1: 0.24 U/mg, C2: 0.14 U/mg birchwood xylan) with higher efficiency than cellulose substrates (C1: 0.002 U/mg, C2: 0.01 U/mg CMC), and could therefore be considered xylanosomes. Interestingly, the MECs did not bind to insoluble birchwood xylan or Avicel® and did not contain glycosylated proteins, which are common features of cellulosomes. This study is, therefore, important in revealing the presence of MECs that differ from the cellulosome and that may have particular application in industries requiring high xylanase activity, such as the paper and pulp industry. The abundant genetic information available on B. subtilis means that this organism could also be used for genetic engineering of cellulolytic/hemicellulolytic MECs.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19758号 / 農博第2154号 / 新制||農||1039(附属図書館) / 学位論文||H28||N4974(農学部図書室) / 32794 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 佐藤 健司, 教授 山下 洋, 准教授 豊原 治彦 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM